WO2021143902A1 - 用于检测HBcAg的方法及抗体 - Google Patents

用于检测HBcAg的方法及抗体 Download PDF

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WO2021143902A1
WO2021143902A1 PCT/CN2021/072483 CN2021072483W WO2021143902A1 WO 2021143902 A1 WO2021143902 A1 WO 2021143902A1 CN 2021072483 W CN2021072483 W CN 2021072483W WO 2021143902 A1 WO2021143902 A1 WO 2021143902A1
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antibody
seq
sequence
antigen
variable region
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PCT/CN2021/072483
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English (en)
French (fr)
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陈自敏
熊君辉
刘嘉祺
王邵娟
葛胜祥
袁权
宋浏伟
孙旭东
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厦门万泰凯瑞生物技术有限公司
厦门大学
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Priority to CA3167671A priority Critical patent/CA3167671A1/en
Priority to US17/791,415 priority patent/US20230069418A1/en
Priority to AU2021208790A priority patent/AU2021208790A1/en
Priority to JP2022543376A priority patent/JP2023510589A/ja
Priority to EP21741247.7A priority patent/EP4092050A4/en
Priority to KR1020227027563A priority patent/KR20220130723A/ko
Publication of WO2021143902A1 publication Critical patent/WO2021143902A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • G01N33/5762—Hepatitis B core antigen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

Definitions

  • the present invention relates to the field of hepatitis B virus (HBV) detection. Specifically, the present invention provides a method for detecting HBcAg using a double antibody sandwich method, as well as antibodies and kits for the above detection. The invention also provides a monoclonal antibody that can be used for HBcAg immunological detection of tissue or cell samples.
  • HBV hepatitis B virus
  • Hepatitis B virus infection is one of the most important public health problems in the world (Rocag JL. Hepatitis B virus infection. N Engl J Med 2008 October 2; 359(14): 1486-1500).
  • HBV serum markers such as: HBsAg, HBsAb, HBeAg, HBeAb, HBcAb, commonly known as "two-and-a-half" detection
  • HBV serum markers such as: HBsAg, HBsAb, HBeAg, HBeAb, HBcAb, commonly known as "two-and-a-half" detection
  • HBsAg, HBsAb, HBeAg, HBeAb, HBcAb commonly known as "two-and-a-half” detection
  • HBV DNA is a direct indicator of HBV replication
  • its dot blot test (or PCR test) is the gold standard for judging the infection and infectivity of hepatitis B patients and HBV carriers. Both PCR detection and dot blot detection can be used as direct indicators of HBV infection and infectivity, but they are not suitable for large-scale census and routine use.
  • HBcAg has always been considered as an antigen directly related to HBV DNA. The detection is based on the quantitative and HBsAg-negative HBV infection and HBV patients have unique diagnostic significance. At present, there is no specific HBcAg detection reagent on the market.
  • HBcAg immunodiagnostic reagents that have been reported or developed are usually pre-processing the sample before detection (lysis of the virus, rupture of the membrane and fire HBcAb) or a circuitous detection of HBcAg-HBcAb immune complex
  • the former is complicated and cumbersome, and it is not easy to be accepted by clinical customers. It is also not suitable for large-scale blood donor screening and epidemiological investigation. The latter is difficult to achieve the desired specificity due to the particularity of the detection method. And sensitivity.
  • the HBcAg detection reagent method mentioned in the patent "a method for combined detection of hepatitis B virus pre-S1 antigen and core antigen and diagnostic kit" reported in 2006 is to use sAg antibody to capture virus particles and then break the membrane. The split virus detects cAg in the nucleus, but the sensitivity is not enough.
  • the inventors unexpectedly discovered an antibody pair that binds to a specific epitope, which is particularly suitable for the detection of HBcAg double antibody sandwich method.
  • the inventors developed a new HBcAg quantitative detection kit and detection method.
  • the sensitivity of the detection method can reach the level of DNA, and it can realize rapid and high-throughput detection, and has great clinical application value.
  • the present invention provides a kit comprising:
  • 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 an antigen-binding fragment thereof that sexually binds to the epitope contained in positions 150-183 of the HBcAg protein; and,
  • 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 binds to the epitope contained in positions 141-154 of the HBcAg protein.
  • the expression "epitope contained in positions 150-183 of the HBcAg protein” or similar expressions means that the epitope exists within or overlaps with amino acids 150-183 of the HBcAg protein.
  • the antibody or antigen-binding fragment thereof capable of specifically binding to the epitope contained in positions 150-183 of HBcAg protein is an antibody or its antigen-binding fragment capable of specifically binding to amino acids 150-183 of HBcAg protein or a fragment thereof Antigen-binding fragments.
  • the HBcAg protein has the sequence shown in SEQ ID NO:17.
  • the second antibody is selected from an antibody or an antigen-binding fragment thereof that can specifically bind to an epitope contained in positions 141-152 of the HBcAg protein.
  • the first antibody is selected from the following antibodies or antigen-binding fragments thereof:
  • An antibody or an antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDR): HCDR1 with the sequence of SEQ ID NO: 3, and the sequence of SEQ ID NO: 4 HCDR2 and HCDR3 of SEQ ID NO: 5; and/or, a light chain variable region (VL) comprising the following 3 complementarity determining regions (CDR): LCDR1 of SEQ ID NO: 6 LCDR2 with the sequence of SEQ ID NO: 7 and LCDR3 with the sequence of SEQ ID NO: 8; or,
  • An antibody or an antigen-binding fragment thereof which comprises: a heavy chain variable region (VH) comprising 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO:1; and/or, comprising SEQ ID
  • the 3 CDRs contained in the chain variable region are defined by the Kabat, Chothia or IMGT numbering system; or,
  • an antibody or an antigen-binding fragment thereof, the antibody is a monoclonal antibody produced by the hybridoma cell line 18B2-2, wherein the hybridoma cell line 18B2-2 is deposited in the Chinese Center for Type Culture Collection (CCTCC), and Has the deposit number CCTCC NO.C2019303.
  • CCTCC Chinese Center for Type Culture Collection
  • the first antibody comprises:
  • VH Heavy chain variable region
  • SEQ ID NO:1 amino acid sequence selected from the following: (i) the sequence shown in SEQ ID NO:1; (ii) compared with the sequence shown in SEQ ID NO:1 A sequence of substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); or (iii) shown in SEQ ID NO:1
  • the sequence has 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 a sequence with 100% sequence identity;
  • the light chain variable region which comprises an amino acid sequence selected from the following: (iv) the sequence shown in SEQ ID NO: 2; (v) compared with the sequence shown in SEQ ID NO: 2 A sequence of substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); or (vi) shown in SEQ ID NO: 2
  • the sequence has 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 a sequence with 100% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • 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.
  • the second antibody is selected from the following antibodies or antigen-binding fragments thereof:
  • An antibody or an antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDR): HCDR1 with a sequence of SEQ ID NO: 11, and a sequence of SEQ ID NO: HCDR2 of 12 and HCDR3 of SEQ ID NO: 13; and/or, a light chain variable region (VL) comprising the following 3 complementarity determining regions (CDR): LCDR1 of SEQ ID NO: 14 LCDR2 with the sequence of SEQ ID NO: 15 and LCDR3 with the sequence of SEQ ID NO: 16; or,
  • An antibody or an antigen-binding fragment thereof which comprises: a heavy chain variable region (VH) comprising 3 CDRs contained in the heavy chain variable region shown in SEQ ID NO: 9; and/or, comprising SEQ ID
  • the light chain variable region (VL) of the 3 CDRs contained in the light chain variable region shown in NO: 10 preferably, the 3 CDRs contained in the heavy chain variable region, and/or the light chain variable region
  • the 3 CDRs contained in the chain variable region are defined by the Kabat, Chothia or IMGT numbering system; or,
  • Antibody or antigen-binding fragment thereof is a monoclonal antibody produced by hybridoma cell line 2A7, wherein the hybridoma cell line 2A7 is deposited in the Chinese Center for Type Culture Collection (CCTCC) and has the deposit number CCTCC NO.C2019302.
  • CTCC Chinese Center for Type Culture Collection
  • the second antibody comprises:
  • Heavy chain variable region which comprises an amino acid sequence selected from the following: (i) the sequence shown in SEQ ID NO: 9; (ii) compared with the sequence shown in SEQ ID NO: 9 A sequence of substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); or (iii) shown in SEQ ID NO: 9
  • the sequence has 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 a sequence with 100% sequence identity;
  • the light chain variable region which comprises an amino acid sequence selected from the following: (iv) the sequence shown in SEQ ID NO: 10; (v) compared with the sequence shown in SEQ ID NO: 10 A sequence of substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); or (vi) and SEQ ID NO: 10
  • the sequence has 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 a sequence with 100% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • 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.
  • the first antibody and/or the second antibody comprise a heavy chain constant region (CH) and a light chain constant region (CL).
  • CH heavy chain constant region
  • CL light chain constant region
  • the first antibody and/or the second antibody comprise a mouse heavy chain constant region and a mouse light chain constant region.
  • the first antibody and/or the second antibody are IgG, IgM, IgE, IgD, or IgA antibodies. In certain embodiments, the first antibody and/or the second antibody are IgG antibodies.
  • the antigen-binding fragment is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).
  • the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.
  • the second antibody bears a detectable label.
  • the kit further includes a third antibody capable of specifically binding to the second antibody, and the third antibody carries a detectable label.
  • the detectable label may be any substance that can be detected by fluorescence, spectroscopy, photochemical, biochemical, immunological, electrical, optical, or chemical means. It is particularly preferable that such a label can be applied to immunological detection (for example, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).
  • immunological detection for example, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.
  • Such labels include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), algae Red protein (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g.
  • enzymes e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.
  • radionuclides For example, 3 H, 125 I, 35 S, 14 C or 32 P
  • fluorescent dyes for example, fluorescein isothi
  • markers covered in the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation calculator, and fluorescent labels can be detected using a light detector to detect the emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate. Calorimetric markers are detected by simple visualization of colored markers.
  • Chemiluminescent substances (such as acridine ester compounds) generally detect the emitted light by providing excitation liquid and/or catalyst to the luminescent substance.
  • Biotin is generally detected by providing biotin with avidin (for example, streptavidin) modified by the above-mentioned label and detecting the label carried by the avidin linked to biotin.
  • avidin for example, streptavidin
  • the detectable label as described above can be attached to the antibody or antigen-binding fragment thereof of the present invention through linkers of different lengths to reduce potential steric hindrance.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent reagent such as acridine ester compound
  • a fluorescent dye such as acridine ester compound
  • biotin biotin
  • the kit may further comprise reagents for allowing the corresponding detectable label to be detected.
  • the detectable label when the detectable label is an enzyme, the kit may also include a color substrate for the corresponding enzyme, such as o-phenylenediamine (OPD) and tetramethylbenzidine for horseradish peroxidase. (TMB), ABTS or luminol compounds, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase.
  • OPD o-phenylenediamine
  • TMB horseradish peroxidase.
  • ABTS horseradish peroxidase.
  • p-NPP p-nitrophenyl phosphate
  • AMPPD p-nitrophenyl phosphate
  • the detectable label when the detectable label is a chemiluminescence reagent (e.g., acridine ester compound), the kit may also include a pre-excitation solution and/
  • the kit further comprises a solid phase carrier.
  • the solid-phase carrier includes a well plate, test tube, beads (e.g., latex) made or coated with a polymer material (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose). Particles) or films (such as nitrocellulose membranes), or magnetic beads pre-coated with functional groups (such as amino, carboxyl, biotin or avidin).
  • the solid phase carrier is selected from magnetic beads or microtiter plates (for example, microtiter plates or microtiter plates).
  • the kit further comprises a coating reagent for coating the first antibody on the solid support, such as a coating buffer (e.g., carbonate buffer, phosphoric acid Salt buffer, Tris-HCL buffer or borate buffer).
  • a coating buffer e.g., carbonate buffer, phosphoric acid Salt buffer, Tris-HCL buffer or borate buffer.
  • the first antibody is coated on the surface of the solid support.
  • the kit includes at least the above-mentioned solid phase carrier and the above-mentioned first antibody in a separate container or in a separate compartment of a single container unit.
  • the kit includes: a first antibody, and a second antibody with a detectable label. In certain exemplary embodiments, the kit includes: a first antibody coated on the surface of a solid-phase carrier, and a second antibody with a detectable label. In certain exemplary embodiments, the kit includes: one or more first antibodies, and a second antibody with a detectable label. In certain exemplary embodiments, the kit includes: one or more first antibodies coated on the surface of a solid-phase carrier, and a second antibody with a detectable label. In certain embodiments, the plurality of first antibodies recognize different epitopes contained in positions 150-183 of the HBcAg protein.
  • the kit further comprises a lytic agent for lysing HBV virus particles.
  • a lysing agent for lysing HBV virus particles refers to any agent capable of lysing Dane particles (ie, destroying the viral envelope) to expose the HBcAg antigen.
  • Such reagents are known to those skilled in the art, such as surfactants such as NP40, LDS or SDS.
  • the lysing agent comprises LDS or SDS.
  • the kit further comprises a neutralizing agent, and the neutralizing agent comprises CHAPS.
  • the lysing agent comprises 20% LDS or 20% SDS.
  • the lysing agent comprises 20% LDS or 20% SDS and the balance water.
  • the neutralizer comprises 10% CHAPS.
  • the neutralizer comprises 10% CHAPS, 20 mM PBS.
  • the neutralizer includes 10% CHAPS, 20 mM PBS, and the balance water.
  • the kit further comprises one or more reagents or devices selected from the following: standards (for example, a series of samples containing different known amounts of HBcAg); positive control samples (for example, containing A sample with a known amount of HBcAg); a negative control sample (for example, a sample that does not contain HBcAg); a lysing agent (and optionally a neutralizing agent) for lysing HBV virus; and, for collecting or storing the sample to be tested Devices (such as blood collection devices).
  • standards for example, a series of samples containing different known amounts of HBcAg
  • positive control samples for example, containing A sample with a known amount of HBcAg
  • a negative control sample for example, a sample that does not contain HBcAg
  • a lysing agent and optionally a neutralizing agent for lysing HBV virus
  • the first antibody and the second antibody described in the first aspect can be prepared by various methods known in the art, for example, obtained by genetic engineering recombination technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into the expression vector and then transfected into the host cell. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present invention is expressed.
  • the antigen-binding fragment described in the first aspect can be obtained by hydrolyzing a complete antibody molecule (see Morimoto et al., J.Biochem.Biophys.Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985). )).
  • these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium.
  • the present invention provides a kit comprising:
  • 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 as in the first aspect Definition; and,
  • 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 as in the first aspect definition.
  • the vector is a cloning vector or an expression vector.
  • the vector is, for example, a plasmid, cosmid, phage, and the like.
  • 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; expressing the second antibody
  • a recombinant cell is a host cell containing an isolated nucleic acid molecule encoding the second antibody or a vector containing the isolated nucleic acid molecule.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the present invention is a mammalian cell, such as CHO (e.g. CHO-K1, CHO-S, CHO DG44).
  • the recombinant cell expressing the first antibody is the hybridoma cell line 18B2-2, which is deposited in the Chinese Center for Type Culture Collection (CCTCC) and has the deposit number CCTCC NO.C2019303; and, express The recombinant cell of the second antibody is the hybridoma cell line 2A7, which is deposited in the Chinese Center for Type Culture Collection (CCTCC), and has the deposit number CCTCC NO.C2019302.
  • the present invention provides a method for detecting the presence or level of HBcAg protein in a sample, which includes the following steps:
  • the method can be used for diagnostic purposes, or for non-diagnostic purposes.
  • the methods of the invention are used for non-diagnostic purposes.
  • the sample to be tested is known to contain HBcAg, that is, before the method of the present invention is applied for detection, the same subject of the sample already has a diagnosis result; therefore, the method of the present invention is useful for the diagnosis of the sample.
  • the steps are not helpful. It can be seen that the direct purpose of the method of the present invention is not to obtain the diagnostic result of the same subject of the sample, but to perform further accurate quantitative detection of the sample with known diagnostic information.
  • the second antibody bears a detectable label.
  • the determination described in step (3) includes the following steps: (3a) detecting the amount of detectable label; (3b) comparing the amount of detectable label obtained in step (3a) with the amount indicating that HBcAg has been The standard curve of the relationship between the known amount and the amount of the detectable label is compared, and the HBcAg content is obtained.
  • the determination described in step (3) includes the following steps: (3a) detecting the amount of detectable label (e.g. luminescence value); (3b) comparing the detectable label obtained in step (3a) The amount (for example, the luminescence value) is compared with the Cutoff value.
  • the sample When the ratio is less than 1, the sample is considered negative, and when the ratio is greater than or equal to 1, the sample is considered HBcAg positive.
  • the Cutoff value when the detectable label is an acridine ester compound, the Cutoff value is 9000.
  • the second antibody does not carry a detectable label.
  • the assay described in step (3) includes the use of a third antibody with a detectable label to detect the antibody-antigen-antibody complex.
  • the third antibody can specifically bind to the second antibody (e.g., can specifically bind to the constant region of the second antibody).
  • the assay described in step (3) may include the following steps: (3a) contacting the antibody-antigen-antibody complex with a third antibody with a detectable label; (3b) detecting The amount of detectable label; (3c) The amount of detectable label obtained in step (3b) is compared with a standard curve representing the relationship between the known amount of HBcAg and the amount of detectable label, and the HBcAg content is obtained.
  • the assay described in step (3) includes the following steps: (3a) contacting the antibody-antigen-antibody complex with a third antibody with a detectable label; (3b) the detection can be The amount of the detectable label (e.g.
  • step (3c) The amount of the detectable label (e.g. luminescence value) obtained in step (3b) is compared with the Cutoff value. When the ratio is less than 1, the sample is considered negative When the ratio is greater than or equal to 1, the sample is considered positive for HBcAg. In some embodiments, when the detectable label is an acridine ester compound, the Cutoff value is 9000.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridine ester compound), a fluorescent dye, or biotin.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent reagent such as acridine ester compound
  • a fluorescent dye such as acridine ester compound
  • biotin biotin
  • the determination is selected from enzyme immunoassay or chemiluminescence immunoassay.
  • the method before step (1), further includes a step of processing the sample, and the processing includes: mixing a lysing agent with the sample to lyse the virus.
  • the treatment further includes the use of a neutralizing agent to terminate the cleavage reaction.
  • the lysing agent, neutralizing agent is as defined in the first aspect.
  • the first antibody is coated on the surface of a solid support.
  • the solid phase carrier is selected from magnetic beads or microtiter plates (for example, microtiter plates or microtiter plates).
  • a washing step is further included before step (2) and/or step (3).
  • the washing step can remove substances that are not involved in the reaction.
  • the sample is selected from whole blood, plasma, and serum.
  • the present invention also relates to the use of the kit described in the first aspect in preparing a detection kit for detecting the presence or level of HBcAg protein in a sample.
  • the kit detects the presence or level of HBcAg protein in the sample by the method described in the third aspect.
  • the anti-HBcAg antibodies currently used in the HBcAg immunological detection of tissue or cell samples are polyclonal antibodies.
  • Polyclonal antibodies can improve the sensitivity of detection, but they often have high background and specificity. It is low, and the results of immunohistochemistry using it are not easy to standardize.
  • anti-HBcAg monoclonal antibodies for HBcAg immunological detection of tissue or cell samples there is currently no report on the use of anti-HBcAg monoclonal antibodies for HBcAg immunological detection of tissue or cell samples.
  • the inventors unexpectedly discovered a monoclonal antibody suitable for HBcAg immunological detection of tissue or cell samples. Based on the detection effect of the monoclonal antibody, the detection effect can reach a level equivalent to that of commercial polyclonal antibodies, which is remarkable. Unexpected and very advantageous technical effect.
  • the present invention also provides a monoclonal antibody or an antigen-binding fragment thereof capable of specifically binding HBcAg, wherein:
  • the monoclonal antibody or antigen-binding fragment thereof includes: a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDR): HCDR1 with a sequence of SEQ ID NO: 11, and a sequence of SEQ ID HCDR2 of NO: 12 and HCDR3 of SEQ ID NO: 13; and/or, a light chain variable region (VL) containing the following 3 complementarity determining regions (CDRs): sequence of SEQ ID NO: 14 LCDR1, LCDR2 with the sequence of SEQ ID NO: 15 and LCDR3 with the sequence of SEQ ID NO: 16; or,
  • the monoclonal antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the three CDRs contained in the heavy chain variable region shown in SEQ ID NO: 9; and/or, The light chain variable region (VL) of the 3 CDRs contained in the light chain variable region shown in SEQ ID NO: 10; preferably, the 3 CDRs contained in the heavy chain variable region, and/or the The 3 CDRs contained in the variable region of the light chain are defined by the Kabat, Chothia or IMGT numbering system; or,
  • the monoclonal antibody is a monoclonal antibody produced by the hybridoma cell line 2A7, wherein the hybridoma cell line 2A7 is deposited in the Chinese Center for Type Culture Collection (CCTCC) and has the deposit number CCTCC NO.C2019302.
  • CTCC Chinese Center for Type Culture Collection
  • the monoclonal antibody or antigen-binding fragment thereof comprises:
  • Heavy chain variable region which comprises an amino acid sequence selected from the following: (i) the sequence shown in SEQ ID NO: 9; (ii) compared with the sequence shown in SEQ ID NO: 9 A sequence of substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); or (iii) shown in SEQ ID NO: 9
  • the sequence has 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 a sequence with 100% sequence identity;
  • the light chain variable region which comprises an amino acid sequence selected from the following: (iv) the sequence shown in SEQ ID NO: 10; (v) compared with the sequence shown in SEQ ID NO: 10 A sequence of substitution, deletion or addition of one or several amino acids (for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); or (vi) and SEQ ID NO: 10
  • the sequence has 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 a sequence with 100% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the monoclonal 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.
  • the monoclonal antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL).
  • the monoclonal antibody is an IgG, IgM, IgE, IgD, or IgA antibody.
  • the antigen-binding fragment is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).
  • the monoclonal antibody is a murine antibody, a chimeric antibody, or a humanized antibody.
  • the present invention also relates to the use of the monoclonal antibody or antigen-binding fragment thereof as described in the fourth aspect in preparing reagents for detecting HBcAg in a sample.
  • the sample is a tissue sample (e.g., a tissue section) or a cell sample.
  • the test is an immunological test.
  • the immunological detection is selected from the group consisting of Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunofluorescence (IF) and Western Blot.
  • the monoclonal antibody or antigen-binding fragment thereof bears a detectable label.
  • the reagent for detecting HBcAg in a sample further comprises a secondary antibody with a detectable label.
  • the secondary antibody is specific for the antibody of the species (for example, mouse) from which the constant region contained in the monoclonal antibody or antigen-binding fragment thereof is derived.
  • the secondary antibody is an anti-immunoglobulin antibody, such as an anti-IgG antibody.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a fluorescent dye, or biotin.
  • the detectable label is selected from enzymes.
  • the detectable label is selected from fluorescent dyes.
  • HBcAg refers to the core antigen of hepatitis B virus (HBV), also known as nucleocapsid protein, which is well known to those skilled in the art (see, for example, NCBIGENBANK database accession number : GU357842.1).
  • HBV hepatitis B virus
  • the HBcAg protein contains an assembly region at its N-terminus that participates in VLP assembly, and an arginine-rich domain (Arginine Rich Domain, ARD) at its C-terminus.
  • amino acid sequence of HBcAg when referring to the amino acid sequence of HBcAg, it uses the sequence shown in SEQ ID NO: 17 for description.
  • amino acid residues 150-183 of HBcAg refers to amino acid residues 150-183 of the polypeptide shown in SEQ ID NO: 17.
  • mutations or variations including but not limited to substitution, deletion and/or addition, such as HBcAg of different genotypes or genotypes
  • the term "HBcAg” shall include all such sequences, including, for example, the sequence shown in SEQ ID NO: 17 and its natural or artificial variants. Moreover, when describing the sequence fragment of HBcAg, it includes not only the sequence fragment of SEQ ID NO: 17, but also the corresponding sequence fragment in its natural or artificial variant. For example, the expression "amino acid residues 150-183 of HBcAg” includes amino acid residues 150-183 of SEQ ID NO: 17, and corresponding fragments in its variants (natural or artificial).
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percent identity, the compared sequences are located in the same position Fragments.
  • HBcAg usually exists in the core of Dane particles. Therefore, in order to detect HBcAg, it is usually necessary to lyse the shell of Dane particles to expose the HBcAg freely.
  • K D value refers to the ratio of kd (specific binding molecule-target molecule interaction dissociation rate; also called koff) to ka (specific binding molecule-target molecule interaction rate of association; also called kon)
  • M molar concentration
  • an antibody that specifically binds to a certain antigen means that the antibody has a concentration of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, The affinity (K D ) of 10 -8 M, 10 -9 M, or 10 -10 M or less binds the antigen.
  • the K D value can be determined by a method well known in the art, for example, measured in a BIACORE instrument using surface plasmon resonance (SPR).
  • immunological detection refers to a determination that uses the specific interaction/binding affinity between an antigen and an antibody. It can generally be used to detect the presence or presence of a specific antigen or antibody in a sample. Level. Such immunological tests are well known to those skilled in the art, and include, but are not limited to, enzyme immunoassay (EIA), chemiluminescence immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA) , Western blotting, immunoturbidimetry, surface plasmon resonance, etc.
  • EIA enzyme immunoassay
  • CLIA chemiluminescence immunoassay
  • RIA radioimmunoassay
  • FFA fluorescence immunoassay
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a light chain (LC) and a heavy chain (HC)).
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL. Constant domains are not directly involved in the binding of antibodies and antigens, but exhibit a variety of effector functions, such as mediating immunoglobulins and host tissues or factors, including various cells of the immune system (for example, effector cells) and classical complement The combination of the first component (C1q) of the system.
  • VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form an antigen binding site.
  • the assignment of amino acids in each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Definition of Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • Each of the variable regions of the heavy chain and the light chain contains three CDRs, named CDR1, CDR2, and CDR3.
  • CDR1, CDR2, and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to 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), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention is preferably determined by the Kabat, Chothia or IMGT numbering system.
  • the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined by the Kabat numbering system.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • antibody is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • 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 It is also called “antigen binding part” for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Recombinant DNA technology can be used. Or through the enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabody, single domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis) and such polypeptides, which contain enough to confer specific antigen binding ability to the polypeptide At least a portion of the antibody.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • full-length antibody means an antibody composed of two “full-length heavy chains” and two “full-length light chains.”
  • full-length heavy chain refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), and a heavy chain in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • HR hinge region
  • heavy chain constant region CH3 domain are composed; and, when the full-length antibody is of the IgE isotype, it optionally also includes the heavy chain constant region CH4 domain.
  • the "full-length heavy chain” is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction.
  • a "full-length light chain” is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
  • the two pairs of full-length antibody chains are connected by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full-length heavy chains.
  • the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
  • the full-length antibody of the present invention includes two antigen binding sites formed by a pair of VH and VL respectively, and the two antigen binding sites specifically recognize/bind the same antigen.
  • the term “Fd” means an antibody fragment composed of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment composed of VH domains (Ward et al., Nature 341:544 546 ( 1989))
  • the term “Fab fragment” means an antibody fragment composed of VL, VH, CL and CH1 domains
  • the term “F(ab') 2 fragment” means two fragments connected by a disulfide bridge on the hinge region An antibody fragment of a Fab fragment
  • the term “Fab'fragment” means a fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and heavy chain Fd Fragment (consisting of VH and CH1 domains).
  • Fv means an antibody fragment composed of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen binding site. It is generally believed that the six CDRs confer the antigen binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind antigen, although its affinity may be lower than the complete binding site.
  • Fc means a disulfide bond formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain.
  • Antibody fragments The Fc fragment of an antibody has many different functions, but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected 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, Volume 113, Roseburg and Moore eds, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • the term "diabody” means that its VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains of the same chain, Thereby forcing the domain to pair with the complementary domain of the other chain and create 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)).
  • single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to the structure of a single monomer variable antibody domain (e.g., a single heavy chain variable antibody). Region), which retains the ability to specifically bind to the same antigen that the full-length antibody binds.
  • Single domain antibodies are also called nanobodies.
  • Each of the aforementioned antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of antibodies e.g., the aforementioned antibody fragments
  • a given antibody e.g., the antibody provided by the present invention
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • chimeric antibody refers to an antibody whose light chain or/and part of its heavy chain is derived from an antibody (which may be derived from a specific species or belong to a certain species).
  • a specific antibody class or subclass), and another part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but no matter However, it still retains the binding activity to the target antigen (USP 4,816,567 to Capability et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from the first antibody (e.g., murine antibody), and the heavy chain and The light chain variable region is derived from a second antibody (e.g., a human antibody).
  • first antibody e.g., murine antibody
  • second antibody e.g., a human antibody
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology with the sequence of a human antibody.
  • CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from human source.
  • Humanized antibodies generally retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc.
  • the donor antibody may be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody with desired properties (e.g., antigen specificity, affinity, reactivity, etc.).
  • the chimeric antibody or humanized antibody of the present invention can be prepared based on the sequence of the murine monoclonal antibody prepared above.
  • DNA encoding the heavy and light chains can be obtained from the target murine hybridoma and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.
  • the murine immunoglobulin variable region can be linked to the human immunoglobulin constant region using methods known in the art.
  • DNA encoding VH is operably linked to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene.
  • the sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ), DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region.
  • DNA encoding VL is operably linked 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 sequence of the human light chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ), DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 fruit fly cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • the molecules are the same at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput.
  • conservative substitution means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • 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 of amino acid residues with similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties, including The ability to form covalent bonds or hydrogen bonds, etc.) is replaced by residues. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • alanine, valine, leucine, isoleucine Acid, proline, phenylalanine, methionine), beta branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well 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), which is incorporated herein by reference).
  • subject includes, but is not limited to, various animals, particularly mammals, such as humans.
  • the invention provides a HBcAg detection kit based on a specific antibody and a double antibody sandwich method based on the establishment of the kit. Compared with the prior art, the technical solution of the present invention can achieve detection sensitivity equivalent to that of DNA, and can realize rapid and high-throughput detection, and has great clinical application value.
  • the present invention also provides an anti-HBcAg monoclonal antibody, which can be used in the field of immunological detection of various tissue or cell samples such as immunohistochemistry and immunofluorescence, and can achieve detection similar to that of commercial polyclonal antibodies. Therefore, it has broad application prospects.
  • Figure 1 shows a schematic diagram of eukaryotic expression plasmids containing HBV antigens of different lengths.
  • Figure 2 shows the western blot analysis results of 2A7 as the primary antibody for HBV antigens of different lengths.
  • Figure 3 shows the detection results of HBcAg in different samples by the enzyme immunoassay method of the present invention.
  • Figure 4 shows the correlation between the chemiluminescence detection method of the present invention and PCR detection results.
  • Figure 5 shows the immunofluorescence detection result of 2A7 as the HBcAg immunofluorescence detection antibody on the cell sample.
  • Figure 6 shows the immunohistochemical detection results of 2A7 as the HBcAg immunohistochemical detection antibody on tissue sections.
  • the present invention relates to the following biological materials that have been preserved in the China Type Culture Collection (CCTCC, Wuhan University, Wuhan, China):
  • Hybridoma cell line 18B2-2 which has the preservation number CCTCC NO.C2019303, and the preservation time is November 28, 2019;
  • Hybridoma cell line 2A7 which has the deposit number CCTCC NO.C2019302, and the preservation time is November 28, 2019.
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compiled Molecular Biology Experiment Guide, 3rd Edition, John Wiley & Sons, Inc., 1995; the restriction enzymes are used in accordance with the conditions recommended by the product manufacturer.
  • the reagents whose sources are not indicated in the examples are all conventional reagents in the field or commercially available reagents.
  • the collected bacteria liquid was ultrasonically broken, the broken liquid was centrifuged at 12000 rpm at 10°C for 10 min, and the supernatant was collected. Then, in a water bath at 65°C for 20 minutes, the supernatant was collected.
  • the immunogen is recombinantly expressed HBcAg (C183 antigen) protein in E. coli. Dilute the recombinant antigen to 0.4mg/mL and mix it with Freund’s adjuvant in equal volume to make it into a water-in-oil emulsion (method to judge whether the mixture is emulsified completely: drop a small drop of the mixed liquid on the surface of the clear water Above, if the mixed liquid does not aggregate, it can be considered that it has been basically mixed). Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for the subsequent booster immunization, and no adjuvant was added for the last booster immunization 72 hours before fusion.
  • mice Use the above immunogen to immunize 6-8 weeks old BALB/c female mice with multiple subcutaneous injections. The injection dose is 500 ⁇ L/mouse/time, and 200 ⁇ L of eyeball venous blood is collected before each immunization. Prepare titer determination. Boost immunization every 2 weeks. The serum titer was measured by indirect ELISA. When the mouse serum titer reached the plateau, the mice stopped immunization and rested for two months before fusion.
  • mice spleen was taken to make a cell suspension and the mouse myeloma cell Sp2/0 was cell-fused to obtain hybridoma cells.
  • feeder cells Prior to this, feeder cells were prepared. In the process of culturing hybridoma cells, a large number of myeloma cells and spleen cells after fusion died one after another in the 1640-HAT medium. Single cells or a few scattered cells are not easy to survive, and other cells must be added to make them survive. The live cells that are added are called feeder cells. This laboratory uses mouse peritoneal macrophages or 13-day-old mouse thymocytes as feeder cells.
  • mouse macrophages proceed as follows: (i) A BALB/c mouse about 6 weeks old was put to death by neck, rinsed with tap water, and immersed in 75% ethanol solution for 5 minutes; Clean the workbench so that the abdomen of the mouse faces upward; lift the skin of the mouse abdomen with tweezers, cut a small cut, and use large tweezers to tear the skin up and down to fully expose the abdomen.
  • mice thymocytes proceed as follows: (i) A BALB/c mouse about 13 days old was put to death by neck, rinsed with tap water, and soaked in 75% ethanol solution for 5 minutes; put into ultra-clean work Place the mouse with the abdomen facing upward; (ii) Lift the skin of the mouse abdomen with tweezers, and cut the outer skin of the abdomen and chest. (iii) Cut the thoracic cavity with another clean pair of scissors, take out the milky white thymus with tweezers, and pass through a 200-mesh cell sieve after grinding to obtain the thymus feeder cell fluid.
  • mouse myeloma cells proceed as follows.
  • the mouse myeloma cell line Sp2/0-Ag14 (Sp2/0) is easy to culture and has a high fusion rate. It is currently the most ideal fusion cell, but the Sp2/0 hybridoma cell line has more changes in culture conditions than NS- 1 Sensitive, poor growth when excessive dilution (density lower than 3 ⁇ 10 5 /mL) and alkaline pH (pH higher than 7.3).
  • Sp2/0 The mouse myeloma cell line Sp2/0-Ag14
  • hybridoma cells using the limiting dilution method, the cells are first diluted in a certain concentration stepwise, and then seeded into each well of a 96-well cell culture plate, so that only one cell grows in the well as much as possible. Monoclonal positive hybridoma cell lines generally need to be cloned 2-3 times until they are 100% positive and confirmed as stable clones.
  • mice Take 2-3 BALB/c mice and inject 0.5 mL of liquid paraffin oil into the abdominal cavity.
  • the hybridoma cells in the logarithmic growth phase were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.
  • the hybridoma cells were suspended in serum-free culture medium, and the number of cells was adjusted to (1-2) ⁇ 10 6 /mL, and 0.5 mL was injected into the intraperitoneal cavity of each mouse.
  • the abdomen of the mouse was obviously enlarged, the mouse was sacrificed by neck, rinsed with tap water, immersed in 75% ethanol for 5 min, and the abdomen of the mouse was fixed with an injection needle on the mouse dissecting table.
  • the following monoclonal antibodies were obtained through the above methods: 1B11, 2A7, 6E1, 14C6, 18B2-2, 14C7, 5H4, 1F9. Among them, the obtained hybridoma cell lines 2A7 and 18B2-2 were preserved as described above in the Chinese Type Culture Collection (CCTCC).
  • CCTCC Chinese Type Culture Collection
  • Example 3 In vitro epitope identification of anti-HBcAg mouse monoclonal antibody
  • the Anti-HBcAg mouse monoclonal antibody obtained in 2.1 was diluted to 1 ⁇ g/mL with a PBS solution containing 20% newborn calf serum for qualitative ELISA detection.
  • Sample reaction Take 36 enzyme-labeled plates that have been coated with peptides, add 100 ⁇ L of diluted samples to each well, and place them in a 37°C incubator for 30 minutes.
  • Enzyme marker reaction After completing the sample reaction step, wash the plate with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20) 5 times, and add 100 ⁇ L HRP-labeled goat anti-mouse IgG (GAM) to each well The reaction solution was placed in a 37°C incubator to react for 30 minutes.
  • PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20) 5 times, and add 100 ⁇ L HRP-labeled goat anti-mouse IgG (GAM) to each well
  • GAM HRP-labeled goat anti-mouse IgG
  • Stop reaction and read value measurement After completing the color reaction step, add 50 ⁇ L of stop solution (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) to each well of the reaction plate, and test each well on the microplate reader. The OD450/630 value of the hole.
  • Judgment of the reactivity of Anti-HBcAg mouse monoclonal antibody with 36 peptides Judgment is based on the reading after the reaction. If the detection value/background value is greater than 5, it is judged as positive.
  • the recognition types of Anti-HBcAg mouse monoclonal antibodies obtained can be divided into 5 groups (according to their recognition properties), namely: sA, sB, sC, sD, sE, and the polypeptide recognized by the sA group antibody is S29/S30 Among them, 2A7, 14C6, and 14C7 belong to the sA group; the polypeptide recognized by the sB group antibody is S31, among which 1F9 and 18B2-2 belong to the sB group; the polypeptide recognized by the sC group antibody is S1; the polypeptide recognized by the sD group antibody is s26, s27 ; And the polypeptides recognized by the sE group antibody are s15 and s16.
  • the specific detection results of antibodies 2A7, 18B2-2 and the corresponding epitopes are shown in Table 3.
  • the epitope of 2A7 is 141-152aa
  • the epitope of 18B2-2 is 150-183aa.
  • Example 4 Epitope identification in vivo
  • 293 ⁇ 5 cells were spread on a 6-well plate, and the cell density reached about 80-90% after 12 hours of attachment for transfection.
  • the constructed eukaryotic expression plasmids were respectively transfected into 293 ⁇ 5 cells using lipo3000 transfection reagent, and replaced 12 hours after transfection Medium (DMEM+10% Gibco FBS), continue culturing for 48 hours, discard the cell supernatant, and wash the cells once with PBS, add 300 ⁇ L of cell lysate to each well, let stand for 1h at 4°C for lysis, collect the lysate in a 1.5ml EP tube Centrifuge at 12000rpm at 4°C for 10min, collect the supernatant into a clean 1.5ml EP tube, and perform western blot analysis on the lysed sample (the secondary antibody is goat anti-mouse-HRP, from Proteintec).
  • Example 5 Screening of magnetic bead-coated monoclonal antibodies and acridinium ester-labeled monoclonal antibodies in the double antibody sandwich method
  • the experimental conditions of the conventional double antibody sandwich method were used to screen the optimal pairing of the magnetic bead-coated monoclonal antibody and the acridinium ester-labeled monoclonal antibody through experiments.
  • the magnetic microparticles are magnetic beads coated with hydrophilic polymers and carboxyl groups, with a particle size of 1.5-3um; the preparation method is as follows: the mass ratio of magnetic microparticles, EDC and NHS is 1:1:1 , And add 50mM MES solution with pH 5.0 to make the concentration of magnetic particles 4mg/mL, and place them on a vertical rotator to activate.
  • the activation environment temperature is 25°C for 20min; the ratio of activated magnetic particles to anti-HBcAg monoclonal antibody Label 15ug of anti-HBcAg monoclonal antibody for each mg of magnetic microparticles, place it on a vertical rotator, and place it on a vertical rotator.
  • the reaction environment temperature is 25°C for 3h; the reacted magnetic microparticles are washed 3 times with a washing solution, and glycine, 0.5% is added.
  • Bovine serum albumin, 0.05% Triton X-100, pH 7.4 phosphate buffer make the magnetic microparticle concentration 4mg/mL, put it on a vertical rotator to stop, the reaction environment temperature is 25°C, time 2h; will stop after The magnetic particles are washed 3 times with a lotion, and added containing 0.5% (W/V) bovine serum albumin, 0.5% (W/V) casein, 0.05% T (W/V) ritonX-100, preservative, pH 7.4 phosphate buffer, make the concentration of magnetic particles 4mg/mL, store at 2-8°C for later use;
  • the monoclonal antibody (18B2-2, 1F9) that recognizes aa150-183 was coated with MS300 magnetic beads by the above method to prepare a magnetic microparticle solution.
  • acridinium ester labeling antibody solution Preparation of acridinium ester labeling antibody solution.
  • the preparation method is as follows: take 50ug of anti-HBcAg monoclonal antibody to be labeled, add NaCl-containing phosphate buffer to a volume of 300 ⁇ L, and then add 5 ⁇ L acridinium ester mother solution, shake and mix, and avoid at room temperature. Light reaction for 30 minutes; after the reaction, add 200 ⁇ L of phosphate buffer containing NaCl and glycine, manually invert 20 times to mix, and react at room temperature for 30 minutes in the dark; after the reaction, transfer the product to a dialysis bag.
  • the dialysate is 20mM PBS with a pH of 7.4 Buffer, dialysis in the dark at 2-8°C, change the PBS buffer every 2h for a total of 3 times to remove the unlabeled acridinium; remove the labeled substance and add 10% (W/V) according to the actual volume
  • the final concentration of bovine serum albumin to bovine serum albumin is 0.1% (V/V 1:100), add an equal volume of glycerin, manually invert and mix, and store in the dark below -15°C for later use.
  • the 6 strains of HBcAg monoclonal antibodies (1B11, 2A7, 6E1, 14C6, 14C7, 5H4) were labeled with acridinium esters by the above method.
  • HBV virus positive (PCR detection) clinical serum samples the positive samples are diluted with 20% NBS to 1*10 7 , 1*10 6 , 1*10 5 , 1*10 4 , 1*10 3 different DNA load, use Different DNA load samples were tested, HBV virus negative (PCR test) clinical serum samples.
  • the above method was used to orthogonally detect the pairing of each magnetic bead-coated monoclonal antibody and the acridinium ester-labeled monoclonal antibody, and calculate the P/N (the ratio of the mean value of positive samples to the mean value of negative samples). The results are shown in the table below.
  • Table 4-1 The P/N value of the paired detection of magnetic bead-coated monoclonal antibody 18B2-2 and acridinium ester-labeled monoclonal antibody.
  • Table 4-2 The P/N value of the paired detection of the magnetic bead-coated monoclonal antibody 1F9 and the acridinium ester-labeled monoclonal antibody.
  • Tables 4-1 and 4-2 show that when the magnetic beads are coated with antibodies that recognize HBcAg aa150-183 (ie, recognize the Arginine Rich Domain (ARD) of HBcAg), and then recognize HBcAg
  • ARD Arginine Rich Domain
  • Tables 4-1 and 4-2 show that when the magnetic beads are coated with antibodies that recognize HBcAg aa150-183 (ie, recognize the Arginine Rich Domain (ARD) of HBcAg), and then recognize HBcAg
  • the monoclonal antibodies with different epitopes in 1-149aa are used as labeled antibodies
  • the results of detecting samples with different DNA loads P/N>3 means positive.
  • the results show that when the three antibodies (2A7, 14C6, 14C7) that recognize the HBcAg 141-154aa epitope are used as labeled antibodies, the detection effect of samples with different HBV DNA loads is significantly better than other antibody pairs.
  • Example 6 Enzyme immunoassay and detection reagents for detecting HBcAg
  • Monoclonal antibody 18B2-2 was diluted with phosphate buffer (20mmol/LPB, pH7.4) and coated on a polyvinyl chloride plate.
  • the monoclonal antibody 2A7 was labeled with horseradish peroxidase (Beijing Wantai Biopharmaceutical Co., Ltd.) Co., Ltd.).
  • the samples to be tested include: C183 antigen dilution with a concentration of 1ug/ml, C149 antigen with a concentration of 1ug/ml (developed by the Laboratory of National Infectious Disease Diagnostic Reagents and Vaccine Engineering Technology Research Center of Xiamen University) dilution, positive sample 1/2 , 1/2 negative sample, and 20% nbs.
  • the samples were processed using the same method as in Example 5 to lyse the virus. Subsequently, 2A7-HRP (1/500 dilution) was added to incubate for 40 minutes, the plate was washed 5 times, and 50 ul each of color developing solution A and B (Beijing Wantai Biopharmaceutical Co., Ltd.) was added and incubated for 15 minutes. Finally, add stop solution (2MH 2 SO 4 ), gently shake and mix, and read the value under the wavelength of the microplate reader at 450-620.
  • the enzyme immunoassay detection reagent of the present invention can specifically detect HBcAg, but does not detect c149 (ie, HBeAg), indicating that it has good specificity.
  • Example 7 Chemiluminescence detection method and detection reagent for detecting HBcAg
  • the magnetic microparticles are magnetic beads coated with hydrophilic polymers and carboxyl groups, with a particle size of 1.5-3um; the preparation method is as follows: the mass ratio of magnetic microparticles, EDC and NHS is 1:1:1, and the pH is added. 5.0 50mM MES solution, make the concentration of magnetic particles 4mg/mL, place it on a vertical rotator to activate, the activation environment temperature is 25°C, and the time is 20min; the ratio of activated magnetic particles to 18B2-2 monoclonal antibody is per mg of magnetic
  • the microparticles are labeled with 15ug of HBcAg monoclonal antibody, placed on a vertical rotator, and the reaction environment temperature is 25°C for 3h; the reacted magnetic microparticles are washed 3 times with a lotion, and added containing glycine, 0.5% bovine serum albumin, 0.05% Triton X-100, pH 7.4 phosphate buffer, make the magnetic particles concentration 4mg/mL, put it
  • the preparation method is as follows: take 50ug of 2A7 monoclonal antibody to be labeled, add NaCl-containing phosphate buffer to a volume of 300 ⁇ L, then add 5 ⁇ L of acridinium ester mother liquor, shake and mix, and react at room temperature for 30min in the dark; after the reaction, add 200 ⁇ L containing The phosphate buffer of NaCl and glycine was mixed by hand 20 times, and the reaction was kept at room temperature for 30 min. After the reaction, the product was transferred to a dialysis bag.
  • the dialysate was a 20mM PBS buffer with a pH of 7.4, protected from light at 2-8°C Dialysis, change the PBS buffer every 2h for a total of 3 times to remove the unlabeled acridinium; remove the marker, add 10% (W/V) bovine serum albumin to bovine serum albumin according to the actual volume The final concentration of is 0.1% (V/V 1:100), add an equal volume of glycerin, manually invert and mix, and store in the dark below -15°C for later use.
  • Liquid preparation Dilute 50ml of concentrated washing solution (20X) with distilled water or deionized water to 1000ml for later use.
  • Lysis The virus was lysed using the same method as in Example 5.
  • Reaction add 50ul magnetic bead-coated monoclonal antibody 18B2-2 to the sample well, mix well, seal the plate with a sealing film and incubate at 37 ⁇ 1°C for 15min; incubate for 15-20min, after the incubation, use 0.05 Wash with ⁇ 0.08% Tween 20 phosphate buffer, then add 50ul acridinium-labeled antibody 2A7, incubate for 10-15 min, after incubation, wash with 0.05 ⁇ 0.08% Tween 20 phosphate buffer, add pre 100 ⁇ 200ul of excitation solution for pre-excitation. Remove the pre-excitation solution, add 100-200ul of excitation solution for excitation and detection.
  • Example 8 Specificity and sensitivity analysis of HBcAg detection kit
  • the luminescence diagnostic kit for detecting HBV core antigen was prepared (luminescence detection reagent method) as in Example 7.
  • Sample number RLU S/C.O. Sample number RLU S/C.O. 1 4042 0.45 41 4250 0.47 2 7762 0.86 42 7862 0.87 3 2415 0.27 43 7690 0.85 4 3289 0.37 44 4921 0.55 5 3934 0.44 45 5134 0.57 6 2659 0.30 46 2610 0.29 7 4208 0.47 47 2011 0.22 8 3353 0.37 48 4551 0.51 9 5665 0.63 49 7942 0.88 10 5729 0.64 50 4328 0.48 11 6535 0.73 51 3660 0.41 12 4042 0.45 52 3398 0.38 13 4074 0.45 53 4108 0.46 14 4845 0.54 54 6807 0.76 15 7663 0.85 55 4818 0.54 16 7635 0.85 56 4525 0.50 17 3915 0.44 57 3793 0.42 18 3263 0.36 58 5096 0.57 19 3359 0.37 59 4639 0.52 20 4642 0.52 60 4899 0.54 twenty one 6425 0.71 61 4482 0.50 twenty two 2001 0.22 62 5347 0.59 twenty
  • the luminescence diagnostic kit for detecting HBV core antigen was prepared (luminescence detection reagent method) as in Example 7.
  • the data S/CO in Table 8 is greater than 1 for positive, and less than 1 for negative, indicating that the sensitivity of antigen detection for c183 is 0.05ng/ml, and the sensitivity for sample detection is about 10 4 copeis/ml (DNA load).
  • 82 hepatitis B virus-infected serum specimens collected from April 2019 to the present are stored frozen at -20°C.
  • Chemiluminescence detection of hepatitis B virus core antigen was performed on each serum specimen.
  • Table 9 Test results of HBsAg-positive hepatitis B virus infection serum specimens
  • S/CO>1 indicates that the core antigen is detected in the sample
  • S/CO ⁇ 1 indicates that the core antigen is not detected in the sample.
  • the correlation analysis between the HBcAg detection result and the DNA load result obtained by the PCR method is carried out. Specifically, the logarithm of the virus content and luminescence intensity of each sample is taken and the linear correlation analysis is carried out. The result is shown in Figure 4, R 2 is 0.8368, this result indicates that the HBcAg detection method of the present invention has good detection performance and can evaluate the DNA load of the sample.
  • HepG2 obtained from the Laboratory of National Infectious Disease Diagnostic Reagents and Vaccine Engineering Technology Research Center of Xiamen University
  • HepG2-N10 cells stably integrated with the HBV1.1 ploid genome obtained from Xiamen University National Infectious Disease Diagnostic Reagents and Vaccine Engineering Technology Research Central laboratory
  • 2A7 has a significant cytoplasmic immune response to HepG2-N10 cells integrated with the HBV genome, but does not bind to cells without the HBV genome, and has good specificity.
  • the above results indicate that 2A7 can be used as an immunofluorescence antibody for HBcAg for accurate detection.
  • the anti-HBcAg antibody used in HBcAg immunohistochemical detection is polyclonal antibody, but polyclonal antibody often has the disadvantages of high background and low specificity, and the immunohistochemical results using it are not easy to standardize, but at present There is no report on the use of anti-HBcAg monoclonal antibody for immunohistochemical detection. This experiment investigates the performance of 2A7 monoclonal antibody as an immunohistochemical detection antibody.
  • liver tissues of HBV transgenic mice HBV-TG obtained from the Laboratory of National Infectious Disease Diagnostic Reagents and Vaccine Engineering Technology Research Center of Xiamen University
  • normal C57BL/6 mice obtained from Shanghai Slack Laboratory Animal Co., Ltd.

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Abstract

在乙型肝炎病毒(Hepatitis B virus,HBV)检测领域，一种采用双抗体夹心法检测HBcAg的方法，以及用于检测HBcAg的抗体及试剂盒；还包括一种能够用于组织或细胞样品的HBcAg免疫学检测的单克隆抗体。

Description

用于检测HBcAg的方法及抗体 技术领域
本发明涉及乙型肝炎病毒(Hepatitis B virus,HBV)检测领域。具体而言，本发明提供了采用双抗体夹心法检测HBcAg的方法，以及用于上述检测的抗体及试剂盒。本发明还提供了能够用于组织或细胞样品的HBcAg免疫学检测的单克隆抗体。
背景技术
乙型肝炎病毒感染，尤其是慢性HBV感染是全球最为重要的公共卫生问题之一(Dienstag JL.Hepatitis B virus infection.N Engl J Med 2008 Oct 2；359(14):1486-1500)。
目前，HBV血清标志物(常规如：HBsAg，HBsAb，HBeAg，HBeAb，HBcAb即俗称“两对半”检测)广泛作为现症HBV感染和既往HBV感染的常规检测标准。但是，高的变异率再加上HBV携带者庞大基数导致常规的“两对半”检测的假阴性高发。并且，普通“两对半”检测无法从量上反映病毒复制性和传染性大小，常出现可怀疑难解释的结果，也无法从“两对半”指标直接确定检测个体并未感染HBV。
HBV DNA是HBV复制的直接指标，其斑点杂交检测(或PCR检测)是乙型肝炎患者和HBV携带者感染与传染性判断的金标准。PCR检测与斑点杂交检测均可作为HBV感染和传染性的直接指标，但是不适合大规模普查和常规使用。
在所有可能与HBV DNA高度相关的血清标志抗原(HBV PreS1、HBcAg、HBxAg、DNAP、HBV PreS2等)中：HBcAg一直被认为是与HBV DNA直接相关的抗原，检测在HBcAg在复制型病毒定量和HBsAg阴性的HBV感染者与HBV患者中特有诊断意义。目前市面上没有开发特异性的HBcAg检测试剂，已报道或者有研发的HBcAg免疫诊断试剂通常采取检测前对样品进行前处理(裂解病毒、破膜并灭火HBcAb)或采用迂回检测HBcAg－HBcAb免疫复合物的方式进行，前者操作复杂程序繁琐，不易被临床客户接受，也不适合用于大规模献血员筛查及流行病学调查，后者则因为检测方法的特殊性，难以达到理想的特异性和灵敏度。2006年有报道的专利“一种联合检测乙型肝炎病毒前S1抗原和核心抗原的方法及诊断试剂盒”中提到的HBcAg的检测试剂方法是采用sAg的抗体捕获病毒颗粒后进行破膜、裂解病毒检测核内的cAg，但是灵敏度不够。
开发一种简单、准确而又高灵敏度的HBcAg发光检测试剂在HBV患者抗病毒疗 效及病程预后方面都有迫切的现实意义。
发明内容
本发明人经过大量的实验研究，出乎意料地发现了特别适用于HBcAg双抗体夹心法检测的结合特定表位的抗体对。在此基础上，本发明人开发了新的HBcAg定量检测试剂盒以及检测方法。该检测方法的灵敏度能够达到与DNA相当水平，且可以实现快速、高通量检测，具备重大临床应用价值。
试剂盒
因此，在第一方面，本发明提供了一种试剂盒，其包含：
(i)第一抗体，编码第一抗体的分离的核酸分子，包含所述分离的核酸分子的载体，或者表达所述第一抗体的重组细胞；其中，所述第一抗体其选自能够特异性结合HBcAg蛋白的第150-183位中所包含的表位的抗体或其抗原结合片段；和，
(ii)第二抗体，编码第二抗体的分离的核酸分子，包含所述分离的核酸分子的载体，或者表达所述第二抗体的重组细胞；其中，所述第二抗体选自能够特异性结合HBcAg蛋白的第141-154位中所包含的表位的抗体或其抗原结合片段。
在本文中，表述“HBcAg蛋白的第150-183位中所包含的表位”或类似表述是指，所述表位存在于HBcAg蛋白的第150-183位氨基酸之内或与之重叠。换言之，所述能够特异性结合HBcAg蛋白的第150-183位中所包含的表位的抗体或其抗原结合片段是能够特异性结合HBcAg蛋白的第150-183位氨基酸或其片段的抗体或其抗原结合片段。
在某些示例性实施方案中，HBcAg蛋白具有如SEQ ID NO:17所示的序列。
在某些实施方案中，所述第二抗体选自能够特异性结合HBcAg蛋白的第141-152位中所包含的表位的抗体或其抗原结合片段。
在某些实施方案中，所述第一抗体选自下列的抗体或其抗原结合片段：
(i)抗体或其抗原结合片段，其包含：包含下述3个互补决定区(CDR)的重链可变区(VH)：序列为SEQ ID NO:3的HCDR1、序列为SEQ ID NO:4的HCDR2、以及序列为SEQ ID NO:5的HCDR3；和/或，包含下述3个互补决定区(CDR)的轻链可变区(VL)：序列为SEQ ID NO:6的LCDR1、序列为SEQ ID NO:7的LCDR2、以及序列为SEQ ID NO:8的LCDR3；或者，
(ii)抗体或其抗原结合片段，其包含：包含SEQ ID NO:1所示的重链可变区中含有的3个CDR的重链可变区(VH)；和/或，包含SEQ ID NO:2所示的轻链可变区 中含有的3个CDR的轻链可变区(VL)；优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号系统定义；或者，
(iii)抗体或其抗原结合片段，所述抗体是杂交瘤细胞株18B2-2所产生的单克隆抗体，其中，杂交瘤细胞株18B2-2保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019303。
在某些实施方案中，所述第一抗体包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(i)SEQ ID NO:1所示的序列；(ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(iv)SEQ ID NO:2所示的序列；(v)与SEQ ID NO:2所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(vi)与SEQ ID NO:2所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述第一抗体包含：具有如SEQ ID NO:1所示的序列的VH和具有如SEQ ID NO:2所示的序列的VL。
在某些实施方案中，所述第二抗体选自下列的抗体或其抗原结合片段：
(i)抗体或其抗原结合片段，其包含：包含下述3个互补决定区(CDR)的重链可变区(VH)：序列为SEQ ID NO:11的HCDR1、序列为SEQ ID NO:12的HCDR2、以及序列为SEQ ID NO:13的HCDR3；和/或，包含下述3个互补决定区(CDR)的轻链可变区(VL)：序列为SEQ ID NO:14的LCDR1、序列为SEQ ID NO:15的LCDR2、以及序列为SEQ ID NO:16的LCDR3；或者，
(ii)抗体或其抗原结合片段，其包含：包含SEQ ID NO:9所示的重链可变区中含 有的3个CDR的重链可变区(VH)；和/或，包含SEQ ID NO:10所示的轻链可变区中含有的3个CDR的轻链可变区(VL)；优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号系统定义；或者，
(iii)抗体或其抗原结合片段，所述抗体是杂交瘤细胞株2A7所产生的单克隆抗体，其中，杂交瘤细胞株2A7保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019302。
在某些实施方案中，所述第二抗体包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(i)SEQ ID NO:9所示的序列；(ii)与SEQ ID NO:9所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(iii)与SEQ ID NO:9所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(iv)SEQ ID NO:10所示的序列；(v)与SEQ ID NO:10所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(vi)与SEQ ID NO:10所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述第二抗体包含：具有如SEQ ID NO:9所示的序列的VH和具有如SEQ ID NO:10所示的序列的VL。
在某些实施方案中，所述第一抗体和/或第二抗体包含重链恒定区(CH)和轻链恒定区(CL)。
在某些实施方案中，所述第一抗体和/或第二抗体包含小鼠重链恒定区和小鼠轻链恒定区。
在某些实施方案中，所述第一抗体和/或第二抗体是IgG、IgM、IgE、IgD或IgA抗体。在某些实施方案中，所述第一抗体和/或第二抗体是IgG抗体。
在某些实施方案中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)。
在某些实施方案中，所述抗体为鼠源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，所述第二抗体带有可检测标记。
在另一些实施方案中，所述试剂盒还包括能够特异性结合所述第二抗体的第三抗体，所述第三抗体带有可检测标记。
在本文中，所述可检测标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。这类标记是本领域熟知的，包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如， 3H、 125I、 35S、 14C或 32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、化学发光物质(如吖啶酯类化合物)、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。本发明中涵盖的标记物可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测。测热标记物通过简单可视化着色标记物来检测。化学发光物质(如吖啶酯类化合物)一般通过给发光物质提供激发液和/或催化剂来检测发射的光。生物素一般通过给生物素提供上述标记物修饰的亲和素(例如，链霉亲和素)及检测与生物素连接的亲和素所携带的标记物来检测。在某些实施方案中，可通过不同长度的接头将如上所述的可检测标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
在某些实施方案中，所述可检测标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。
在某些实施方案中，所述试剂盒可以进一步包含用于使相应可检测标记被检测到的试剂。例如，当所述可检测标记为酶时，所述试剂盒还可以包含相应酶的显色底物，例如用于辣根过氧化物酶的邻苯二胺(OPD)、四甲基联苯胺(TMB)、ABTS或鲁米诺类化合物，或用于碱性磷酸酶的对硝基苯磷酸酯(p-NPP)或AMPPD。例如当所述可检测标记为化学发光试剂(例如吖啶酯类化合物)时，所述试剂盒还可以包含用 于化学发光的预激发液和/或激发液。
在某些实施方案中，所述试剂盒进一步包含固相载体。在某些实施方案中，所述固相载体包括由聚合物材料(例如聚氯乙烯、聚苯乙烯、聚丙酰胺或纤维素)制成或包被的凹孔平板、试管、珠粒(例如乳胶颗粒)或薄膜(例如硝酸纤维素膜)，或由功能基团(例如氨基、羧基、生物素或亲和素)预包被的磁珠。在某些实施方案中，所述固相载体选自磁珠或微量滴定板(例如微孔板或酶标板)。
在某些实施方案中，所述试剂盒还包含用于将所述第一抗体包被于所述固相载体上的包被试剂，例如包被缓冲液(例如，碳酸盐缓冲液、磷酸盐缓冲液、Tris-HCL缓冲液或硼酸盐缓冲液)。将蛋白或多肽包被于固相载体上的方法是本领域熟知的，例如物理吸附、通过氨基化或羧基化表面实现的共价偶联或通过亲和素-生物素系统、聚赖氨酸预包被表面、蛋白A或蛋白G预包被表面实现的介导结合。
在某些实施方案中，所述第一抗体包被在固相载体的表面。
在某些实施方案中，所述试剂盒在单独的容器中或在单个容器单元的分开的隔室中至少包含上述固相载体，以及上述第一抗体。
在某些示例性实施方案中，所述试剂盒包含：第一抗体，和带有可检测标记的第二抗体。在某些示例性实施方案中，所述试剂盒包含：包被于固相载体表面的第一抗体，和带有可检测标记的第二抗体。在某些示例性实施方案中，所述试剂盒包含：一种或多种第一抗体，和带有可检测标记的第二抗体。在某些示例性实施方案中，所述试剂盒包含：包被于固相载体表面的一种或多种第一抗体，和带有可检测标记的第二抗体。在某些实施方案中，所述多种第一抗体识别HBcAg蛋白的第150-183位中所包含的不同表位。
在某些实施方案中，所述试剂盒进一步包含用于裂解HBV病毒颗粒的裂解剂。在本文中，用于裂解HBV病毒颗粒的裂解剂是指能够裂解Dane颗粒(即，破坏病毒包膜)以暴露HBcAg抗原的任何试剂。这类试剂是本领域技术人员已知的，例如表面活性剂，例如NP40，LDS或SDS。
在某些实施方案中，所述裂解剂包含LDS或SDS。在某些实施方案中，所述试剂盒进一步包含中和剂，所述中和剂包含CHAPS。在某些实施方案中，所述裂解剂包含20％LDS或20％SDS。在某些实施方案中，所述裂解剂包含20％LDS或20％SDS以及余量的水。在某些实施方案中，所述中和剂包含10％CHAPS。在某些实施方案中，所述中和剂包含10％CHAPS、20mM PBS。在某些实施方案中，所述中和剂包含 10％CHAPS、20mM PBS以及余量的水。
在某些实施方案中，所述试剂盒还包含一种或多种选自下列的试剂或装置：标准品(例如，含有不同已知量的HBcAg的系列样品)；阳性对照样品(例如，含有已知量的HBcAg的样品)；阴性对照样品(例如，不含有HBcAg的样品)；用于裂解HBV病毒的裂解剂(以及任选地中和剂)；和，用于收集或贮存待测样品的装置(例如采血装置)。
抗体的制备
第一方面所述的第一抗体和第二抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
第一方面所述的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’) 2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’) 2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
因此，在第二方面，本发明提供了一种试剂盒，其包含：
(i)第一抗体，编码第一抗体的分离的核酸分子，包含所述分离的核酸分子的载体，或者表达所述第一抗体的重组细胞；其中，所述第一抗体如第一方面中定义；和，
(ii)第二抗体，编码第二抗体的分离的核酸分子，包含所述分离的核酸分子的载体，或者表达所述第二抗体的重组细胞；其中，所述第二抗体如第一方面中定义。
在某些实施方案中，所述载体是克隆载体或表达载体。在某些实施方案中，所述载体是例如质粒，粘粒，噬菌体等。
在某些实施方案中，表达所述第一抗体的重组细胞是包含编码所述第一抗体的分离的核酸分子或包含所述分离的核酸分子的载体的宿主细胞；表达所述第二抗体的重组细胞是包含编码所述第二抗体的分离的核酸分子或包含所述分离的核酸分子的载体 的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。在某些实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DG44)。
在某些实施方案中，表达所述第一抗体的重组细胞是杂交瘤细胞株18B2-2，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019303；和，表达所述第二抗体的重组细胞是杂交瘤细胞株2A7，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019302。
检测方法及用途
在第三方面，本发明提供了一种检测HBcAg蛋白在样品中的存在或其水平的方法，其包括以下步骤：
(1)将所述样品与第一抗体接触，以形成抗体-抗原复合物，所述第一抗体如第一方面中所定义；
(2)使所述抗体-抗原复合物与第二抗体接触，以形成抗体-抗原-抗体复合物，所述第二抗体如第一方面中所定义；和
(3)测定所述抗体-抗原-抗体复合物的量。
所述方法可以用于诊断目的，或者非诊断目的。在某些实施方案中，本发明的方法用于非诊断目的。在此类实施方案中，由于待测的样品已知含有HBcAg，也即在施用本发明的方法进行检测前，该样品的同一主体已具备诊断结果；因此，本发明的方法对于该样品的诊断步骤而言并无任何帮助。由此可见，本发明的方法的直接目的并不在于获得该样品的同一主体的诊断结果，而是在于对该已知诊断信息的样品进行进一步的精确定量检测。
在一些实施方案中，所述第二抗体带有可检测标记。在某些实施方案中，步骤(3)中所述的测定包括以下步骤：(3a)检测可检测标记的量；(3b)将步骤(3a)所获得的可检测标记的量与表示HBcAg已知量与所述可检测标记的量的关系的标准曲线比较，并获得HBcAg的含量。在某些实施方案中，步骤(3)中所述的测定包括以下步骤：(3a)检测可检测标记的量(例如发光值)；(3b)将步骤(3a)所获得的可检测标记的量(例如发光值)与Cut off值比较，当所述比值小于1时，认为该样品为阴性，当所述比值大于等于1时，认为该样品为HBcAg阳性。在某些实施方案中，当所述可检测标记为吖啶酯 类化合物时，所述Cut off值为9000。
在另一些实施方案中，所述第二抗体不带有可检测标记。在此类实施方案中，步骤(3)中所述的测定包括使用带有可检测标记的第三抗体来检测所述抗体-抗原-抗体复合物。在某些实施方案中，所述第三抗体能够特异性结合所述第二抗体(例如，能够特异性结合所述第二抗体的恒定区)。在某些实施方案中，步骤(3)中所述的测定可以包括以下步骤：(3a)使所述抗体-抗原-抗体复合物与带有可检测标记的第三抗体接触；(3b)检测可检测标记的量；(3c)将步骤(3b)所获得的可检测标记的量与表示HBcAg已知量与所述可检测标记的量的关系的标准曲线比较，并获得HBcAg的含量。在某些实施方案中，步骤(3)中所述的测定包括以下步骤：(3a)使所述抗体-抗原-抗体复合物与带有可检测标记的第三抗体接触；(3b)检测可检测标记的量(例如发光值)；(3c)将步骤(3b)所获得的可检测标记的量(例如发光值)与Cut off值比较，当所述比值小于1时，认为该样品为阴性，当所述比值大于等于1时，认为该样品为HBcAg阳性。在某些实施方案中，当所述可检测标记为吖啶酯类化合物时，所述Cut off值为9000。
在某些实施方案中，所述可检测标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。
在某些实施方案中，在步骤(3)中，所述测定选自酶免疫测定法或化学发光免疫分析法。
在某些实施方案中，在步骤(1)之前，所述方法还包括处理所述样品的步骤，所述处理包括：将裂解剂与所述样品混合以裂解病毒。在某些实施方案中，所述处理还包括使用中和剂终止所述裂解反应。
在某些实施方案中，所述裂解剂、中和剂如第一方面中定义。
在某些实施方案中，所述第一抗体包被于固相载体的表面。在某些实施方案中，所述固相载体选自磁珠或微量滴定板(例如微孔板或酶标板)。
在某些实施方案中，在步骤(2)和/或步骤(3)之前还包括洗涤步骤。所述洗涤步骤可以去除未参与反应的物质。
在某些实施方案中，所述样品选自全血、血浆和血清。
在另一方面，本发明还涉及第一方面所述的试剂盒在制备用于检测HBcAg蛋白在样品中的存在或其水平的检测试剂盒中的用途。
在某些实施方案中，所述试剂盒通过第三方面所述的方法来检测HBcAg蛋白在样品中的存在或其水平。
2A7单抗及其用途
目前在组织或细胞样品的HBcAg免疫学检测(例如免疫组化或免疫荧光)中所采用的抗HBcAg抗体为多克隆抗体，多克隆抗体可以提高检测的灵敏度，但往往具有背景较高而特异性较低、并且应用其进行的免疫组化结果不易标准化等缺点。然而，目前尚无抗HBcAg单克隆抗体用于组织或细胞样品的HBcAg免疫学检测的报道。
本发明人出乎意料地发现了适用于组织或细胞样品的HBcAg免疫学检测的单克隆抗体，基于该单克隆抗体的检测效果能够达到与商业化多克隆抗体相当的水平，这是显著的、意料不到的且非常有利的技术效果。
因此，在第四方面，本发明还提供了一种能够特异性结合HBcAg的单克隆抗体或其抗原结合片段，其中，
(i)所述单克隆抗体或其抗原结合片段包含：包含下述3个互补决定区(CDR)的重链可变区(VH)：序列为SEQ ID NO:11的HCDR1、序列为SEQ ID NO:12的HCDR2、以及序列为SEQ ID NO:13的HCDR3；和/或，包含下述3个互补决定区(CDR)的轻链可变区(VL)：序列为SEQ ID NO:14的LCDR1、序列为SEQ ID NO:15的LCDR2、以及序列为SEQ ID NO:16的LCDR3；或者，
(ii)所述单克隆抗体或其抗原结合片段包含：包含SEQ ID NO:9所示的重链可变区中含有的3个CDR的重链可变区(VH)；和/或，包含SEQ ID NO:10所示的轻链可变区中含有的3个CDR的轻链可变区(VL)；优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号系统定义；或者，
(iii)所述单克隆抗体是杂交瘤细胞株2A7所产生的单克隆抗体，其中，杂交瘤细胞株2A7保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019302。
在某些实施方案中，所述单克隆抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(i)SEQ ID NO:9所示的序列；(ii)与SEQ ID NO:9所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(iii)与SEQ ID NO:9所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少 99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(iv)SEQ ID NO:10所示的序列；(v)与SEQ ID NO:10所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(vi)与SEQ ID NO:10所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述单克隆抗体或其抗原结合片段包含：具有如SEQ ID NO:9所示的序列的VH和具有如SEQ ID NO:10所示的序列的VL。
在某些实施方案中，所述单克隆抗体包含重链恒定区(CH)和轻链恒定区(CL)。在某些实施方案中，所述单克隆抗体是IgG、IgM、IgE、IgD或IgA抗体。
在某些实施方案中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)。在某些实施方案中，所述单克隆抗体为鼠源抗体、嵌合抗体或人源化抗体。
在另一方面，本发明还涉及第四方面所述的单克隆抗体或其抗原结合片段在制备用于检测样品中的HBcAg的试剂中的用途。
在某些实施方案中，所述样品是组织样品(例如组织切片)或细胞样品。
在某些实施方案中，所述检测是免疫学检测。在某些实施方案中，所述免疫学检测选自免疫组织化学Immunohistochemistry(IHC)、免疫细胞化学Immunocytochemistry(ICC)、免疫荧光Immunofluorescence(IF)和Western Blot。
在一个实施方案中，所述单克隆抗体或其抗原结合片段带有可检测的标记。
在另一个实施方案中，所述用于检测样品中的HBcAg的试剂还包含带有可检测的标记的二级抗体。
在某些实施方案中，所述二级抗体对所述单克隆抗体或其抗原结合片段所包含的恒定区所来自的物种(例如小鼠)的抗体是特异的。
在某些实施方案中，所述二级抗体是抗-免疫球蛋白抗体，例如抗IgG抗体。
在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、荧光染料或生物素。
在某些示例性实施方案中，当所述免疫学检测选自免疫组织化学(IHC)、免疫细胞化学(ICC)、或Western Blot时，所述可检测的标记选自酶。
在某些示例性实施方案中，当所述免疫学检测选自免疫荧光(IF)时，所述可检测的标记选自荧光染料。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“HBcAg”是指，乙型肝炎病毒(HBV)的核心抗原，也称为核衣壳蛋白，其是本领域技术人员公知的(参见，例如NCBI GENBANK数据库登录号：GU357842.1)。HBcAg蛋白包含位于其N端的参与VLP组装的装配区，以及位于其C端的富含精氨酸的结构域(Arginine Rich Domain，ARD)。
如本文中所使用的，当提及HBcAg的氨基酸序列时，其使用SEQ ID NO：17所示的序列来进行描述。例如，表述“HBcAg的第150-183位氨基酸残基”是指，SEQ ID NO：17所示的多肽的第150-183位氨基酸残基。然而，本领域技术人员理解，在HBcAg的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同基因型或基因亚型的HBcAg)，而不影响其生物学功能。因此，在本发明中，术语“HBcAg”应包括所有此类序列，包括例如SEQ ID NO：17所示的序列以及其天然或人工的变体。并且，当描述HBcAg的序列片段时，其不仅包括SEQ ID NO：17的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“HBcAg的第150-183位氨基酸残基”包括，SEQ ID NO：17的第150-183位氨基酸残基，以及其变体(天然或人工)中的相应片段。根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
如本文中所使用的，术语“Dane颗粒”又称为大球形颗粒，是指完整的具有传染性的乙肝病毒颗粒，具有双层衣壳结构。HBcAg通常存在于Dane颗粒的核心。因此为检测HBcAg，通常需要先裂解Dane颗粒外壳，以使HBcAg暴露游离。
如本文中所使用的，术语“特异性结合”是指，两分子(即结合分子与靶分子)之 间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。两分子之间的结合亲和力可用K D值描述。K D值是指由kd(特定的结合分子-靶分子相互作用的解离速率；亦称为koff)与ka(特定结合分子-靶分子相互作用的缔合速率；亦称为kon)之比得到的解离常数，或者指表示为摩尔浓度(M)的kd/ka。K D值越小，两分子结合越紧密，亲和力越高。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。K D值可通过本领域熟知的方法确定，例如使用表面等离子体共振术(SPR)在BIACORE仪中测定。
如本文中所使用的，术语“免疫学检测”是指，利用抗原-抗体之间的特异性相互作用/结合亲和力来进行的测定，其一般可用于检测特定抗原或者抗体在样品中的存在或水平。此类免疫学检测是本领域技术人员公知的，包括但不限于，酶免疫测定法(EIA)、化学发光免疫分析法(CLIA)、放射免疫测定法(RIA)、荧光免疫测定法(FIA)、Western印迹法、免疫比浊法、表面等离子共振法等。关于免疫学检测的详细描述，可参见例如，Fundamental Immunology,Ch.7Paul,W.,ed.，第2版，Raven Press,N.Y.(1989)。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia &  Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat编号系统确定。
如本文中所使用的，术语“构架区(framework region)”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’) 2、Fd、Fv、互补决定区(CDR)片段、scFv、双抗体(diabody)、单域抗体(single domain antibody)、嵌合抗体、线性抗体(linear antibody)、纳米抗体(技术来自Domantis)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126- 1136中。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”和两条“全长轻链”组成的抗体。其中，“全长重链”是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种，例如人；也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’) 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’) 2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和 Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。
如本文中所使用的，术语“双抗体”意指，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力。单域抗体也称为纳米抗体(nanobody)。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA, 81:6851 6855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链可变区来自第二抗体(例如人抗体)。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性等)的小鼠、大鼠、兔或非人灵长类动物(例如，食蟹猴)抗体。
本发明的嵌合抗体或人源化抗体可以根据上述制备的鼠单克隆抗体的序列进行制备。编码重链和轻链的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
为制备嵌合抗体，可使用本领域已知的方法将鼠免疫球蛋白可变区连接至人免疫球蛋白恒定区。例如，将编码VH的DNA可操作的连接至编码重链恒定区的另一DNA分子以获得全长重链基因。人重链恒定区基因的序列是本领域已知的(参见例如Kabat,E.A.等人(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)，包含这些区的DNA片段可以通过标准PCR扩增获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区，但是通常优选为IgG1或IgG4恒定区。例如，将编码VL的DNA可操作的连接至编码轻链恒定区CL的另一DNA分子以获得全长轻链基因(以及Fab轻链基因)。人轻链恒定区基因的序列是本领域已知的(参见例如Kabat，E.A.等人(1991)Sequences of Proteins of Immunological Interest，Fifth Edition，U.S.Department of Health and Human Services，NIH Publication No.91-3242)，包含这些区的DNA片段可以通过标准PCR扩增获得。轻链恒定区可以是κ或λ恒定区，但通常优选为κ恒定区。
为制备人源化抗体，可以使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539；Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370；以及Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸 序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“受试者”包括但不限于各种动物，特别是哺乳动物，例如人。
有益效果
本发明提供了一种基于特定抗体的HBcAg检测试剂盒以及基于该试剂盒的建立的双抗体夹心法。与现有技术相比，本发明的技术方案能够达到与DNA相当水平的检测灵敏度，且可以实现快速、高通量检测，具备重大临床应用价值。
此外，本发明还提供了一种抗HBcAg单克隆抗体，其能够用于免疫组化、免疫荧光等多种组织或细胞样品的免疫学检测领域，且能够达到与商业化多克隆抗体类似的 检测效果，因此具有广阔的应用前景。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了包含不同长度的HBV抗原的真核表达质粒示意图。
图2分别显示了2A7作为一抗对不同长度的HBV抗原的western blot分析结果。
图3显示了本发明的酶免检测法对不同样本中HBcAg的检测结果。
图4显示了本发明的化学发光检测法与PCR检测结果的相关性。
图5显示了2A7作为HBcAg免疫荧光检测抗体对细胞样品的免疫荧光检测结果。
图6显示了2A7作为HBcAg免疫组化检测抗体对组织切片的免疫组化检测结果。
序列信息
本申请涉及的部分序列的信息描述于下面的表中。
Figure PCTCN2021072483-appb-000001
Figure PCTCN2021072483-appb-000002
关于生物材料保藏的说明
本发明涉及下列已在中国典型培养物保藏中心(CCTCC,Wuhan University,Wuhan,China)进行保藏的生物材料：
1)杂交瘤细胞株18B2-2，其具有保藏号CCTCC NO.C2019303，且保藏时间为2019年11月28日；
2)杂交瘤细胞株2A7，其具有保藏号CCTCC NO.C2019302，且保藏时间为2019年11月28日。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley & Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明来源的试剂均是本领域的常规试剂或市售可得的试剂。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：c183抗原的制备
1.1合成构建C183克隆(其序列如SEQ ID NO:17所示)，利用大肠杆菌表达体系，制备C183B抗原。
1.2纯化C183抗原
收集菌液打超声破碎，将破碎液12000rpm，10℃离心10min，留取上清。而后65℃水浴20min，留取上清。
样品对1×PB7.4透析。准备中压DEAE-FF层析(GE介质)
收集穿透峰溶液(目的蛋白)。而后过Capto Core700分子筛纯化。收集第一个样品峰即可。
实施例2：anti-HBcAg小鼠单克隆抗体的制备
2.1小鼠免疫
2.1.1免疫原的准备：免疫原为大肠杆菌重组表达的HBcAg(C183抗原)蛋白。取重组抗原稀释至0.4mg/mL，与弗氏佐剂等体积混合，使其成油包水状乳剂(判断混合液是否乳化完全的方法：可将一小滴混合液滴在清水的液面上，若混合液凝聚不散，则可认为已基本上混匀)。初次免疫使用弗氏完全佐剂，后续加强免疫使用弗氏不完全佐剂，并且融合前72h的最后一次加强免疫不加佐剂。
2.1.2小鼠的基础免疫：使用上述免疫原对6-8周龄BALB/c雌鼠进行皮下多点注射免疫，注射剂量为500μL/只/次，每次免疫前采集200μL眼球静脉血以备滴度测定。每隔2周加强免疫一次。用间接ELISA测定血清滴度，当小鼠血清滴度达到平台期后，小鼠停止免疫并休息两个月后进行融合。
2.1.3融合前72小时的免疫加强(Final boost)：小鼠脾脏细胞与小鼠骨髓瘤细胞融合前72h进行脾脏免疫加强，此次加强的免疫原不含佐剂，取100μl 0.5mg/mL的重组蛋白进行注射。其中脾脏免疫前，需先用乙醚麻醉小鼠，剖开腹腔外皮取出脾脏，沿脾脏纵向注射100μL抗原，迅速手术缝合腹皮切口。
2.2融合杂交瘤的制备与筛选
融合前72h加强免疫后，取小鼠脾脏制成细胞悬液与小鼠骨髓瘤细胞Sp2/0进行细胞融合，以获得杂交瘤细胞。在此之前先制备饲养细胞。在杂交瘤细胞的培养过程中，融合后的大量骨髓瘤细胞和脾细胞在1640-HAT培养液中相继死亡，单个细胞或少数分散的细胞不易存活，必须加入其他细胞方能使之生存，这种被加入的活细胞称为饲养细胞。本实验室使用小鼠腹腔巨噬细胞或13天龄的小鼠胸腺细胞作为饲养细胞。
2.2.1小鼠巨噬细胞的制备：分如下步骤进行：(i)将一只6周龄左右的BALB/c小鼠引颈处死，自来水冲洗，浸泡于75％乙醇溶液中5min；放入超净工作台，使小鼠 腹部朝上；用镊子提起小鼠腹部皮肤，剪一小口，用大镊子向上下方向撕拉开皮肤，充分暴露腹部。(ii)用无菌眼科镊子提起腹膜，然后用5mL注射器向腹腔内注入适量培养液，用另一无菌眼科镊子轻微提动小鼠四肢，最后利用注射器吸出培养液于离心管中。(iii)将腹腔细胞液溶解于HAT培养液或HT培养液中，使成浓度为2×10 5/mL的巨噬饲养细胞。(iv)加入96孔细胞培养板，每孔0.1mL，置培养箱培养；也可直接与融合细胞混合后添加到96孔细胞培养板中。
2.2.2小鼠胸腺细胞的制备：分如下步骤进行(i)将一只13天龄左右的BALB/c小鼠引颈处死，自来水冲洗，浸泡于75％乙醇溶液中5min；放入超净工作台，使小鼠腹部朝上；(ii)用镊子提起小鼠腹部皮肤，将腹部和胸部的外皮剪开。(iii)用另一把干净的剪刀剪开胸腔，用镊子取出乳白色的胸腺，经研磨后通过200目细胞筛，得到胸腺饲养细胞液。
2.2.3小鼠骨髓瘤细胞的制备：分如下步骤进行。(i)小鼠骨髓瘤细胞株Sp2/0-Ag14(Sp2/0)易培养，融合率高，是目前最理想的融合细胞，但Sp2/0杂交瘤细胞系对培养条件的变化比NS-1敏感，过度稀释(密度低于3×10 5/mL)和pH碱性(pH高于7.3)时生长不良。(ii)选择对数生长期的细胞进行融合。(iii)融合前从培养瓶移出骨髓瘤细胞于离心管，用RPMI-1640培养液洗3次(1000rpm×5min)。用RPMI-1640培养液重悬细胞，计数。(iv)一般融合前5天开始复苏小鼠骨髓瘤细胞，每次融合约需6瓶35cm 2Sp2/0细胞。
2.2.4免疫脾细胞的制备：分如下步骤进行。(i)取待融合的BALB/C小鼠，摘除眼球放血致小鼠死亡，收集的血液制成抗血清，可作抗体检测的阳性对照。自来水冲洗，浸泡于75％乙醇溶液中5min，随即放入超净台内小鼠解剖板上，右侧卧位。(ii)无菌手术打开腹腔取出脾脏，用剪刀剪成小块放于200目细胞筛网上，再用研磨棒(注射器内芯)挤压研磨，同时用吹管滴加RPMI-1640培养液。(iii)补加适量RPMI-1640培养液，静置3-5min，取上2/3部分悬液移入50mL塑料离心管中。上述过程反复2-3次。(iv)用RPMI-1640培养液洗细胞3次(1000rpm×10min)。(v)用RPMI-1640培养液重悬细胞，计数。
2.2.5使用PEG促融剂融合制备杂交瘤：分如下步骤进行。(1)融合前先将1mL的PEG-1500和10mL的RPMI-1640无血清培养基和200mL完全培养基预温至37℃。(ii)将制备的骨髓瘤细胞与脾细胞混合于50mL离心管内(1×10 8脾细胞+1×10 7骨髓瘤细胞，约10:1)，离心1500rpm×8min，离心后，轻轻弹击管底，使细胞疏松成 糊状。(iii)1mL吸管吸取0.8mL(按1×10 8脾细胞+0.8mL PEG)加入离心管内，边加边轻轻搅拌，PEG平均在60s内加完，随即加入10mL预温至37℃的RPMI-1640完全培养液，搅拌要温和。最后补加RPMI-1640培养液至40mL，离心1000rpm×5min。(iv)弃上清液，取少量HT培养液将细胞小心吹散，将细胞移入准备好的HT培养液中，加入96孔细胞培养板，每孔0.1mL，放入CO2孵育箱中培养。(v)12h后，配置适量的HAT完全培养基，每个孔中滴加0.1mL；5天后，用HT完全培养基给孔中的细胞上清进行50-100％的换液；大约9-14天后，吸取上清检测。
2.2.6杂交瘤的筛选：间接ELISA筛选，包被重组抗原100ng/mL，每孔包被0.1mL，加入细胞上清50uL检测，并挑选出阳性克隆孔。
2.2.7杂交瘤细胞的克隆化：采用有限稀释法，先把细胞按一定浓度进行梯度稀释，然后接种到96孔细胞培养板的每个孔中，尽可能使孔内只有一个细胞生长。杂交瘤单克隆阳性细胞株一般需要重复克隆2-3次，直到100％阳性后确认为稳定克隆株。
2.3单抗腹水的生产
取BALB/c小鼠2-3只，经腹腔注入0.5mL液体石蜡油。1周后，将对数生长期的杂交瘤细胞以1000rpm离心5min，弃上清液。杂交瘤细胞用无血清培养液悬浮，并将细胞数调至(1-2)×10 6/mL，每只小鼠腹腔注射0.5mL。7-10d后，小鼠腹部明显膨大后，引颈处死小鼠，自来水冲洗，浸洗，浸泡于75％乙醇中5min，使小鼠腹部朝上用注射针头固定四肢于小鼠解剖台板。用镊子提起小鼠腹部皮肤，剪一小口，然后从两边向小鼠背部方向剪开，用大镊子向上下方向撕开皮肤，充分暴露腹部。用无菌眼科镊子提取腹膜，将腹膜中央处剪一小口，然后用1mL吸管通过小口吸出腹腔内全部腹水。收集的腹水可以混合，置离心管离心，3000rpm，20min。离心后收集上清。
2.4单抗腹水的纯化
经硫酸铵沉淀和Protein A亲和层析纯化(购自美国GE公司)，获得纯化的单克隆抗体。
通过上述方法共获得一下单克隆抗体：1B11、2A7、6E1、14C6、18B2-2、14C7、5H4、1F9。其中，所获得的杂交瘤细胞株2A7和18B2-2在中国典型培养物保藏中心(CCTCC)进行了如上文中所描述的保藏。
实施例3：anti-HBcAg小鼠单克隆抗体的体外表位鉴定
3.1多肽合成
以HBV序列GenBank ID：CAA59669.1作为参考序列，合成31条多肽(委托上海生工生物科技有限公司合成)。这31条多肽(s1-s31)一起覆盖了HBcAg的全长183个氨基酸。S1-S31的多肽信息如下表1所示，HBcAg的全长氨基酸序列如GenBank:GU357842.1所示。
表1：S1-S31的多肽信息
Figure PCTCN2021072483-appb-000003
Figure PCTCN2021072483-appb-000004
3.2 Anti-HBcAg小鼠单克隆抗体与多肽的S1-S31的反应性分析
3.2.1反应板的制备
将多肽用pH9.6的50mM CB缓冲液(NaHCO 3/Na 2CO 3缓冲液，终浓度为50mM，pH值为9.6)稀释，终浓度为5μg/mL；在96孔酶标板每孔中加入100μL的包被液，2～8℃包被16～24小时后再37℃包被2小时；用PBST洗涤液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤1次；然后每孔加入200μL的封闭液(含有20％小牛血清及1％酪蛋白的pH值为7.4的20mM Na 2HPO 4/NaH 2PO 4缓冲液溶液)，放入37℃封闭2小时；弃去封闭液。干燥后装入铝箔袋2-8℃保存备用。
3.2.2 Anti-HBcAg小鼠单克隆抗体的ELISA检测
取2.1中获得的Anti-HBcAg小鼠单克隆抗体以含20％新生牛血清的PBS溶液稀释至1μg/mL，进行定性ELISA检测。
样品反应：取36种已包被多肽的酶标板，每孔加入100μL已稀释的样品，置于37℃温箱反应30分钟。
酶标记物反应：完成样品反应步骤后，将酶标板用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤5遍，每孔加入100μL HRP标记的羊抗鼠IgG(GAM)反应液，置于37℃温箱反应30分钟。
显色反应：完成酶标记物反应步骤后，将酶标板用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤5遍，每孔加入TMB显色剂(购自北京万泰生物药业股份有限公司)各50μL，置于37℃温箱反应15分钟。
终止反应及读值测量：完成显色反应步骤后，在反应完的酶标板中每孔加入终止液(购自北京万泰生物药业股份有限公司)50μL，并于酶标仪上检测各孔的OD450/630值。
Anti-HBcAg小鼠单克隆抗体与36种多肽的反应性判定：根据反应后的读值进行判定。若检测值/本底值大于5，则判定为阳性。
3.2.3 Anti-HBcAg小鼠单克隆抗体的识别性质分析
结果如表2所示。所获得的Anti-HBcAg小鼠单克隆抗体的识别类型可分为5组(根据其识别性质)，分别为：sA、sB、sC、sD、sE，其中sA组抗体识别的多肽为S29/S30，其中2A7、14C6、14C7属于sA组；sB组抗体识别的多肽为S31，其中1F9、18B2-2属于sB组中；sC组抗体识别的多肽为S1；sD组抗体识别的多肽为s26、s27；和sE组抗体识别的多肽为s15、s16。
表2：单抗多肽识别性质分析
分组 单抗名称 单抗亚型 识别的多肽
sA HBc－2A7 IgG2B S29/S30
sA HBc－14C6 IgG1 S29/S30
sC HBc－5H4 IgG1 S1
sA HBc－14C7 IgG1 S29/S30
sB HBc－18B2-2 IgG1 S31
sD HBc－1B11 IgG1 S26/S27
sE HBc－6E1 IgG2A S15/S16
sB HBc－1F9 IgG2B S31
抗体2A7、18B2-2与相应表位的具体检测结果如表3所示，2A7的表位在141-152aa，18B2-2的表位在150-183aa。
表3：2A7/18B2-2具体表位的反应情况
分组 单抗名称 识别多肽 反应情况OD 450(1ug/ml)
sA 2A7 S30 2.111
sB 18B2-2 s31 2.220
实施例4：体内的表位鉴定
4.1真核质粒构建方法
我们将HBV基因的HBcAg序列及其N端-29至-1共222个氨基酸段的序列分别构建到真核表达载体(EHRP载体，获得自厦门大学国家传染病诊断试剂与疫苗工程技术研究中心)CMV启动子下游，其结构示意图如图1所示，其中C149至C183是HBcAg序列(从+1位置开始)分别从C端进行截短至数字对应的氨基酸位置，pca-C183为-29至183段，pcb-C183为-20至183段，pcc-C183为-10至183段。
4.2真核表达以及western blot评估
293β5细胞铺6孔板，待12h贴壁后细胞密度达到80-90％左右进行转染，使用lipo3000转染试剂将构建好的真核表达质粒分别转染293β5细胞，并于转染后12h更换培养基(DMEM+10％Gibco FBS)，继续培养48h后弃细胞上清，并用PBS洗涤一次细胞，每孔添加300μL细胞裂解液，4℃静置1h裂解，收集裂解液于1.5ml EP管中，12000rpm 4℃离心10min，收集上清至干净的1.5ml EP管中，并对裂解样品进行western blot分析(二抗为羊抗鼠－HRP，来源于proteintec公司)。
4.3结果分析
利用体内真核构建的不同长度的抗原评估单抗，结果如图2所示，结果验证了2A7-21识别位点为141-152aa。
实施例5：双抗体夹心法中磁珠包被单抗和吖啶酯标记单抗的筛选
本实施例使用常规双抗体夹心法实验条件、通过实验筛选磁珠包被单抗和吖啶酯标记单抗的最佳配对。
5.1 HBcAg磁珠包被单抗制备
磁性微粒子溶液制备，磁性微粒子为表面覆有亲水性聚合物和羧基的磁珠，粒径为1.5～3um；其制备方法为：将磁性微粒子、EDC与NHS的质量比为1:1:1，并加入pH为5.0的50mM MES溶液，使磁性微粒子浓度为4mg/mL，放置在垂直旋转仪上活化，活化环境温度25℃，时间20min；将活化后的磁性微粒子与抗HBcAg单克隆抗体比例为每毫克磁性微粒子标记15ug的抗HBcAg单克隆抗体，放置在垂直旋转仪上标记，反应环境温度25℃，时间3h；将反应后的磁性微粒子用洗液洗涤3次，加入含有甘氨酸、0.5％牛血清白蛋白、0.05％Triton X-100、pH为7.4的磷酸盐缓冲液，使磁性微粒子浓度为4mg/mL，放置在垂直旋转仪上终止，反应环境温度25℃，时间2h；将终止后的磁性微粒子用洗液洗涤3次，加入含有0.5％(W/V)牛血清白蛋白、0.5％(W/V)酪蛋白、0.05％T(W/V)ritonX-100、防腐剂、pH为7.4的磷酸盐缓冲液， 使磁性微粒子浓度为4mg/mL，2-8℃保存备用；
以上述方法对识别aa150-183的单克隆抗体(18B2-2、1F9)进行MS300磁珠包被，制备磁性微粒子溶液。
5.2 HBcAg吖啶酯标记单抗制备
吖啶酯标记抗体溶液制备，其制备方法为：取待标记抗HBcAg单克隆抗体50ug，加入含NaCl的磷酸盐缓冲液至体积为300μL，再加入5μL吖啶酯母液，振荡混匀，室温避光反应30min；反应后加入200μL含NaCl和甘氨酸的磷酸盐缓冲液，手工颠倒20次混匀，室温避光反应30min；反应后将产物转移至透析袋中，透析液为pH为7.4的20mM PBS缓冲液，2-8℃避光透析，每隔2h换一次PBS缓冲液，共更换3次，以除去未标记的吖啶酯；将标记物取出，按实际体积加入10％(W/V)牛血清白蛋白至牛血清白蛋白的终浓度为0.1％(V/V 1:100)，加入等体积甘油，手工颠倒混匀后，-15℃以下避光保存备用。
以上述方法对6株HBcAg单克隆抗体(1B11、2A7、6E1、14C6、14C7、5H4)进行吖啶酯标记。
5.3实验方法
5.3.1标本：
HBV病毒阳性(PCR检测)临床血清标本，阳性样本利用20％NBS稀释至1*10 7、1*10 6、1*10 5、1*10 4、1*10 3不同的DNA载量，利用不同的DNA载量样本开展检测，HBV病毒阴性(PCR检测)临床血清标本。
5.3.2加样：
取25ul样本，加入12.5ul 20％LDS，混匀后37℃孵育30min，加入30ul10％CHAPS中和，混匀后加入50ul磁珠包被单抗，37℃孵育15min，孵育结束后，用含0.05～0.08％的吐温20磷酸盐缓冲液洗涤，再加入50ul吖啶酯标记单抗，震荡混匀后37℃孵育10min，孵育结束后，用含0.05～0.08％的吐温20磷酸盐缓冲液洗涤，加入预激发液100～200ul，进行预激发。去除预激发液，加入激发液100～200ul，进行激发和检测。
以上述方法正交检测各磁珠包被单抗与吖啶酯标记单抗配对，求P/N(阳性标本检测均值与阴性标本检测均值比值)值。结果见下表。
表4-1：磁珠包被单抗18B2-2与吖啶酯标记单抗配对检测的P/N值。
Figure PCTCN2021072483-appb-000005
表4-2：磁珠包被单抗1F9与吖啶酯标记单抗配对检测的P/N值。
Figure PCTCN2021072483-appb-000006
表4-1及4-2显示了当以识别HBcAg aa150-183(即，识别HBcAg的富含精氨酸的结构域(Arginine Rich Domain,ARD))的抗体包被磁珠，并以识别HBcAg的1-149aa中的不同表位的单抗作为标记抗体时，检测不同的DNA载量的样本(P/N>3代表阳性)的结果。结果显示，当以识别HBcAg 141-154aa表位的三株抗体(2A7、14C6、14C7)作为标记抗体时，对不同HBV DNA载量样本的检测效果均显著优于其他抗体配对。
实施例6:用于检测HBcAg的酶免检测法及检测试剂
单克隆化抗体18B2-2用磷酸盐缓冲液(20mmol/LPB，pH7.4)，稀释后包被于聚氯乙烯板上，单抗2A7标记辣根过氧化物酶(北京万泰生物药业股份有限公司)。待测样品包括：浓度为1ug/ml的C183抗原稀释液、浓度为1ug/ml的C149抗原(厦门大学国家传染病诊断试剂与疫苗工程技术研究中心实验室研发)稀释液、阳性样本1/2、阴性样本1/2、以及20％nbs。
使用与实施例5相同的方法处理样品裂解病毒。随后加入2A7-HRP(1/500稀释)温育40min，清洗板5次，加入显色液A、B各50ul(北京万泰生物药业股份有限公司)温育15min。最后加入终止液(2MH 2SO 4)，轻轻震荡混匀，酶标仪波长450-620下读值。结果如图3所示，本发明的酶免检测法检测试剂可以特异性检测HBcAg，对c149(即，HBeAg)不检出，表明其具备良好的特异性。
实施例7:用于检测HBcAg的化学发光检测法及检测试剂
7.1检测试剂盒的制备
7.1.1磁珠包被单抗制备
磁性微粒子为表面覆有亲水性聚合物和羧基的磁珠，粒径为1.5～3um；其制备方法为：将磁性微粒子、EDC与NHS的质量比为1:1:1，并加入pH为5.0的50mM MES溶液，使磁性微粒子浓度为4mg/mL，放置在垂直旋转仪上活化，活化环境温度25℃，时间20min；将活化后的磁性微粒子与18B2-2单克隆抗体比例为每毫克磁性微粒子标记15ug的HBcAg单克隆抗体，放置在垂直旋转仪上标记，反应环境温度25℃，时间3h；将反应后的磁性微粒子用洗液洗涤3次，加入含有甘氨酸、0.5％牛血清白蛋白、0.05％Triton X-100、pH为7.4的磷酸盐缓冲液，使磁性微粒子浓度为4mg/mL，放置在垂直旋转仪上终止，反应环境温度25℃，时间2h；将终止后的磁性微粒子用洗液洗涤3次，加入含有0.5％(W/V)牛血清白蛋白、0.5％(W/V)酪蛋白、0.05％T(W/V)ritonX-100、防腐剂、pH为7.4的磷酸盐缓冲液，使磁性微粒子浓度为4mg/mL，2-8℃保存备用。
7.1.2吖啶酯标记单抗制备
其制备方法为：取待标记2A7单克隆抗体50ug，加入含NaCl的磷酸盐缓冲液至体积为300μL，再加入5μL吖啶酯母液，振荡混匀，室温避光反应30min；反应后加入200μL含NaCl和甘氨酸的磷酸盐缓冲液，手工颠倒20次混匀，室温避光反应30min； 反应后将产物转移至透析袋中，透析液为pH为7.4的20mM PBS缓冲液，2-8℃避光透析，每隔2h换一次PBS缓冲液，共更换3次，以除去未标记的吖啶酯；将标记物取出，按实际体积加入10％(W/V)牛血清白蛋白至牛血清白蛋白的终浓度为0.1％(V/V 1:100)，加入等体积甘油，手工颠倒混匀后，-15℃以下避光保存备用。
7.2检测方法
1、准备：将7.1获得的试剂盒放置室温(18-30℃)下平衡15-30min。
2、配液：将50ml浓缩洗涤液(20X)用蒸馏水或去离子水稀释至1000ml备用。
3、加样：分别在对应孔中加入25ul待测标本。
4、裂解：使用与实施例5相同的方法裂解病毒。
5、反应：向标本孔中加入50ul磁珠包被单抗18B2-2,混匀后用封板膜封板后置于37±1℃孵育15min；孵育15～20min，孵育结束后，用含0.05～0.08％的吐温20磷酸盐缓冲液洗涤，再加入50ul吖啶酯标记抗体2A7，孵育10～15min，孵育结束后，用含0.05～0.08％的吐温20磷酸盐缓冲液洗涤，加入预激发液100～200ul，进行预激发。去除预激发液，加入激发液100～200ul，进行激发和检测。
结果判定
临界值：Cut Off(C.O.)＝9000
结果判定：(S＝每孔的发光值)
阴性结果：(S/C.O.<1):标本的发光值小于Cut Off值为阴性，代表该标本中未检测出HBV核心抗原
阳性结果：(S/C.O.≥1):标本的发光值大于等于Cut Off值为阳性，代表该标本中检测出HBV核心抗原。
实施例8：HBcAg检测试剂盒的特异性和灵敏度分析
8.1核心抗原检测试剂盒的特异性分析
8.1.1试剂盒准备
用于检测HBV核心抗原的发光诊断试剂盒制备(发光检测试剂法)如实施例7方法。
8.1.2检测标本
收集自2019年4月至今的80份乙肝两对半5项全阴样本，-20℃冷冻保存。
8.1.3检测项目
对每份血清标本进行乙型肝炎病毒核心抗原化学发光检测，方法如实施例7所示。
8.1.4检测结果
完成所有样本检测后统计结果，分析试剂盒的特异性。各样本检测结果如下表所示。
表7：检测结果
样本编号 RLU S/C.O. 样本编号 RLU S/C.O.
1 4042 0.45 41 4250 0.47
2 7762 0.86 42 7862 0.87
3 2415 0.27 43 7690 0.85
4 3289 0.37 44 4921 0.55
5 3934 0.44 45 5134 0.57
6 2659 0.30 46 2610 0.29
7 4208 0.47 47 2011 0.22
8 3353 0.37 48 4551 0.51
9 5665 0.63 49 7942 0.88
10 5729 0.64 50 4328 0.48
11 6535 0.73 51 3660 0.41
12 4042 0.45 52 3398 0.38
13 4074 0.45 53 4108 0.46
14 4845 0.54 54 6807 0.76
15 7663 0.85 55 4818 0.54
16 7635 0.85 56 4525 0.50
17 3915 0.44 57 3793 0.42
18 3263 0.36 58 5096 0.57
19 3359 0.37 59 4639 0.52
20 4642 0.52 60 4899 0.54
21 6425 0.71 61 4482 0.50
22 2001 0.22 62 5347 0.59
23 6185 0.69 63 7227 0.80
24 6711 0.75 64 5773 0.64
25 4260 0.47 65 4618 0.51
26 3622 0.40 66 6705 0.75
27 2002 0.22 67 5271 0.59
28 6600 0.73 68 3337 0.37
29 3373 0.37 69 5560 0.62
30 3506 0.39 70 4115 0.46
31 4236 0.47 71 6320 0.70
32 5977 0.66 72 5610 0.62
33 2003 0.22 73 4806 0.53
34 4441 0.49 74 3181 0.35
35 5622 0.62 75 7675 0.85
36 3348 0.37 76 6643 0.74
37 2247 0.25 77 4273 0.47
38 5231 0.58 78 6221 0.69
39 2703 0.30 79 6740 0.75
40 4687 0.52 80 6391 0.71
8.1.5结果与分析
从表7的结果可以看出，S/C.O.<1表示该标本中未检测出HBV核心抗原，特异性良好。
8.2核心抗原检测试剂盒的灵敏度分析
8.2.1试剂盒准备
用于检测HBV核心抗原的发光诊断试剂盒制备(发光检测试剂法)如实施例7方法。
8.2.2检测标本
8.2.2.1.选取1份利用乙型肝炎病毒核酸定量PCR检测试剂检测DNA载量为1.60E+08copies/mL的新鲜血清样本利用20％NBS以3倍为梯度进行线性稀释11个点，并利用20％NBS作为阴性对照，按照实施例8所述方法进行检测，并做参考曲线。
8.2.2.2.将C183B抗原利用20％NBS释至1ug/ml,并以3倍为梯度进行稀释11个 点，并利用不含C183B抗原的20％NBS作为阴性对照，按照实施例8所述方法进行检测，并利用上述参考曲线换算出抗原各个点检测相对应的值。结果如下表所示。
表8：检测结果
C183B ng/ml RLU1 S/C.O. DNA载量 RLU2 S/C.O.
1000.00 10272307 1141.37 5.19E+08 1233807 137.09
333.33 3047322 338.59 1.73E+08 906003 100.67
111.11 1297299 144.14 5.77E+07 389994 43.33
37.04 459415 51.05 1.92E+07 164105 18.23
12.35 177478 19.72 6.41E+06 64594 7.18
4.12 85184 9.46 2.14E+06 25633 2.85
1.37 39003 4.33 7.12E+05 14108 1.57
0.46 19941 2.22 2.37E+05 10453 1.16
0.15 11277 1.25 7.91E+04 9244 1.03
0.05 9368 1.04 2.64E+04 6975 0.78
0.02 6532 0.73 8.79E+03 3558 0.40
20％nbs 2780 0.31 20％nbs 2195 0.24
8.2.2.3.结果与分析
表8中数据S/C.O大于1代表阳性，小于1代表阴性，说明对c183的抗原检测灵敏度在0.05ng/ml，对样本检测灵敏度大约在10 4copeis/ml(DNA载量)左右。
实施例9：HBcAg检测试剂盒与PCR检测法的比较
9.1试剂盒准备
9.1.1用于检测HBV核心抗原的发光诊断试剂盒制备(发光检测试剂法)如实施例7的方法。
9.1.2乙型肝炎病毒核酸定量PCR检测试剂，购自深圳匹基生物工程有限公司。
9.2检测标本
自2019年4月至今收集的82份的乙型肝炎病毒感染血清标本，-20℃冷冻保存。
9.3检测项目
对每份血清标本进行乙型肝炎病毒核酸定量PCR检测。
对每份血清标本进行乙型肝炎病毒核心抗原化学发光检测。
9.4检测结果
完成所有项目检测后统计结果，比较HBV核心抗原与HBV病毒核酸检测的相关性。结果如下表所示。
表9：HBsAg阳性的乙型肝炎病毒感染血清标本检测结果
样本编号 DNA载量 lg(DNA载量) RLU S/C.O. lg(RLU)
1 5.22E+02 2.72 7058 0.78 3.85
2 5.54E+02 2.74 9611 1.07 3.98
3 8.61E+02 2.94 5454 0.61 3.74
4 9.35E+02 2.97 7074 0.79 3.85
5 1.10E+03 3.04 7360 0.82 3.87
6 1.22E+03 3.09 7795 0.87 3.89
7 1.57E+03 3.20 6627 0.74 3.82
8 1.65E+03 3.22 8167 0.91 3.91
9 2.40E+03 3.38 6574 0.73 3.82
10 2.68E+03 3.43 5818 0.65 3.76
11 2.85E+03 3.45 8450 0.94 3.93
12 3.23E+03 3.51 9279 1.03 3.97
13 1.33E+04 4.12 9822 1.09 3.99
14 1.53E+04 4.18 11797 1.31 4.07
15 1.57E+04 4.20 11252 1.25 4.05
16 3.03E+04 4.48 18600 2.07 4.27
17 4.24E+04 4.63 11099 1.23 4.05
18 6.94E+04 4.84 13330 1.48 4.12
19 2.08E+05 5.32 5862 0.65 3.77
20 2.60E+05 5.41 29362 3.26 4.47
21 9.62E+05 5.98 25736 2.86 4.41
22 1.19E+06 6.08 51702 5.74 4.71
23 4.93E+06 6.69 17820 1.98 4.25
24 2.11E+07 7.32 99756 11.08 5.00
25 2.68E+07 7.43 283919 31.55 5.45
26 3.47E+07 7.54 152230 16.91 5.18
27 3.74E+07 7.57 1067414 118.60 6.03
28 4.79E+07 7.68 455741 50.64 5.66
29 1.01E+08 8.00 1434375 159.38 6.16
30 1.05E+08 8.02 74442 8.27 4.87
31 1.26E+08 8.10 160285 17.81 5.20
32 1.48E+08 8.17 1017496 113.06 6.01
33 1.54E+08 8.19 205803 22.87 5.31
34 1.64E+08 8.21 252638 28.07 5.40
35 3.28E+08 8.52 1503273 167.03 6.18
36 5.38E+08 8.73 2669259 296.58 6.43
37 1.15E+03 3.06 9936 1.10 4.00
38 9.14E+07 7.96 170179 18.91 5.23
39 1.26E+08 8.10 940798 104.53 5.97
40 5.39E+07 7.73 110586 12.29 5.04
41 9.02E+07 7.96 124868 13.87 5.10
42 1.96E+04 4.29 10500 1.17 4.02
43 2.04E+03 3.31 9001 1.00 3.95
44 5.19E+08 8.72 1053667 117.07 6.02
45 1.09E+06 6.04 14822 1.65 4.17
46 4.09E+04 4.61 9546 1.06 3.98
47 1.25E+03 3.10 4749 0.53 3.68
48 1.92E+03 3.28 9042 1.00 3.96
49 2.96E+08 8.47 810430 90.05 5.91
50 1.36E+08 8.13 327209 36.36 5.51
51 9.75E+03 3.99 7899 0.88 3.90
52 4.38E+03 3.64 4702 0.52 3.67
53 6.35E+03 3.80 9578 1.06 3.98
54 1.26E+05 5.10 22687 2.52 4.36
55 1.73E+05 5.24 16642 1.85 4.22
56 1.73E+05 5.24 10389 1.15 4.02
57 2.11E+03 3.32 8117 0.90 3.91
58 1.96E+03 3.29 7350 0.82 3.87
59 2.83E+06 6.45 22523 2.50 4.35
60 1.25E+03 3.10 10673 1.19 4.03
61 1.06E+09 9.03 1432767 159.20 6.16
62 9.48E+06 6.98 28928 3.21 4.46
63 1.35E+04 4.13 9640 1.07 3.98
64 1.88E+04 4.27 9524 1.06 3.98
65 8.82E+07 7.95 292615 32.51 5.47
66 8.82E+07 7.95 110758 12.31 5.04
67 3.29E+03 3.52 6017 0.67 3.78
68 7.18E+06 6.86 27373 3.04 4.44
69 7.75E+04 4.89 9986 1.11 4.00
70 3.03E+05 5.48 13518 1.50 4.13
71 7.43E+07 7.87 233848 25.98 5.37
72 3.84E+07 7.58 148066 16.45 5.17
73 1.33E+08 8.12 439697 48.86 5.64
74 2.36E+05 5.37 18871 2.10 4.28
75 2.72E+08 8.43 460449 51.16 5.66
76 3.78E+04 4.58 10103 1.12 4.00
77 6.50E+07 7.81 255839 28.43 5.41
78 7.35E+05 5.87 15580 1.73 4.19
79 3.57E+07 7.55 255944 28.44 5.41
80 8.58E+04 4.93 9963 1.11 4.00
81 1.50E+04 4.18 12018 1.34 4.08
82 4.29E+03 3.63 6577 0.73 3.82
9.5结果分析
在表9中，S/C.O.>1表示该样本中检测出核心抗原，S/C.O.<1表示该样本中未检测出核心抗原。将HBcAg的检测结果与PCR方法获得的DNA载量结果进行相关性分析，具体地，对各样本的病毒含量及发光强度分别取对数后进行线性相关性分析。结果如图4所示，R 2为0.8368，这一结果表明本发明的HBcAg检测方法检测性能良好，可以评估样本的DNA载量。
实施例10：2A7单抗的其他免疫学检测的应用
10.1 2A7作为HBcAg免疫荧光检测抗体的用途
将HepG2(获自厦门大学国家传染病诊断试剂与疫苗工程技术研究中心实验室)及稳定整合HBV1.1倍体基因组的HepG2-N10细胞(获自厦门大学国家传染病诊断试剂与疫苗工程技术研究中心实验室)以每孔60000个细胞铺24孔板，待12h细胞贴壁后，吸掉培养基，用20mM PBS洗一次，4％多聚甲醛固定15min后再以0.02％Triton x-100通透细胞10min，2％BSA封闭1h后将2A7单抗(1mg/ml)以1：1000的稀释比例稀释到2％BSA中，室温孵育1h后PBS洗4次，荧光二抗为羊抗鼠-Alexa488(Beyotime，产品编号：A0428)，室温孵育40min后PBS洗4次并用DAPI染料对细胞核进行染色，实验完成后用Opera phenix激光共聚焦高内涵成像系统63倍水镜拍摄。
结果如图5所示，2A7对整合了HBV基因组的HepG2-N10细胞有明显的胞质免疫反应，但对未整合HBV基因组的细胞没有结合，具备良好的特异性。上述结果说明2A7能够作为HBcAg的免疫荧光抗体进行准确检测。
10.2 2A7作为HBcAg免疫组化检测单抗的用途
目前在HBcAg免疫组化检测中所采用的抗HBcAg抗体为多克隆抗体，但是多克隆抗体往往具有背景较高而特异性较低，并且应用其进行的免疫组化结果不易标准化等缺点，但是目前尚无抗HBcAg单克隆抗体用于免疫组化检测的报道。本实验考察2A7单抗作为免疫组化检测抗体的性能。
取HBV转基因鼠HBV-TG(获自厦门大学国家传染病诊断试剂与疫苗工程技术研究中心实验室)和正常C57BL/6小鼠(获自上海斯莱克实验动物有限责任公司)的肝 组织石蜡切片，进行脱蜡、复水、抗原修复、清洗、封闭，而后加入anti-HBc商品化多克隆抗体和2A7单抗，室温反应1h，而后清洗，加入二抗室温反应10min，再清洗后加入显色液染色反应，最后进行盐酸分化、复染、封片。
结果见图6，与商业化多克隆抗体类似，2A7单抗能够准确检测HBV转基因鼠的组织石蜡切片，并且对正常小鼠的组织石蜡切片无结合，表明2A7也能够作为HBcAg的免疫组化抗体进行准确检测。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (26)

  1. 一种试剂盒，其包含：
    (i)第一抗体，其选自能够特异性结合HBcAg蛋白的第150-183位中所包含的表位的抗体或其抗原结合片段；和，
    (ii)第二抗体，其选自能够特异性结合HBcAg蛋白的第141-154位中所包含的表位的抗体或其抗原结合片段。
  2. 权利要求1所述的试剂盒，其中，所述第二抗体选自能够特异性结合HBcAg蛋白的第141-152位中所包含的表位的抗体或其抗原结合片段。
  3. 权利要求1或2所述的试剂盒，其中，所述第一抗体选自下列的抗体或其抗原结合片段：
    (i)抗体或其抗原结合片段，其包含：包含下述3个互补决定区(CDR)的重链可变区(VH)：序列为SEQ ID NO:3的HCDR1、序列为SEQ ID NO:4的HCDR2、以及序列为SEQ ID NO:5的HCDR3；和/或，包含下述3个互补决定区(CDR)的轻链可变区(VL)：序列为SEQ ID NO:6的LCDR1、序列为SEQ ID NO:7的LCDR2、以及序列为SEQ ID NO:8的LCDR3；或者，
    (ii)抗体或其抗原结合片段，其包含：包含SEQ ID NO:1所示的重链可变区中含有的3个CDR的重链可变区(VH)；和/或，包含SEQ ID NO:2所示的轻链可变区中含有的3个CDR的轻链可变区(VL)；优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号系统定义；或者，
    (iii)抗体或其抗原结合片段，所述抗体是杂交瘤细胞株18B2-2所产生的单克隆抗体，其中，杂交瘤细胞株18B2-2保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019303。
  4. 权利要求3所述的试剂盒，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：(i)SEQ ID NO:1所示的序列；(ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添 加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(iv)SEQ ID NO:2所示的序列；(v)与SEQ ID NO:2所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(vi)与SEQ ID NO:2所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:1所示的序列的VH和具有如SEQ ID NO:2所示的序列的VL。
  5. 权利要求1-4任一项所述的试剂盒，其中，所述第二抗体选自下列的抗体或其抗原结合片段：
    (i)抗体或其抗原结合片段，其包含：包含下述3个互补决定区(CDR)的重链可变区(VH)：序列为SEQ ID NO:11的HCDR1、序列为SEQ ID NO:12的HCDR2、以及序列为SEQ ID NO:13的HCDR3；和/或，包含下述3个互补决定区(CDR)的轻链可变区(VL)：序列为SEQ ID NO:14的LCDR1、序列为SEQ ID NO:15的LCDR2、以及序列为SEQ ID NO:16的LCDR3；或者，
    (ii)抗体或其抗原结合片段，其包含：包含SEQ ID NO:9所示的重链可变区中含有的3个CDR的重链可变区(VH)；和/或，包含SEQ ID NO:10所示的轻链可变区中含有的3个CDR的轻链可变区(VL)；优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号系统定义；或者，
    (iii)抗体或其抗原结合片段，所述抗体是杂交瘤细胞株2A7所产生的单克隆抗体，其中，杂交瘤细胞株2A7保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019302。
  6. 权利要求5所述的试剂盒，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：(i)SEQ ID NO:9所示的序列；(ii)与SEQ ID NO:9所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(iii)与SEQ ID NO:9所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(iv)SEQ ID NO:10所示的序列；(v)与SEQ ID NO:10所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(vi)与SEQ ID NO:10所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:9所示的序列的VH和具有如SEQ ID NO:10所示的序列的VL。
  7. 权利要求1-6任一项所述的试剂盒，其中，所述第一抗体和/或第二抗体包含重链恒定区(CH)和轻链恒定区(CL)；
    优选地，所述第一抗体和/或第二抗体包含小鼠重链恒定区和小鼠轻链恒定区；
    优选地，所述第一抗体和/或第二抗体是IgG、IgM、IgE、IgD或IgA抗体。
  8. 权利要求1-7任一项所述的试剂盒，其中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)；和/或，所述抗体为鼠源抗体、嵌合抗体或人源化抗体。
  9. 权利要求1-8任一项所述的试剂盒，其中，所述第二抗体带有可检测标记，或者，所述试剂盒还包括能够特异性结合所述第二抗体的第三抗体，所述第三抗体带有可检 测标记；
    优选地，所述可检测标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。
  10. 权利要求1-9任一项所述的试剂盒，其中，所述试剂盒进一步包含固相载体；
    优选地，所述固相载体选自磁珠或微量滴定板(例如微孔板或酶标板)；
    优选地，所述第一抗体包被在固相载体的表面。
  11. 权利要求1-10任一项所述的试剂盒，其中，所述试剂盒还包含一种或多种选自下列的试剂或装置：标准品(例如，含有不同已知量的HBcAg的系列样品)；阳性对照样品(例如，含有已知量的HBcAg的样品)；阴性对照样品(例如，不含有HBcAg的样品)；用于裂解HBV病毒的裂解剂；和，用于收集或贮存待测样品的装置(例如采血装置)。
  12. 一种试剂盒，其包含：
    (i)第一抗体，编码第一抗体的分离的核酸分子，包含所述分离的核酸分子的载体，或者表达所述第一抗体的重组细胞；其中，所述第一抗体如权利要求1-8任一项中定义；和，
    (ii)第二抗体，编码第二抗体的分离的核酸分子，包含所述分离的核酸分子的载体，或者表达所述第二抗体的重组细胞；其中，所述第二抗体如权利要求1-8任一项中定义。
  13. 权利要求12所述的试剂盒，其中，
    表达所述第一抗体的重组细胞是包含编码所述第一抗体的分离的核酸分子或包含所述分离的核酸分子的载体的宿主细胞，
    表达所述第二抗体的重组细胞是包含编码所述第二抗体的分离的核酸分子或包含所述分离的核酸分子的载体的宿主细胞。
  14. 权利要求12所述的试剂盒，其中，
    表达所述第一抗体的重组细胞是杂交瘤细胞株18B2-2，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019303；和
    表达所述第二抗体的重组细胞是杂交瘤细胞株2A7，其保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019302。
  15. 检测HBcAg蛋白在样品中的存在或其水平的方法，其包括以下步骤：
    (1)将所述样品与第一抗体接触，以形成抗体-抗原复合物，所述第一抗体如权利要求1-8任一项中所定义；
    (2)使所述抗体-抗原复合物与第二抗体接触，以形成抗体-抗原-抗体复合物，所述第二抗体如权利要求1-8任一项中所定义；和
    (3)测定所述抗体-抗原-抗体复合物的量。
  16. 权利要求15所述的方法，其中，所述第二抗体带有可检测标记；或者，步骤(3)中所述的测定包括使用带有可检测标记的第三抗体；
    优选地，所述可检测标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物)、荧光染料或生物素。
  17. 权利要求15或16所述的方法，其中，在步骤(3)中，所述测定选自酶免疫测定法或化学发光免疫分析法。
  18. 权利要求15-17任一项所述的方法，其中，所述第一抗体包被于固相载体的表面；
    优选地，所述固相载体选自磁珠或微量滴定板(例如微孔板或酶标板)。
  19. 权利要求15-18任一项所述的方法，其中，所述样品选自全血、血浆和血清。
  20. 权利要求15-19任一项所述的方法，其中，
    在步骤(1)之前，所述方法还包括处理所述样品的步骤，所述处理包括：将裂解剂与所述样品混合以裂解病毒；和/或
    在步骤(2)和/或步骤(3)之前，所述方法还包括洗涤步骤。
  21. 权利要求1-14任一项所述的试剂盒在制备用于检测HBcAg蛋白在样品中的存 在或其水平的检测试剂盒中的用途。
  22. 能够特异性结合HBcAg的单克隆抗体或其抗原结合片段，其中，
    (i)所述单克隆抗体或其抗原结合片段包含：包含下述3个互补决定区(CDR)的重链可变区(VH)：序列为SEQ ID NO:11的HCDR1、序列为SEQ ID NO:12的HCDR2、以及序列为SEQ ID NO:13的HCDR3；和/或，包含下述3个互补决定区(CDR)的轻链可变区(VL)：序列为SEQ ID NO:14的LCDR1、序列为SEQ ID NO:15的LCDR2、以及序列为SEQ ID NO:16的LCDR3；或者，
    (ii)所述单克隆抗体或其抗原结合片段包含：包含SEQ ID NO:9所示的重链可变区中含有的3个CDR的重链可变区(VH)；和/或，包含SEQ ID NO:10所示的轻链可变区中含有的3个CDR的轻链可变区(VL)；优选地，所述重链可变区中含有的3个CDR，和/或所述轻链可变区中含有的3个CDR，由Kabat、Chothia或IMGT编号系统定义；或者，
    (iii)所述单克隆抗体是杂交瘤细胞株2A7所产生的单克隆抗体，其中，杂交瘤细胞株2A7保藏于中国典型培养物保藏中心(CCTCC)，且具有保藏号CCTCC NO.C2019302。
  23. 权利要求22所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：(i)SEQ ID NO:9所示的序列；(ii)与SEQ ID NO:9所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(iii)与SEQ ID NO:9所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(iv)SEQ ID NO:10所示的序列；(v)与SEQ ID NO:10所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或(vi)与SEQ ID NO:10所示的序列具有至少80％、至少85％、至少90％、至少91％、至 少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述单克隆抗体或其抗原结合片段包含：具有如SEQ ID NO:9所示的序列的VH和具有如SEQ ID NO:10所示的序列的VL。
  24. 权利要求22或23所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体包含重链恒定区(CH)和轻链恒定区(CL)；
    优选地，所述单克隆抗体是IgG、IgM、IgE、IgD或IgA抗体。
  25. 权利要求22-24任一项所述的单克隆抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)；和/或，所述单克隆抗体为鼠源抗体、嵌合抗体或人源化抗体。
  26. 权利要求22-25任一项所述的单克隆抗体或其抗原结合片段在制备用于检测样品中的HBcAg的试剂中的用途；
    优选地，所述样品是组织样品(例如组织切片)或细胞样品；
    优选地，所述检测是免疫学检测；优选地，所述免疫学检测选自免疫组织化学(IHC)、免疫细胞化学(ICC)、免疫荧光(IF)和Western Blot；
    优选地，所述单克隆抗体或其抗原结合片段带有可检测的标记，或者，所述试剂还包含带有可检测的标记的二级抗体；
    优选地，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、荧光染料或生物素；
    优选地，所述二级抗体是抗-免疫球蛋白抗体。
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Publication number Priority date Publication date Assignee Title
CN116120436A (zh) * 2022-11-23 2023-05-16 福州迈新生物技术开发有限公司 抗HBcAg蛋白的单克隆抗体及其细胞株、制备方法和应用
CN116120437A (zh) * 2022-11-23 2023-05-16 福州迈新生物技术开发有限公司 抗HBsAg蛋白的单克隆抗体及其细胞株、制备方法和应用
CN116120437B (zh) * 2022-11-23 2023-10-27 福州迈新生物技术开发有限公司 抗HBsAg蛋白的单克隆抗体及其细胞株、制备方法和应用
CN116120436B (zh) * 2022-11-23 2023-10-27 福州迈新生物技术开发有限公司 抗HBcAg蛋白的单克隆抗体及其细胞株、制备方法和应用

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