WO2024074114A1 - 一种多表位HCV core抗体组合及检测试剂盒 - Google Patents

一种多表位HCV core抗体组合及检测试剂盒 Download PDF

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WO2024074114A1
WO2024074114A1 PCT/CN2023/122268 CN2023122268W WO2024074114A1 WO 2024074114 A1 WO2024074114 A1 WO 2024074114A1 CN 2023122268 W CN2023122268 W CN 2023122268W WO 2024074114 A1 WO2024074114 A1 WO 2024074114A1
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antibody
antigen
hepatitis
optionally
virus
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French (fr)
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潘少丽
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菲鹏生物股份有限公司
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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
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • 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
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • 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
    • 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
    • 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
    • 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
    • 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
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/186—Hepatitis C; Hepatitis NANB
    • 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

Definitions

  • the present disclosure relates to the field of virus detection. Specifically, it relates to a multi-epitope HCV core antibody combination and a detection kit.
  • HCV hepatitis C virus
  • HCV is a small enveloped RNA virus belonging to the Flaviviridae family. Its genome contains a single-stranded, positive-strand RNA molecule of approximately 9.6 kb in length. The virus has a lipid envelope bound to lipids or immunoglobulins from the host. Inside the envelope is a dense core particle composed of a combination of core protein and viral nucleic acid.
  • the HCV genome has only one open reading frame, located in the center of the genome, encoding a viral precursor polypeptide of approximately 3,000 amino acids (AA); it is then cleaved by host cell signal peptidases and the virus's own proteases to form at least 10 viral proteins, including structural proteins and non-structural proteins.
  • Structural proteins include core proteins and two glycoproteins (E1 and E2), while non-structural proteins include functional proteins such as NS2, NS3, NS4A, NS4B, NS5A and NS5B.
  • HCV infection is an important basis for the diagnosis of hepatitis C, which mainly includes the examination of specific antibodies (HCV antibodies), the detection of HCV genes and HCV core antigens, and the examination of liver cell damage.
  • HCV antibody positivity is indirect evidence of HCV infection, while positive viral gene and antigen detection is direct evidence of the presence of the virus; liver cell damage is often reflected by liver enzyme tests, especially alanine aminotransferase (ALT).
  • the present disclosure provides an antibody combination for detecting hepatitis C virus core antigen, comprising a first antibody, a second antibody, a third antibody and a fourth antibody, wherein the first antibody specifically binds to the amino acid sequence at positions 28-35 of the hepatitis C virus core antigen, the second antibody specifically binds to the amino acid sequence at positions 100-120 of the hepatitis C virus core antigen, the third antibody specifically binds to the amino acid sequence at positions 35-53 of the hepatitis C virus core antigen, and the fourth antibody specifically binds to the amino acid sequence at positions 60-72 of the hepatitis C virus core antigen.
  • the antibody comprises a capture antibody and a labeling antibody.
  • the capture antibody is selected from any one, any two or any three of the first to fourth antibodies, and the labeling antibody is selected from all antibodies of the first to fourth antibodies except the capture antibody.
  • the capture antibodies are the first antibody and the second antibody;
  • the labeling antibodies are the third antibody and the fourth antibody;
  • the labeled antibodies are the first antibody and the second antibody; and the capture antibodies are the third antibody and the fourth antibody.
  • the capture antibody is bound to a solid phase.
  • the solid phase is selected from one or more of a carrier, microparticles or a microwell plate.
  • the solid phase is selected from one or more of magnetic particles, latex, microspheres, microtiter plates, nitrocellulose membranes, glass cellulose membranes, nylon membranes or microfluidic chips.
  • the labeled antibody is labeled with a detectable marker.
  • the detectable label is selected from one or more of metal particles, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radioactive labels, or enzyme labels.
  • the detectable label is selected from colloidal gold, colloidal silver, colloidal selenium, colloidal carbon, colloidal tellurium, fluorophore, rhodamine, luciferase, luciferin, acridinium ester, radioisotope, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, triple ruthenium, luminol, Eu chelate, spin label, phage label.
  • the capture antibody or the label antibody is independently linked to one of the binding partners
  • the binding partner is selected from one or more of biotin/avidin, biotin or its derivatives/avidin or its derivatives, receptor/ligand, digoxigenin/digoxigenin and carbohydrate/lectin.
  • the binding partner is selected from biotin/streptavidin.
  • the present disclosure provides a kit for detecting hepatitis C, comprising the above-mentioned antibody combination for detecting hepatitis C virus core antigen.
  • the kit further comprises a reagent for detecting hepatitis C virus antibodies.
  • the hepatitis C virus antibody is at least one of a core antibody, an E1 antibody, an E2 antibody, a NS2 antibody, a NS3 antibody, a NS4 antibody or a NS5 antibody.
  • the reagent for detecting hepatitis C virus antibodies comprises an antigen.
  • the detection of hepatitis C virus antibodies adopts a double antigen sandwich method or an indirect method.
  • the double antigen sandwich method comprises using a first antigen and a second antigen.
  • the first antigen and the second antigen are respectively selected from a fragment of amino acids 1 to 56 of HCV core antigen, a fragment of amino acids 1201 to 1490 of NS3, a fragment of amino acids 1883 to 1925 of NS4; or a combination or chimeric sequence of the above fragments.
  • the first antigen and the second antigen are a capture antigen and a labeling antigen, respectively.
  • the capture antigen is bound to a solid phase.
  • the solid phase is a membrane carrier, microparticles or a microplate; for example, magnetic microparticles, latex, microspheres, microtiter plates, nitrocellulose membranes, glass cellulose membranes, nylon membranes or microfluidic chips.
  • the labeled antigen is labeled with a detectable marker.
  • the detectable label is selected from metal particles, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radioactive labels, or enzyme labels, such as colloidal gold, colloidal silver, colloidal selenium, colloidal carbon, colloidal tellurium, fluorophores, rhodamine, luciferase, luciferin, acridinium esters, radioisotopes, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, triple ruthenium, luminol, Eu chelates, spin labels, and phage labels.
  • colloidal gold colloidal silver, colloidal selenium, colloidal carbon, colloidal tellurium, fluorophores, rhodamine, luci
  • the capture antigen or the labelled antigen is independently linked to one of the binding partners.
  • the binding partner is selected from biotin/avidin, biotin or its derivatives/avidin or its derivatives, receptor/ligand, digoxigenin/digoxigenin or carbohydrate/lectin.
  • the kit further comprises reagents for immunoassay
  • the immunoassay is selected from ELISA, chemiluminescence, immunochromatography, indirect immunofluorescence assay IFA, radioimmunoassay RIA and other non-enzyme-linked antibody binding tests or methods.
  • the kit further comprises a virus lysis solution.
  • the present disclosure provides use of the above antibody combination in preparing a kit for detecting hepatitis C.
  • the kit further comprises a reagent for detecting hepatitis C virus antibodies.
  • the present disclosure provides a method for detecting hepatitis C, comprising contacting a sample of a subject with the above-mentioned antibody combination for detecting hepatitis C virus core antigen, and detecting antigen-antibody immune response signals.
  • the method further comprises contacting the subject's sample with a reagent for detecting hepatitis C virus antibodies to detect antigen-antibody immune response signals.
  • the present disclosure also provides the above-mentioned antibody combination for detecting hepatitis C virus core antigen, which is used for detecting hepatitis C.
  • the present disclosure provides a method for preparing the above-mentioned antibody combination for detecting hepatitis C virus core antigen, comprising: selecting the following fragments 1 to 4 as antigenic parts in the immunogen, immunizing an animal with the immunogen, and obtaining antibodies that specifically bind to fragments 1 to 4, respectively:
  • Fragment 1 amino acid sequence 28-35 of hepatitis C virus core antigen
  • Fragment 2 amino acid sequence of hepatitis C virus core antigen at positions 100-120;
  • Fragment 3 amino acid sequence 35-53 of hepatitis C virus core antigen
  • Fragment 4 amino acid sequence 60-72 of hepatitis C virus core antigen.
  • HCV hepatitis C virus
  • core protein As used in this article, unless otherwise specified, core protein, core, and core antigen all refer to HCV core protein (HCV core protein).
  • core antibody means an antibody against HCV core protein
  • E1 antibody means an antibody against E1
  • E2 antibody means an antibody against protein E2
  • NS2 antibody means an antibody against protein NS2
  • NS3 antibody means an antibody against protein NS3
  • NS4 antibody means an antibody against protein NS4
  • NS5 antibody means an antibody against protein NS5.
  • HCV antibody detection is the current mainstream serological detection method.
  • Kuo et al. established the anti-C-100 radioimmunoassay method (RIA), and then Ortho successfully developed an enzyme-linked immunosorbent assay (ELISA) to detect anti-C-100.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Both methods use recombinant yeast-expressed viral antigens (C-100-3, a protein encoded by NS4, containing 363 amino acids), which are purified and coated with micro-plastic plate wells, and then the test serum is added.
  • the viral antigen binds to the anti-C-100 in the test serum, and finally isotope- or enzyme-labeled mouse anti-human IgG monoclonal antibodies are added, and the substrate is added for color development to determine the results.
  • the second-generation enzyme-linked immunosorbent assay detects anti-HCV using the HCV Core region-encoded protein C-22-3 and the non-structural region NS3-encoded protein C-33-3 and C-100-3 coating vector.
  • ELISA enzyme-linked immunosorbent assay
  • the double antibody sandwich method for detecting HCV antigen can effectively increase the detection window period and can be used for immune dysfunction Auxiliary diagnosis of HCV infection in the population, differentiation of past HCV infection and current HCV infection, auxiliary monitoring of hepatitis C treatment, etc.
  • the core antigen is rich in arginine, lysine, and proline, has strong hydrophilicity, strong antigenicity, high specificity, and strong affinity, and is the key segment for HCV diagnosis.
  • HCV core antigen determination is a new determination method that has emerged internationally in recent years. Foreign research data show that it has a good correlation with HCV RNA detection and can be used for early diagnosis of HCV. However, due to the low level of free core antigen in serum, a highly sensitive multi-epitope antibody combination is required to meet higher activity detection requirements.
  • the inventors analyzed various antibodies that bind to HCV antigen fragments, and through experimental screening and verification, obtained an antibody combination that can be used for HCV core antigen detection.
  • the antibodies in the combination recognize different HCV core fragments and can fully complement each other, achieving highly sensitive and highly active detection of HCV core antigen, reducing the risk of missed detection and shortening the window period.
  • the present disclosure provides an antibody combination for detecting hepatitis C virus core antigen, which has improved sensitivity, detection rate and detection activity, and can be used to prepare a kit for detecting hepatitis C, thereby shortening the detection window period.
  • an antibody combination for detecting hepatitis C virus core antigen may include a first antibody, a second antibody, a third antibody and a fourth antibody, wherein the first antibody specifically binds to the amino acid sequence 28-35 of the hepatitis C virus core antigen, the second antibody specifically binds to the amino acid sequence 100-120 of the hepatitis C virus core antigen, the third antibody specifically binds to the amino acid sequence 35-53 of the hepatitis C virus core antigen, and the fourth antibody specifically binds to the amino acid sequence 60-72 of the hepatitis C virus core antigen.
  • amino acid sequence of positions 1-175 of the hepatitis C virus core antigen is as shown in SEQ ID NO: 1: MSTNPKPQRKTKRNTNRRPEDVKFPGGGQIVGGVYLLPRRGPRLGVRTTRKTSERSQPRGRRQPIPKDRRSTGKAWGKPGRPWPLYGNEGLGWAGWLLSPRGSRPSWGPTDPRHRSRNVGKVIDTLTCGFADLMGYIPVVGAPLSGAARAVAHGVRVLEDGVNYATGNLPGFPFS.
  • the first antibody, the second antibody, the third antibody or the fourth antibody can be a monoclonal antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody or an antigen-binding fragment of an antibody, as long as they exhibit the desired biological activity, such as an amino acid fragment that specifically binds to an HCV antigen.
  • the first antibody, the second antibody, the third antibody or the fourth antibody may be an antibody fragment, i.e., a portion of a full-length antibody, preferably including its antigen binding region, variable region or CDR.
  • antibody fragment i.e., a portion of a full-length antibody, preferably including its antigen binding region, variable region or CDR.
  • F(ab')2, Fab', Fab, Fd, Fv, dAb, scFv bivalent antibody or domain antibody.
  • “specific binding” may refer to an antibody selectively or preferentially binding to the amino acid sequence, or a major epitope on the amino acid sequence.
  • any suitable in vitro assay, cell-based assay, in vivo assay, animal assay, the antibody disclosed herein can be tested for its binding ability, activity, specificity, or sensitivity, etc., using a biological model, etc.
  • the assay can include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, etc.
  • the EC50 value of the binding of the antibody disclosed herein (or its antigen-binding fragment) to the antigen for example, in ELISA or FACS.
  • standard assays such as surface plasmon resonance technology (e.g. ) to determine the binding affinity.
  • the ability of the antibody to bind to the amino acid sequence can also be detected by enzyme-linked immunosorbent assay.
  • HRP is used to label goat anti-mouse IgG
  • the amino acid sequence is coated with an enzyme-labeled reaction plate
  • the reaction value of the antibody in the sample is measured to indicate the binding ability.
  • a ratio of the reaction value to the control value (negative) of ⁇ 2.0 is considered positive, and a positive indicates specific binding.
  • the epitopes of the first antibody, the second antibody, the third antibody, and the fourth antibody are complementary to obtain an improved detection rate.
  • the first antibody, the second antibody, the third antibody, and the fourth antibody are coated or labeled.
  • the above four antibodies can be divided into capture antibodies and labeled antibodies.
  • the capture antibody can be selected from any one, any two, or any three of the first to fourth antibodies, and the labeled antibody can be selected from all antibodies other than the capture antibody in the first to fourth antibodies.
  • the first antibody can be a capture antibody
  • the second, third, and fourth antibodies can be labeled antibodies.
  • the second antibody can be a capture antibody, and the first, third, and fourth antibodies can be labeled antibodies; and so on.
  • the capture antibody can be the first antibody and the second antibody; and the labeled antibody can be the third antibody and the fourth antibody.
  • the capture antibody can be the first antibody and the third antibody; and the labeled antibody can be the second antibody and the fourth antibody; and so on.
  • the first antibody, the second antibody, and the third antibody can be a capture antibody
  • the fourth antibody can be a labeled antibody.
  • the first antibody, the second antibody, and the fourth antibody may be capture antibodies, and the third antibody may be a labeling antibody; and so on.
  • the capture antibody is also called a coating antibody.
  • the capture antibody can be bound to a solid phase, and in some embodiments, the capture antibody can be used to coat a solid phase; in some embodiments, the solid phase is not particularly limited, and it can be, for example, a membrane carrier, a microparticle or a microwell plate; for example, magnetic particles, latex, microspheres, microtiter plates, nitrocellulose membranes, glass cellulose membranes, nylon membranes or microfluidic chips.
  • the labeled antibody can be labeled with a detectable marker.
  • the detectable marker can be, for example, metal particles, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radioactive labels, or enzyme labels, such as colloidal gold, colloidal silver, colloidal selenium, colloidal carbon, colloidal tellurium, fluorophores, rhodamine, luciferase, luciferin, acridinium esters, radioisotopes, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, triple ruthenium, luminol, Eu chelates, spin labels, and phage labels.
  • the capture antibody or the label antibody can be independently linked to one of the binding partners; By binding to a solid phase or a detectable label in an assay reaction; or by binding to a solid phase or a detectable label in an indirect manner by a binding partner.
  • the binding partner can be selected from biotin/avidin, biotin or its derivatives/avidin or its derivatives (such as streptavidin), receptor/ligand, digoxigenin/digoxigenin, carbohydrate/lectin.
  • an antibody is linked to biotin, for example, an antibody is linked to streptavidin.
  • the present disclosure provides a kit for detecting hepatitis C, comprising the antibody combination for detecting hepatitis C virus core antigen, to achieve sensitive detection of hepatitis C virus core antigen.
  • the kit also includes a reagent for detecting hepatitis C virus antibodies.
  • the kit of the present disclosure can be used for the combined detection of hepatitis C virus antigens and antibodies.
  • the kit of the present invention improves the detection rate of hepatitis C virus and reduces the missed diagnosis rate during the hepatitis C antibody detection window period by combining the detection of HCV surface antibodies and HCV core antigens.
  • the hepatitis C virus antibody can be at least one of a core antibody, an E1 antibody, an E2 antibody, an NS2 antibody, an NS3 antibody, an NS4 antibody, or an NS5 antibody.
  • the detection of hepatitis C virus antibodies can be performed by a double antigen sandwich method or an indirect method.
  • the double antigen sandwich method detects hepatitis C virus antibodies, using at least two antigens, including a first antigen and a second antigen, to sandwich the antibody to be detected, wherein the first antigen and the second antigen can be selected from a fragment of amino acids 1 to 56 of the HCV core antigen, a fragment of amino acids 1201 to 1490 of NS3, and a fragment of amino acids 1883 to 1925 of NS4; or a combination or chimeric sequence of the above fragments; for example, the first antigen and the second antigen can be amino acids 1 to 35 of the HCV core antigen, amino acids 1223 to 1426 of NS3, and amino acids 1890 to 1923 of NS4.
  • amino acid sequence from positions 1201 to 1490 of the hepatitis C virus NS3 is as shown in SEQ ID NO: 2:
  • amino acid sequence of amino acids 1883 to 1925 of the hepatitis C virus NS4 is as shown in SEQ ID NO:3: VINLLPGILSPGALVVGVICAAILRRHVGPGEGAVQWMNRLIA.
  • the first antigen and the second antigen are from different positions of the same hepatitis C virus antigen, for example, from the amino acid sequence of positions 7-48 of the hepatitis C virus core antigen, for example, from the amino acid sequence of positions 7-21 of the hepatitis C virus core antigen, and/or the amino acid sequence of positions 29-48.
  • the first antigen and the second antigen can be used as a capture antigen and a labeled antigen, for example, the first antigen is a capture antigen and the second antigen is a labeled antigen, or the first antigen is a labeled antigen.
  • the second antigen is a capture antigen.
  • the capture antigen can be bound to a solid phase.
  • the capture antigen can be used to coat a solid phase; in some embodiments, the solid phase is not particularly limited, and can be, for example, a membrane carrier, a microparticle or a microplate; for example, a magnetic particle, latex, a microsphere, a microtiter plate, a nitrocellulose membrane, a glass cellulose membrane, a nylon membrane or a microfluidic chip.
  • the labeled antigen is labeled with a detectable label
  • the detectable label is such as metal particles, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radioactive labels, or enzyme labels, such as colloidal gold, colloidal silver, colloidal selenium, colloidal carbon, colloidal tellurium, fluorophores, rhodamine, luciferase, luciferin, acridinium esters, radioisotopes, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, triple ruthenium, luminol, Eu chelate, spin label, phage label.
  • the capture antigen or the labeled antigen can be independently connected to one of the binding partners; connected to a solid phase or a detectable label in an assay reaction through a binding partner; or indirectly connected to a solid phase or a detectable label through a binding partner.
  • the binding partner is selected from biotin/avidin, biotin or its derivatives/avidin or its derivatives (such as streptavidin), receptor/ligand, digoxigenin/digoxigenin, carbohydrate/lectin.
  • the antigen is linked to biotin, for example, the antigen is linked to streptavidin.
  • the kit may also include reagents suitable for performing immunoassays.
  • the kit of the present disclosure may be used to perform immunoassays, such as ELISA, chemiluminescence, immunochromatography, indirect immunofluorescence assay IFA, radioimmunoassay RIA, and other non-enzyme-linked antibody binding assays or methods.
  • the capture antibody can be coated with a solid phase such as magnetic beads or microplates to capture the HCV core antigen in the sample, and then the labeled antibody is used to bind to the antigen bound to the magnetic beads or the plate again, and the result is read after color development.
  • the capture antibody can be fixed to a solid surface, such as plastic, a membrane such as nitrocellulose membrane, glass, magnetic beads or metal supports, etc.
  • the sample from the subject can be contacted with the capture antibody in the solid phase, and then contacted with a labeled antibody with a detectable label, and a recognizable signal is developed or emitted by the detectable label.
  • a blocking agent such as bovine serum albumin, milk powder solution, gelatin, PVP, Superblock can be used to block non-specific sites to reduce the background caused by non-specific binding.
  • a diluent can be used, such as using BSA and phosphate buffered saline (PBS)/Tween to dilute the sample to be tested, which helps to reduce non-specific background.
  • the kit may further include a virus lysis solution.
  • the virus lysis solution may include, for example, a denaturant, a surfactant, a protective protein, ammonium sulfate, and anhydrous ethanol.
  • the virus lysis solution may be a buffer, such as a phosphate buffer.
  • the lysis solution does not require antigen-antibody dissociation, and by adjusting a mild lysis solution, the core antigen in the virus can be released, thereby achieving an efficient reaction of the antigen and antibody and improving the virus detection rate.
  • the present disclosure provides use of an antibody combination for detecting hepatitis C virus core antigen in preparing a kit for detecting hepatitis C.
  • the present disclosure also provides a method for detecting hepatitis C, comprising contacting a sample from a subject with an antibody combination for detecting hepatitis C virus core antigen, and detecting an antigen-antibody immune response signal.
  • the sample from the subject may include biological tissues, cells, or body fluids in a healthy or pathological state, such as a blood sample, such as plasma, serum, blood products, such as semen or vaginal secretions.
  • a blood sample such as plasma, serum, blood products, such as semen or vaginal secretions.
  • the antibodies of the present disclosure are prepared by methods known in the art.
  • the amino acid sequence at positions 28-35 of the hepatitis C virus core antigen can be used as the antigenic portion in the immunogen, and the immunogen can be used to immunize an animal to obtain the first antibody; in some embodiments, the amino acid sequence at positions 100-120 of the hepatitis C virus core antigen can be used as the antigenic portion in the immunogen, and the immunogen can be used to immunize an animal to obtain the second antibody; in some embodiments, the amino acid sequence at positions 35-53 of the hepatitis C virus core antigen can be used as the antigenic portion in the immunogen, and the immunogen can be used to immunize an animal to obtain the third antibody; in some embodiments, the amino acid sequence at positions 60-72 of the hepatitis C virus core antigen can be used as the antigenic portion in the immunogen, and the
  • the full length of the hepatitis C virus core antigen can be used as the antigenic part in the immunogen, the immunogen can be used to immunize animals, and then the first antibody is screened with the amino acid sequence of the 28th to 35th position of the hepatitis C virus core antigen; the second antibody is screened with the amino acid sequence of the 100th to 120th position of the hepatitis C virus core antigen; the third antibody is screened with the amino acid sequence of the 35th to 53rd position of the hepatitis C virus core antigen; and the fourth antibody is screened with the amino acid sequence of the 60th to 72nd position of the hepatitis C virus core antigen.
  • the immunogen in addition to the antigenic part, can also contain a suitable carrier protein.
  • suitable carrier proteins are known to those skilled in the art, and can be, for example, KLH and BSA.
  • the present disclosure provides a method for preparing an antibody combination for detecting hepatitis C virus core antigen, comprising: selecting the following fragments 1 to 4 as the antigenic portion in the immunogen, using the immunogen to immunize an animal, and obtaining antibodies that specifically bind to fragments 1 to 4, respectively: Fragment 1: amino acid sequence 28-35 of hepatitis C virus core antigen; Fragment 2: amino acid sequence 100-120 of hepatitis C virus core antigen; Fragment 3: amino acid sequence 35-53 of hepatitis C virus core antigen; and Fragment 4: amino acid sequence 60-72 of hepatitis C virus core antigen.
  • amino acid sequence of positions 1-175 of the hepatitis C virus core antigen is as shown in SEQ ID NO: 1:
  • the combination of four antibodies against different epitopes provided in the present disclosure improves the overall detection rate of samples without increasing false positives, and is particularly suitable for clinical detection applications.
  • HCV core antigen (provided by Feipeng Biotechnology Co., Ltd.) was mixed with an equal amount of Freund's complete adjuvant and injected subcutaneously and intraperitoneally into BALB/c mice at multiple points. 14 days after the first immunization, intraperitoneal booster immunization was performed. After four booster injections, tail blood was collected for titer detection. The titer reached the fusion requirement. Three days after the last immunization of the mice, the spleen was removed under sterile conditions for fusion.
  • BALB/c mouse peritoneal macrophages were used as feeder cells.
  • BALB/c mice were killed by pulling the neck, and the whole body was immersed in 75% alcohol.
  • the abdominal skin was cut open with scissors under sterile operation to expose the peritoneum.
  • 5 mL of RPMI 1640 basal culture medium was injected into the peritoneal cavity with a syringe, and the cells were repeatedly rinsed.
  • the rinsing fluid was recovered and centrifuged at 1000 rpm for 5 minutes.
  • the precipitate was retained and resuspended with RPMI 1640 screening culture medium (RPMI 1640 complete culture medium containing HAT), and the cell concentration was adjusted to 1 ⁇ 10 5 cells/mL.
  • the cells were added to a 96-well plate at 150 ⁇ L/well and cultured overnight at 37°C and 5% CO 2 .
  • mice Three days after the last immunization of mice, the spleen was removed under sterile conditions, placed in a plate, rinsed once with RPMI 1640 basal culture medium, placed on a nylon mesh in a small beaker, ground and filtered to make a cell suspension. Centrifuged, the supernatant was discarded, and the cell suspension was resuspended in RPMI 1640 basal culture medium. This was repeated three times and counted.
  • Mouse myeloma cells Sp2/0 (stored by Feipeng Biotechnology Co., Ltd.) were selected with 8-azaguanine and cultured to the logarithmic growth phase. Two large bottles were used to prepare cell suspension, which was centrifuged and the supernatant was discarded. The cells were resuspended in RPMI 1640 basal culture medium. This process was repeated three times and the cells were counted.
  • Myeloma cells and immune spleen cells were mixed at a ratio of 1:10, washed once with RPMI 1640 basal culture medium in a 50mL plastic centrifuge tube, and centrifuged at 1200rpm for 8 minutes. Discard the supernatant, mix the cells, slowly add 1mL 50% PEG1500 for fusion, and add 15mL RPMI 1640 basal culture medium to terminate cell fusion after 1 minute of fusion. Centrifuge at 1000rpm for 5 minutes. Discard the supernatant, gently suspend with 50mL RPMI 1640 screening culture medium, divide equally into 10 96-well plates with feeder cells, 50 ⁇ L/well, 37°C, 5% CO 2 culture. Culture to the sixth day, change HT culture medium (RPMI 1640 complete culture medium containing HT) twice.
  • the enzyme-labeled reaction plate was coated with HCV core antigen at 4°C overnight and then incubated with 10% calf serum or 1% skim milk. 0.02M pH7.2 PBS, 0.15ml/well, blocked at 37°C for 2 hours, added cell culture supernatant at 37°C, 30 minutes later, added 2000-fold diluted horseradish peroxidase-labeled goat anti-mouse IgG (produced by Feipeng Biological Co., Ltd., item number BA-PAB-MU0001), 37°C, 30 minutes later, added 100 ⁇ l of 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid phosphate buffer to each well, 37°C, 15 minutes, added dilute sulfuric acid solution, 50 ⁇ l per well, measured 450nm absorbance.
  • RPMI 1640 complete culture fluid was used as a negative control, and the ratio of the measured value to the control value was ⁇ 2.0 for positive cell wells.
  • a total of 14 cell lines that stably secreted HCV core antibodies were obtained by fusion three times, and the titers determined by ELISA were all between 105 and 107.
  • microwells were coated with 10 HCV short peptide antigens A1 to A10, respectively.
  • PBS + 20% NBS was used as the diluent to dilute the 14 monoclonal antibodies (as primary antibodies) obtained in Example 1 to a concentration of 1 ⁇ g/ml.
  • Sheep anti-mouse IgG-HRP was used as the secondary antibody.
  • the monoclonal antibody epitopes were determined based on the reaction of each monoclonal antibody to different antigens. The results are shown in Table 1 below.
  • Example 2 The detection effect of different antibody epitope pairings was tested on the ELISA platform, and the antibodies identified in Example 2 were used as HRP-labeled antibody and coating antibody.
  • the ELISA test method is as follows:
  • monoclonal antibodies 5E-7, 8F-16, 13C-10, 3F-21, and 6B-25 were used as coating antibodies
  • monoclonal antibodies 10D-2, 11C-13, 5G-8, 6D-33, and 12B-3 were used as HRP enzyme-labeled antibodies.
  • Table 2 The results are shown in Table 2 below.
  • the antibody binding to HCV core 28-35aa and the antibody binding to HCV core 60-72aa can detect HCV core antigen as low as 2ng/ml
  • the antibody binding to HCV core 35-53aa and the antibody binding to HCV core 100-120aa can detect HCV core antigen as low as 2ng/ml.
  • the detection sensitivity of these two antibody pairing combinations is higher than that of other pairing combinations.
  • test results are shown in Table 5 below.
  • the background values of clinical negative samples tested by the three combinations are similar.
  • the signal value of positive samples detected by the antibody combination disclosed in the present invention is relatively the highest overall.
  • the control combination 2 is slightly lower than the antibody combination disclosed in the present invention, and the control combination 1 is significantly lower than the antibody combination disclosed in the present invention.
  • RNA test positive 35 clinical positive samples (RNA test positive) and 400 clinical negative samples (RNA test negative) were tested on the ELISA platform using the disclosed antibody combination and the control combination.
  • the results are shown in Table 6 below, and the disclosed antibody combination achieved 0 missed detection and also had a fairly high specificity, demonstrating that the disclosed antibody combination has significant technical progress.
  • the present disclosure provides a hepatitis C virus detection kit.
  • the present disclosure provides a hepatitis C virus detection kit, which includes a first antibody, a second antibody, a third antibody and a fourth antibody for detecting a core antigen of the hepatitis C virus.
  • the kit disclosed in the present disclosure has high sensitivity, good stability, and simple operation, and can be used for rapid detection of early acute hepatitis C, and therefore has excellent practicality.

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Abstract

一种丙型肝炎病毒检测试剂盒。丙型肝炎病毒检测试剂盒，其包括检测丙型肝炎病毒核心抗原的第一抗体、第二抗体、第三抗体和第四抗体。试剂盒灵敏度高，稳定性好，操作简单，可用于早期急性丙型肝炎的快速检测。

Description

一种多表位HCV core抗体组合及检测试剂盒
相关申请的交叉引用
本公开要求于2022年10月08日提交中国国家知识产权局的公开号为202211222339.7、名称为“一种多表位HCV core单抗组合试剂盒制备方法”的中国专利公开的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及病毒检测领域。具体而言，涉及一种多表位HCV core抗体组合及检测试剂盒。
背景技术
根据WHO统计，全世界多达1.7亿人被丙型肝炎病毒(Hepatitis C virus,HCV)感染，即肝脏的一种病毒性感染。75％-85％的感染者会发展成慢性感染，这些病例中大约20％会发展慢性丙型肝炎的并发症，包括肝硬化或肝癌。当前推荐HCV感染的治疗是干扰素和利巴韦林药物的结合，但是，该治疗并不是所有情况下都是有效的，并且在丙型肝炎相关的末期肝病中指示肝移植。因此，及早检出HCV对于HCV感染者来说非常重要。
HCV是一种小包膜RNA病毒，属黄病毒科，基因组含一条约9.6kb长的单股、正链RNA分子。病毒有一脂质包膜，结合着宿主来源的脂质或免疫球蛋白。包膜内部是密度较高的核心颗粒，由核心蛋白和病毒核酸相结合构成。HCV基因组只有一个开放阅读框，位于基因组中央，编码一条约3000个氨基酸(AA)的病毒前体多肽；后经宿主细胞信号肽酶和病毒自身蛋白酶的剪切形成至少10种病毒蛋白，包括结构蛋白和非结构蛋白。结构蛋白包括核心蛋白和两种糖蛋白(E1和E2)，而非结构蛋白则包括NS2、NS3、NS4A、NS4B、NS5A和NS5B等功能蛋白。
HCV感染的实验室诊断是丙型肝炎诊断的重要依据，主要包括特异性抗体(HCV抗体)的检查、HCV基因和HCV核心抗原的检测以及肝细胞损害的检查。HCV抗体阳性是HCV感染的间接证据，而病毒基因和抗原检测阳性是病毒存在的直接证据；肝细胞损害的检查往往以肝脏酶学检查，特别是丙氨酸氨基转移酶(ALT)来反映。
发明内容
本公开提供了一种检测丙型肝炎病毒核心抗原的抗体组合，包括第一抗体、第二抗体、第三抗体和第四抗体，所述第一抗体特异性结合丙型肝炎病毒核心抗原第28-35位氨基酸序列，所述第二抗体特异性结合丙型肝炎病毒核心抗原第100-120位氨基酸序列，所述第三抗体特异性结合丙型肝炎病毒核心抗原第35-53位氨基酸序列，所述第四抗体特异性结合丙型肝炎病毒核心抗原第60-72位氨基酸序列。
可选地，所述抗体包括捕获抗体和标记抗体。
可选地，所述捕获抗体选自所述第一抗体至第四抗体中的任意一种、任意两种或任意三种，所述标记抗体选自所述第一抗体至第四抗体中的除捕获抗体的其他所有抗体。
可选地，所述捕获抗体为第一抗体和第二抗体；所述标记抗体为第三抗体和第四抗体；
可选地，所述标记抗体为第一抗体和第二抗体；所述捕获抗体为第三抗体和第四抗体。
可选地，所述捕获抗体结合至固相。
可选地，所述固相选自载体、微粒或微孔板的一种或多种。
可选地，所述固相选自磁微粒、乳胶、微球、微量滴定板、硝酸纤维素膜、玻璃纤维素膜、尼龙膜或微流控芯片的一种或多种。
可选地，所述标记抗体用可检测标记物标记。
可选地，所述可检测标记物选自金属粒子、荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，或酶标记的一种或多种。
可选地，所述可检测标记物选自胶体金、胶体银、胶体硒、胶体碳、胶体碲、荧光团、罗丹明、荧光素酶、荧光素、吖啶酯、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶、葡萄糖-6-磷酸脱氢酶、三联钌、鲁米诺类、Eu螯合物、自旋标记、噬菌体标记。
可选地，所述捕获抗体或所述标记抗体独立地连接至结合配偶体之一；
可选地，所述结合配偶体选自生物素/抗生物素蛋白、生物素或其衍生物/亲和素或其衍生物、受体/配体，地高辛/地高辛配基和碳水化合物/凝集素的一种或多种。
可选地，所述结合配偶体选自生物素/链霉亲和素。
本公开提供了一种检测丙型肝炎试剂盒，包括上述的检测丙型肝炎病毒核心抗原的抗体组合。
可选地，所述试剂盒还包括检测丙型肝炎病毒抗体的试剂。
可选地，所述丙型肝炎病毒抗体为core抗体、E1抗体、E2抗体、NS2抗体、NS3抗体、NS4抗体或NS5抗体中的至少一种。
可选地，所述检测丙型肝炎病毒抗体的试剂包含抗原。
可选地，所述检测丙型肝炎病毒抗体采用双抗原夹心法或间接法。
可选地，所述双抗原夹心法包括使用第一抗原和第二抗原。
可选地，所述第一抗原和第二抗原分别选自HCV核心抗原第1位至56位氨基酸中的片段，NS3第1201位至1490位氨基酸中的片段，NS4的1883位至1925位氨基酸中的片段；或者选自以上片段的组合或嵌合的序列。
可选地，所述第一抗原和第二抗原分别为捕获抗原和标记抗原。
可选地，所述捕获抗原结合至固相。
可选地，所述固相是膜载体、微粒或微孔板；例如是磁微粒，乳胶、微球、微量滴定板、硝酸纤维素膜、玻璃纤维素膜、尼龙膜或微流控芯片。
可选地，所述标记抗原用可检测标记物标记。
可选地，所述可检测标记物选自金属粒子、荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，或酶标记，例如胶体金，胶体银，胶体硒，胶体碳，胶体碲、荧光团，罗丹明，荧光素酶，荧光素，吖啶酯，放射性同位素，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖类氧化酶，葡萄糖氧化酶，半乳糖氧化酶，葡萄糖-6-磷酸脱氢酶，三联钌、鲁米诺类、Eu螯合物，自旋标记，噬菌体标记。
可选地，所述捕获抗原或所述标记抗原独立地连接至结合配偶体之一。
可选地，所述结合配偶体选自生物素/抗生物素蛋白、生物素或其衍生物/亲和素或其衍生物、受体/配体，地高辛/地高辛配基或碳水化合物/凝集素。
可选地，所述试剂盒还包括用于免疫测定的试剂；
可选地，所述免疫测定选自ELISA，化学发光、免疫层析、间接免疫荧光测定IFA，放射免疫测定RIA以及其它非酶联抗体结合试验或方法。
可选地，所述试剂盒还包括病毒裂解液。
本公开提供上述抗体组合在制备检测丙型肝炎试剂盒中的用途。
可选地，所述试剂盒还包括检测丙型肝炎病毒抗体的试剂。
本公开提供一种检测丙型肝炎的方法，包括用上述检测丙型肝炎病毒核心抗原的抗体组合接触受试者的样品，检测抗原抗体免疫反应信号。
可选地，所述方法还包括用检测丙型肝炎病毒抗体的试剂接触受试者的样品，检测抗原抗体免疫反应信号。
本公开还提供上述检测丙型肝炎病毒核心抗原的抗体组合，用于检测丙型肝炎。
本公开提供了一种制备上述的检测丙型肝炎病毒核心抗原的抗体组合的方法，包括：选自下述片段1-片段4作为免疫原中的抗原性部分，利用免疫原免疫动物，获得分别特异性结合片段1-片段4的抗体：
片段1：丙型肝炎病毒核心抗原第28-35位氨基酸序列；
片段2：丙型肝炎病毒核心抗原第100-120位氨基酸序列；
片段3：丙型肝炎病毒核心抗原第35-53位氨基酸序列；和
片段4：丙型肝炎病毒核心抗原第60-72位氨基酸序列。
具体实施方案
下面主要结合具体实施例对多表位HCV core抗体组合及HCV core抗原检测试剂盒及其制备作进一步详细的说明。以下实施例仅仅是例证，本公开并不局限于这些实施例。
术语定义：
如本文所用，除非特别指出，HCV与丙型肝炎病毒可互换使用。
如本文所用，除非特别指出，核心蛋白、core、核心抗原均指HCV核心蛋白(HCV core protein)。
如本文所用，除非特别指出，core抗体意指针对HCV核心蛋白的抗体；E1抗体意指针对E1的抗体；E2抗体意指针对蛋白E2的抗体；NS2抗体意指针对蛋白NS2的抗体；NS3抗体意指针对蛋白NS3的抗体；NS4抗体意指针对蛋白NS4的抗体；NS5抗体意指针对蛋白NS5的抗体。
在丙型肝炎检测领域，用PCR技术检测HCV基因，虽然能在感染后2-3天即可检测到阳性结果，实现早期病原学诊断，且特异性强，可以判断传播途径，但对设备和人员操作要求高，极易出现污染及操作误差，因此在基层医疗机构普及存在困难。
HCV抗体检测是当前主流的血清学检测方法。1989年，Kuo等建立了抗-C-100放射免疫试验方法(RIA)，随后Ortho公司又研制成功酶联免疫试验方法(ELISA)检测抗-C-100。这两种方法均用重组酵母表达的病毒抗原(C-100-3，为NS4编码的蛋白，含363个氨基酸)，经纯化后包被微量塑料板孔，然后加被检血清，该病毒抗原即与被检血清中抗-C-100结合，最后加同位素或酶标记的鼠抗人lgG单克隆抗体，加底物显色判断结果。第二代酶联免疫试验法(ELISA)检测抗HCV采用HCV Core区编码蛋白C-22-3和非结构区NS3编码蛋白C-33-3和C-100-3包被载体。虽然抗HCV抗体检测方法不断迭代，极大的提高检出率，但由于抗体检测存在窗口期，因此，检测HCV抗原才能更早的检测出病毒感染。
采用双抗体夹心法检测HCV抗原可以有效提高检出窗口期，并且可用于免疫功能障碍 人群HCV感染的辅助诊断，鉴别HCV既往感染与现症感染，丙肝治疗的辅助监测等方面，其中，核心抗原富含精氨酸、赖氨酸、脯氨酸，亲水性强，抗原性强，特异性高，亲和力强，是HCV诊断的重点区段。HCV核心抗原测定是近年来国际上出现的一种新的测定方法，国外的研资料表明它与HCV RNA检测存在着很好的相关性，可用于HCV早期诊断。但是，由于血清中的游离核心抗原水平较低，因此需要一种高灵敏的多表位抗体组合，来满足更高的活性检测要求。
发明人经过大量研究和探索，对各种结合HCV抗原片段的抗体进行分析，通过实验筛选和验证，获得能够用于HCV核心抗原检测的抗体组合，并且组合中的抗体识别不同的HCV core片段，能够充分互补，实现HCV core抗原的高灵敏、高活性检测，降低漏检风险和缩短窗口期。
因此，在一些实施方案中，本公开提供了一种检测丙型肝炎病毒核心抗原的抗体组合，具备改善的灵敏度、检出率和检测活性，可用于制备检测丙型肝炎试剂盒，缩短了检测窗口期。
在一些实施方案中，一种检测丙型肝炎病毒核心抗原的抗体组合可以包括第一抗体、第二抗体、第三抗体和第四抗体，所述第一抗体特异性结合丙型肝炎病毒核心抗原第28-35位氨基酸序列，所述第二抗体特异性结合丙型肝炎病毒核心抗原第100-120位氨基酸序列，所述第三抗体特异性结合丙型肝炎病毒核心抗原第35-53位氨基酸序列，所述第四抗体特异性结合丙型肝炎病毒核心抗原第60-72位氨基酸序列。
可选地，所述丙型肝炎病毒核心抗原的1-175位氨基酸序列如SEQ ID NO:1所示：MSTNPKPQRKTKRNTNRRPEDVKFPGGGQIVGGVYLLPRRGPRLGVRTTRKTSERSQPRGRRQPIPKDRRSTGKAWGKPGRPWPLYGNEGLGWAGWLLSPRGSRPSWGPTDPRHRSRNVGKVIDTLTCGFADLMGYIPVVGAPLSGAARAVAHGVRVLEDGVNYATGNLPGFPFS。
在一些实施方案中，第一抗体、第二抗体、第三抗体或第四抗体可以是单克隆抗体、双特异性抗体、多特异性抗体、嵌合抗体或者抗体的抗原结合片段，只要他们展示所需的生物学活性，如特异性结合HCV抗原的氨基酸片段。
在一些实施方案中，第一抗体、第二抗体、第三抗体或第四抗体可以是抗体片段，即可以是全长抗体的部分，优选地包括其抗原结合区、可变区或CDR。例如F(ab’)2、Fab’、Fab、Fd、Fv、dAb、scFv、双价抗体或结构域抗体。在一些实施方案中，当用于本文中时，"特异性结合"可以指抗体选择性地或优先地结合所述氨基酸序列，或者所述氨基酸序列上的主要表位。
在一些实施方案中，可以使用任何适当的体外测定、基于细胞的测定、体内测定、动 物模型等检测本公开抗体的效果如结合能力、活性、特异性、或灵敏度等。在一些实施方案中，所述测定可以包括例如ELISA，FACS结合测定，Biacore，竞争性结合测定等。在一些实施方案中，例如在ELISA或FACS中本公开的抗体(或其抗原结合片段)与抗原结合的EC50值。在一些实施方案中，可以使用标准测定法如表面等离子共振技术(例如)确定结合亲和力。在一些实施方案中，也可以使用酶联免疫吸附法检测抗体结合所述氨基酸序列的能力。例如在一些实施方案中，采用HRP标记羊抗鼠IgG，所述氨基酸序列包被酶标反应板，测定样品(如血清)中抗体的反应值，来表示结合能力。例如在一些实施方案中，以反应值与对照值(阴性)比值≥2.0为阳性，阳性表示特异性结合。
在一些实施方案中，第一抗体、第二抗体、第三抗体、第四抗体之间表位互补，获得改善的检出率。在一些实施方案中，对第一抗体、第二抗体、第三抗体、第四抗体进行包被或者标记。在一些实施方案中，可以将上述四种抗体分为捕获抗体和标记抗体。例如捕获抗体可以选自所述第一抗体至第四抗体中的任意一种、任意两种或任意三种，标记抗体可以选自所述第一抗体至第四抗体中的除捕获抗体的其他所有抗体。在一些实施方案中，第一抗体可以为捕获抗体，第二抗体、第三抗体、第四抗体可以为标记抗体。又或者，在一些实施方案中，第二抗体可以为捕获抗体，第一抗体、第三抗体、第四抗体可以为标记抗体；以此类推。在一些实施方案中，所述捕获抗体可以为第一抗体和第二抗体；所述标记抗体为第三抗体和第四抗体。又或者，在一些实施方案中，所述捕获抗体可以为第一抗体和第三抗体；所述标记抗体可以为第二抗体和第四抗体；以此类推。在一些实施方案中，第一抗体、第二抗体、第三抗体可以为捕获抗体，第四抗体可以为标记抗体。又或者，在一些实施方案中，第一抗体、第二抗体、第四抗体可以为捕获抗体，第三抗体可以为标记抗体；以此类推。在一些实施方案中，捕获抗体又称为包被抗体。
在一些实施方案中，捕获抗体可以结合至固相，在一些实施方案中，所述捕获抗体可以用于包被固相；在一些实施方案中，固相没有特别限制，其可以是例如是膜载体、微粒或微孔板；例如是磁微粒，乳胶、微球、微量滴定板、硝酸纤维素膜、玻璃纤维素膜、尼龙膜或微流控芯片。
在一些实施方案中，所述标记抗体可以用可检测标记物标记，在一些实施方案中，可检测标记物可以例如金属粒子、荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，或酶标记，可以例如胶体金，胶体银，胶体硒，胶体碳，胶体碲、荧光团，罗丹明，荧光素酶，荧光素，吖啶酯，放射性同位素，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖类氧化酶，葡萄糖氧化酶，半乳糖氧化酶，葡萄糖-6-磷酸脱氢酶，三联钌、鲁米诺类、Eu螯合物，自旋标记，噬菌体标记。
在一些实施方案中，所述捕获抗体或所述标记抗体可以独立地连接至结合配偶体之一； 通过结合配偶体在测定反应中连接至固相或者可检测标记物；又或者通过结合配偶体以间接方式连接至固相或者可检测标记物。在一些实施方案中，结合配偶体可以选自生物素/抗生物素蛋白、生物素或其衍生物/亲和素或其衍生物(如链霉亲和素)、受体/配体，地高辛/地高辛配基，碳水化合物/凝集素。在一些实施方案中，例如抗体连接至生物素，例如抗体连接至链霉亲和素。
在一些实施方案中，本公开提供一种检测丙型肝炎试剂盒，包括所述检测丙型肝炎病毒核心抗原的抗体组合，实现丙型肝炎病毒核心抗原的灵敏检测。
在一些实施方案中，试剂盒还包括检测丙型肝炎病毒抗体的试剂。在一些实施方案中，本公开的试剂盒可用于丙肝病毒抗原和抗体的联合检测。本发明的试剂盒，通过对HCV表面抗体和HCV核心抗原的联合检测，提高了丙肝病毒的检出率，并减少了丙肝抗体检测窗口期的漏诊率。
在一实施方案中，所述丙型肝炎病毒抗体可以为core抗体，E1抗体、E2抗体、NS2抗体、NS3抗体、NS4抗体或NS5抗体中的至少一种。在一些实施方案中，检测丙型肝炎病毒抗体可以采用双抗原夹心法或间接法。在一些实施方案中，双抗原夹心法检测丙型肝炎病毒抗体，采用至少两个抗原，包括第一抗原和第二抗原，对待检测抗体进行夹心，其中第一抗原和第二抗原可以选自HCV核心抗原第1位至56位氨基酸中的片段，NS3第1201位至1490位氨基酸中的片段，NS4的1883位至1925位氨基酸中的片段；或者选自以上片段的组合或嵌合的序列；例如第一抗原和第二抗原可以是HCV核心抗原第1位至35位氨基酸，NS3第1223位至1426位氨基酸，NS4的1890位至1923位氨基酸。
可选地，所述丙型肝炎病毒NS3的第1201位至1490位氨基酸序列如SEQ ID NO:2所示：
可选地，所述丙型肝炎病毒NS4的1883至1925位氨基酸氨基酸序列如SEQ ID NO:3所示：VINLLPGILSPGALVVGVICAAILRRHVGPGEGAVQWMNRLIA。
例如第一抗原和第二抗原来自同一丙型肝炎病毒抗原的不同位置，例如来自丙型肝炎病毒核心抗原第7-48位氨基酸序列，例如来自丙型肝炎病毒核心抗原第7-21位氨基酸序列，和/或第29-48位氨基酸序列。在一些实施方案中，第一抗原和第二抗原可以用作捕获抗原和标记抗原，例如第一抗原为捕获抗原，第二抗原为标记抗原，或者第一抗原为标记抗原， 第二抗原为捕获抗原。在一些实施方案中，所述捕获抗原可以结合至固相。在一些实施方案中，所述捕获抗原可以用于包被固相；在一些实施方案中，固相没有特别限制，其可以是例如是膜载体、微粒或微孔板；例如是磁微粒，乳胶、微球、微量滴定板、硝酸纤维素膜、玻璃纤维素膜、尼龙膜或微流控芯片。在一些实施方案中，所述标记抗原用可检测标记物标记，在一些实施方案中，可检测标记物例如金属粒子、荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，或酶标记，例如胶体金，胶体银，胶体硒，胶体碳，胶体碲、荧光团，罗丹明，荧光素酶，荧光素，吖啶酯，放射性同位素，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖类氧化酶，葡萄糖氧化酶，半乳糖氧化酶，葡萄糖-6-磷酸脱氢酶，三联钌、鲁米诺类、Eu螯合物，自旋标记，噬菌体标记。在一些实施方案中，所述捕获抗原或所述标记抗原可以独立地连接至结合配偶体之一；通过结合配偶体在测定反应中连接至固相或者可检测标记物；又或者通过结合配偶体以间接方式连接至固相或者可检测标记物。在一些实施方案中，结合配偶体选自生物素/抗生物素蛋白、生物素或其衍生物/亲和素或其衍生物(如链霉亲和素)、受体/配体，地高辛/地高辛配基，碳水化合物/凝集素。在一些实施方案中，例如抗原连接至生物素，例如抗原连接至链霉亲和素。
在一些实施方案中，试剂盒还可以包括适合进行免疫测定的试剂。在一些实施方案中，本公开的试剂盒可以用于进行免疫测定，例如ELISA，化学发光、免疫层析、间接免疫荧光测定IFA，放射免疫测定RIA以及其它非酶联抗体结合试验或方法。
在一些实施方案中，例如在ELISA实验方案中，可以将捕获抗体包被固相如磁珠或者微孔板，捕获样品中的HCV核心抗原，然后用标记抗体与结合在磁珠或者板上的抗原再次结合，经显色后读取结果。在一些实施方案中，捕获抗体可以固定到固相表面，例如塑料、膜如硝酸纤维素膜、玻璃、磁珠或金属支持物等等。在一些实施方案中，来自受试者的样品可以与所述固相中的捕获抗体接触，然后接触带有可检测标记的标记抗体，通过可检测标记显色或发出可识别信号。在一些实施方案中，可以采用封闭剂如牛血清白蛋白、奶粉溶液、明胶、PVP、Superblock封闭非特异性位点，減少非特异性结合造成的背景。在一些实施方案中，可以采用稀释剂，如应用BSA和磷酸缓冲盐液(PBS)/吐温稀释待测样品，有助于减少非特异性背景。
在一些实施方案中，试剂盒还可以包括病毒裂解液。在一些实施方案中，病毒裂解液可以包含例如变性剂，表面活性剂，保护蛋白，硫酸铵和无水乙醇。在一些实施方案中，所述病毒裂解液可以是缓冲液，例如磷酸盐缓冲液。在一些实施方案中，所述裂解液不需要进行抗原抗体的解离，通过调整温和的裂解液，能够把病毒中的核心抗原释放出来，从而实现抗原抗体高效的反应，提高病毒检出率。
在一些实施方案中，本公开提供检测丙型肝炎病毒核心抗原的抗体组合在制备检测丙型肝炎试剂盒中的用途。
在一些实施方案中，本公开还提供一种检测丙型肝炎的方法，包括用检测丙型肝炎病毒核心抗原的抗体组合接触受试者的样品，检测抗原抗体免疫反应信号。
在一些实施方案中，来自受试者的样品可以包括健康或病理状态的生物组织、细胞或体液，例如血液样品，例如血浆、血清、血制品，例如精液或阴道分泌物。
在一些实施方案中，采用本领域已知的方法制备本公开的抗体，例如第一抗体、第二抗体、第三抗体或第四抗体。在一些实施方案中，可以利用丙型肝炎病毒核心抗原第28-35位氨基酸序列作为免疫原中的抗原性部分，利用免疫原免疫动物，获得第一抗体；在一些实施方案中，可以利用丙型肝炎病毒核心抗原第100-120位氨基酸序列作为免疫原中的抗原性部分，利用免疫原免疫动物，获得第二抗体；在一些实施方案中，可以利用丙型肝炎病毒核心抗原第35-53位氨基酸序列作为免疫原中的抗原性部分，利用免疫原免疫动物，获得第三抗体；在一些实施方案中，可以利用丙型肝炎病毒核心抗原第60-72位氨基酸序列作为免疫原中的抗原性部分，利用免疫原免疫动物，获得第四抗体。在一些实施方案中，可以利用丙型肝炎病毒核心抗原全长作为免疫原中的抗原性部分，利用免疫原免疫动物，再用丙型肝炎病毒核心抗原第28-35位氨基酸序列筛选第一抗体；用丙型肝炎病毒核心抗原第100-120位氨基酸序列筛选第二抗体；用丙型肝炎病毒核心抗原第35-53位氨基酸序列筛选第三抗体；用丙型肝炎病毒核心抗原第60-72位氨基酸序列筛选第四抗体。在一些实施方案中，免疫原中除了抗原性部分，还可以包含适当的载体蛋白。在一些实施方案中，适当的载体蛋白是本领域技术人员已知的，其可以是例如KLH和BSA等。
在一些实施方案中，本公开提供制备检测丙型肝炎病毒核心抗原的抗体组合的方法，包括：选自下述片段1-片段4作为免疫原中的抗原性部分，利用免疫原免疫动物，获得分别特异性结合片段1-片段4的抗体：片段1：丙型肝炎病毒核心抗原第28-35位氨基酸序列；片段2：丙型肝炎病毒核心抗原第100-120位氨基酸序列；片段3：丙型肝炎病毒核心抗原第35-53位氨基酸序列；和片段4：丙型肝炎病毒核心抗原第60-72位氨基酸序列。
可选地，所述丙型肝炎病毒核心抗原的1-175位氨基酸序列如SEQ ID NO:1所示：
本公开提供的4个针对不同表位的抗体组合提高了样本整体检出率，并且还不提高假阳性，特别适合临床检测应用。
实施例
实施例1 HCV core抗体制备
1.1免疫小鼠
取1m HCV core抗原(菲鹏生物股份有限公司提供)与等量弗氏完全佐剂混合，采用皮下、腹腔多点注射BALB/c小鼠，第一次免疫14天后腹腔增强免疫，增强免疫到四针后，采尾血进行效价检测，效价达到融合要求。小鼠末次免疫后三天，在无菌条件下取出脾脏供融合用。
1.2杂交瘤细胞系的制备
(1)饲养细胞的制备
以BALB/c鼠腹腔巨噬细胞作饲养细胞。在融合前1天，BALB/c鼠拉颈处死，75％酒精全身浸泡，超净台内，无菌操作下用剪刀剪开腹部皮肤，暴露腹膜，用注射器腹腔注入RPMI 1640基础培养液5mL，反复冲洗，回收冲洗液，1000rpm，离心5分钟，留沉淀，用RPMI 1640筛选培养液(含HAT的RPMI 1640完全培养液中)重悬，调整细胞浓度1×105个/mL，加入96孔板，150μL/孔，37℃，5％CO2培养过夜。
(2)免疫脾细胞的制备
小鼠末次免疫后三天，在无菌条件下取出脾脏，置于平皿中，RPMI 1640基础培养液冲洗一次，放于小烧杯的尼龙网上磨碎过滤，制成细胞悬液。离心，弃上清，RPMI 1640基础培养液重悬，如此重复三次，计数。
(3)骨髓瘤细胞的制备
小鼠骨髓瘤细胞Sp2/0(菲鹏生物股份有限公司保存)经8-氮鸟嘌呤筛选后，培养至对数生长期，取两大瓶制成细胞悬液，离心，弃上清，用RPMI 1640基础培养液重悬，如些重复三次，计数。
(4)细胞融合及HAT选择杂交瘤
将骨髓瘤细胞与免疫脾细胞按1:10比例混合，在50mL塑胶离心管内用RPMI 1640基础培养液洗1次，1200rpm，离心8分钟。弃上清，将细胞混匀，缓慢加入1mL 50％的PEG1500融合，融合1分钟后加入15mL的RPMI 1640基础培养液终止细胞融合。1000rpm，离心5分钟。弃上清，用50mL的RPMI 1640筛选培养液轻轻混悬，平分于10块铺有饲养细胞的96孔板，50μL/孔，37℃，5％CO2培养。培养至第六天，换HT培养液(含HT的RPMI 1640完全培养液)两次。
1.3抗体的筛选
以HCV core抗原包被酶标反应板，4℃包被过夜后，用含10％小牛血清或1％脱脂奶 粉的0.02M pH7.2 PBS，0.15ml/孔，37℃封闭2小时，加入细胞培养上清37℃、30分钟后加入2000倍稀释的辣根过氧化酶标记的羊抗鼠IgG(菲鹏生物股份有限公司生产，货号BA-PAB-MU0001)，37℃、30分钟后，每孔加入100μl含0.1％(M/V)邻苯二胺，0.1％(V/V)双氧水，pH5.0柠檬酸磷酸缓冲液，37℃、15分钟，加入稀硫酸溶液，每孔50μl，测450nm吸光度。RPMI 1640完全培养液作为阴性对照，以测定值与对照值得比≧2.0为阳性细胞孔。融合三次共获得14株稳定分泌HCV core抗体的细胞株，其ELISA法测定的效价均在105～107之间。
实施例2 HCV core抗体表位鉴定
用10种HCV短肽抗原A1～A10分别包被微孔，以PBS+20％NBS为稀释液，稀释实施例1所得14株单抗(作为一抗)浓度到1μg/ml，以羊抗鼠IgG-HRP为二抗，依据各单抗对不同抗原的反应情况来确定单抗表位，结果如下表1所示。
表1
实施例3 HCV core抗体表位配对
在ELISA平台测试不同抗体表位配对的检测效果，取实施例2所鉴定抗体分别作为 HRP酶标抗体和包被抗体。
HRP酶标抗体制备工艺如下：
(1)将12mg抗体用20mM PH9.51 CB缓冲液透析，4℃透析过夜；换液
(2)取24mgHRP用缓冲液成10mg/ml的HRP溶液，
(3)向HRP溶液中加入5ml新配的10mg/ml的NaIO4溶液，4℃下避光搅拌40分钟；
(4)加入20μl乙二醇避光搅拌60分钟；
(5)将上述处理好的HRP溶液加入到抗体溶液中，用20mM PH9.51的CB缓冲液透析，4℃透析过夜；
(6)加入20μl的NaBH4终止反应；
(7)用G25脱盐柱进行纯化，收集主峰测浓度后加入甘油保存至-20℃备用。
ELISA测试方法如下：
(1)取包被抗体分别按照1μg/ml用量分别加入到pH 9.51，50mM CB中混匀后，每孔100μl,包被到ELISA聚苯乙烯微孔板中，4℃包被18-22小时；
(2)取出包被板平衡置室温，用洗涤液洗板1次拍干后，每孔加220μl封闭液37℃封闭2小时；甩干置于湿度小于30％的干燥房或者是电子干燥箱中干燥24h后待用。
(3)将上述制得的HRP酶标抗体稀释为酶标抗体工作液，待用。
取100μl待测样品按照1:1稀释，在37度恒温箱反应90min，洗板5次，每次浸泡1min；
拍干后加入200μl HRP酶标抗体工作液，37℃反应30min，洗板5次，每次浸泡1min；
加入TMB显色液，显色10min，
加入终止液50μl，用450nm～630nm双波长读值。
其中单抗5E-7、8F-16、13C-10、3F-21、6B-25分别作为包被抗体，而单抗10D-2、11C-13、5G-8、6D-33、12B-3分别作为HRP酶标抗体。结果如下表2所示，结合HCV core 28-35aa的抗体与结合HCV core 60-72aa抗体配对夹心能检测到低至2ng/ml的HCV core抗原，结合HCV core 35-53aa的抗体与结合HCV core 100-120aa抗体配对夹心能检测到低至2ng/ml的HCV core抗原。这两种抗体配对组合比其它配对组合的检测灵敏度更高。
表2.

实施例4不同表位的抗体配对检测比较
依据实施例3所述的ELISA测试方法，用不同的抗体与针对表位60-72aa的标记抗体配对，进行测试，结果如表3所示。
表3.
实施例5用四种HCV core抗体进行表位配对
依据实施例3所述的ELISA测试方法，用4种抗体配对夹心组合对临床样本进行测试，配对模式如表4所示。
表4.

测试结果如下表5所示，三种组合测试临床阴性样本的本底值差不多。而采用本公开抗体组合，检测阳性样本的信号值总体会相对最高，对照组合2稍微低于本公开抗体组合，对照组合1明显低于本公开抗体组合。
表5.

实施例6临床样本测试
在ELISA平台分别用本公开抗体组合和对照组合测试35份临床阳性样本(RNA测试阳性)和400份临床阴性样本(RNA测试阴性)。结果如下表6所示，本公开抗体组合实现了0漏检，并且还具有相当高的特异性，证明本公开抗体组合具有显著的技术进步。
表6
以上所述仅为本公开的可选的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之。
工业实用性
本公开提供一种丙型肝炎病毒检测试剂盒。本公开提供丙型肝炎病毒检测试剂盒，其包括检测丙型肝炎病毒核心抗原的第一抗体、第二抗体、第三抗体和第四抗体。本公开的试剂盒灵敏度高，稳定性好，操作简单，可用于早期急性丙型肝炎的快速检测，因此具备优异的实用性。

Claims (16)

  1. 一种检测丙型肝炎病毒核心抗原的抗体组合，包括第一抗体、第二抗体、第三抗体和第四抗体，所述第一抗体特异性结合丙型肝炎病毒核心抗原第28-35位氨基酸序列，所述第二抗体特异性结合丙型肝炎病毒核心抗原第100-120位氨基酸序列，所述第三抗体特异性结合丙型肝炎病毒核心抗原第35-53位氨基酸序列，所述第四抗体特异性结合丙型肝炎病毒核心抗原第60-72位氨基酸序列。
  2. 根据权利要求1所述的抗体组合，包括捕获抗体和标记抗体；
    可选地，所述捕获抗体选自所述第一抗体至第四抗体中的任意一种、任意两种或任意三种，标记抗体选自所述第一抗体至第四抗体中的除捕获抗体的其他所有抗体；
    可选地，所述捕获抗体为第一抗体和第二抗体；所述标记抗体为第三抗体和第四抗体；
    可选地，所述标记抗体为第一抗体和第二抗体；所述捕获抗体为第三抗体和第四抗体。
  3. 根据权利要求2所述的抗体组合，其中捕获抗体结合至固相；
    可选地，所述固相是膜载体、微粒或微孔板；
    可选地，所述固相是磁微粒、乳胶、微球、微量滴定板、硝酸纤维素膜、玻璃纤维素膜、尼龙膜或微流控芯片。
  4. 根据权利要求1-3中任一项所述的抗体组合，其中所述标记抗体用可检测标记物标记；
    可选地，所述可检测标记物选自金属粒子、荧光标记、发色团标记、电子致密标记、化学发光标记、放射性标记或酶标记；
    可选地，所述可检测标记物选自胶体金、胶体银、胶体硒、胶体碳、胶体碲、荧光团、罗丹明、荧光素酶、荧光素、吖啶酯、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶、葡萄糖-6-磷酸脱氢酶、三联钌、鲁米诺类、Eu螯合物、自旋标记、噬菌体标记。
  5. 根据权利要求1-4中任一项所述的抗体组合，其中所述捕获抗体或所述标记抗体独立地连接至结合配偶体之一；
    可选地，所述结合配偶体选自生物素/抗生物素蛋白、生物素或其衍生物/亲和素或其衍生物、受体/配体，地高辛/地高辛配基或碳水化合物/凝集素；
    可选地，所述结合配偶体选自生物素/链霉亲和素。
  6. 一种检测丙型肝炎试剂盒，包括权利要求1-5任一项所述的检测丙型肝炎病毒 核心抗原的抗体组合。
  7. 根据权利要求6所述的试剂盒，还包括检测丙型肝炎病毒抗体的试剂。
  8. 根据权利要求7所述的试剂盒，所述丙型肝炎病毒抗体为core抗体、E1抗体、E2抗体、NS2抗体、NS3抗体、NS4抗体或NS5抗体中的至少一种。
  9. 根据权利要求7或8所述的试剂盒，所述检测丙型肝炎病毒抗体的试剂包含抗原；
    可选地，所述检测丙型肝炎病毒抗体采用双抗原夹心法或间接法；
    可选地，所述双抗原夹心法包括使用第一抗原和第二抗原；
    可选地，所述第一抗原和第二抗原分别选自HCV核心抗原第1位至56位氨基酸中的片段，NS3第1201位至1490位氨基酸中的片段，NS4的1883位至1925位氨基酸中的片段；或者选自以上片段的组合或嵌合的序列；
    可选地，所述第一抗原和第二抗原分别为捕获抗原和标记抗原；
    可选地，所述捕获抗原结合至固相；
    可选地，所述固相是膜载体、微粒或微孔板；例如是磁微粒，乳胶、微球、微量滴定板、硝酸纤维素膜、玻璃纤维素膜、尼龙膜或微流控芯片；
    可选地，所述标记抗原用可检测标记物标记；
    可选地，所述可检测标记物选自金属粒子、荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，或酶标记，例如胶体金，胶体银，胶体硒，胶体碳，胶体碲、荧光团，罗丹明，荧光素酶，荧光素，吖啶酯，放射性同位素，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖类氧化酶，葡萄糖氧化酶，半乳糖氧化酶，葡萄糖-6-磷酸脱氢酶，三联钌、鲁米诺类、Eu螯合物，自旋标记，噬菌体标记；
    可选地，所述捕获抗原或所述标记抗原独立地连接至结合配偶体之一；
    可选地，所述结合配偶体选自生物素/抗生物素蛋白、生物素或其衍生物/亲和素或其衍生物、受体/配体，地高辛/地高辛配基或碳水化合物/凝集素。
  10. 根据权利要求6-9中任一项所述的试剂盒，还包括用于免疫测定的试剂；
    可选地，所述免疫测定选自ELISA，化学发光、免疫层析、间接免疫荧光测定IFA，放射免疫测定RIA以及其它非酶联抗体结合试验或方法。
    可选地，所述试剂盒还包括病毒裂解液。
  11. 如权利要求1-5任一项所述的抗体组合在制备检测丙型肝炎试剂盒中的用途。
  12. 根据权利要求11所述的用途，所述试剂盒还包括检测丙型肝炎病毒抗体的试剂。
  13. 一种检测丙型肝炎的方法，包括用权利要求1-5任一项所述的检测丙型肝炎病毒核心抗原的抗体组合接触受试者的样品，检测抗原抗体免疫反应信号。
  14. 根据权利要求13所述的方法，还包括用检测丙型肝炎病毒抗体的试剂接触受试者的样品，检测抗原抗体免疫反应信号。
  15. 权利要求1-5任一项所述的检测丙型肝炎病毒核心抗原的抗体组合用于检测丙型肝炎。
  16. 制备权利要求1所述的检测丙型肝炎病毒核心抗原的抗体组合的方法，包括：选自下述片段1-片段4作为免疫原中的抗原性部分，利用免疫原免疫动物，获得分别特异性结合片段1-片段4的抗体：
    片段1：丙型肝炎病毒核心抗原第28-35位氨基酸序列；
    片段2：丙型肝炎病毒核心抗原第100-120位氨基酸序列；
    片段3：丙型肝炎病毒核心抗原第35-53位氨基酸序列；和
    片段4：丙型肝炎病毒核心抗原第60-72位氨基酸序列。
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