WO2020221098A1 - 一种丙型肝炎病毒检测试剂盒 - Google Patents

一种丙型肝炎病毒检测试剂盒 Download PDF

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WO2020221098A1
WO2020221098A1 PCT/CN2020/086437 CN2020086437W WO2020221098A1 WO 2020221098 A1 WO2020221098 A1 WO 2020221098A1 CN 2020086437 W CN2020086437 W CN 2020086437W WO 2020221098 A1 WO2020221098 A1 WO 2020221098A1
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antigen
hepatitis
antibody
virus
amino acids
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PCT/CN2020/086437
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English (en)
French (fr)
Inventor
潘少丽
李瑞净
于秀玲
程珍珠
武云波
陈一尘
池朗山
龚航
欧阳岁燕
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广东菲鹏生物有限公司
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Priority to CA3138571A priority Critical patent/CA3138571C/en
Priority to US17/606,059 priority patent/US20220252614A1/en
Publication of WO2020221098A1 publication Critical patent/WO2020221098A1/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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • 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 invention relates to the field of virus detection. Specifically, the present invention relates to a detection method and kit for hepatitis C virus.
  • Hepatitis C is one of the infectious diseases that seriously threaten human health, and there is no effective vaccine to prevent its spread.
  • DAAs direct antiviral drugs
  • the main plan for HCV infection in my country to receive antiviral treatment is still the treatment plan based on interferon.
  • hepatitis C has milder symptoms, slower development, and less severe disease, which is not easy to attract the attention of clinicians and patients.
  • HCV is mainly spread through blood transfusion and transfusion of blood products. Hepatitis C virus can cause acute or chronic infection.
  • Acute hepatitis C virus infection usually has no symptoms and only in very rare cases can it cause life-threatening illness. About 15%-45% of infected people can clear the virus by themselves within 6 months of infection without any treatment. The remaining 60%–80% of infected people will develop chronic hepatitis C virus infection. Among these chronic hepatitis C virus infections, the risk of cirrhosis within 20 years is 15%-30%. Hepatitis C is difficult to treat, long course of treatment, poor curative effect, and high cost, so it is very important to choose the ideal detection method to detect HCV as soon as possible.
  • HCV is a positive single-stranded RNA virus with a spherical envelope, belonging to the flavivirus family, with a total length of about 9500 bases.
  • the HCV genome is divided into 5'and 3'non-coding regions on both sides, and the open reading frame (ORF) in the middle, which is divided into structural and non-structural regions.
  • the structural region includes a core protein region (C) and two envelope protein regions (E1, E2), which encode core protein and envelope protein, respectively.
  • Non-structural protein regions include NS2, NS3, NS4 and NS5 regions, which encode functional proteins such as proteases (NS2, NS3 and NS4A regions), helicase (NS3) and RNA-dependent RNA polymerase (NS5B region).
  • the hepatitis C virus core protein contains about 190 aa, which plays a very important role in virus replication.
  • genetic engineering is used to express the above-mentioned structural and non-structural proteins as coating antigens to establish an anti-HCV detection ELISA method.
  • the HCV genome has significant heterogeneity, and the degree of variation in different segments of the same genome has significant differences.
  • the 5'non-coding region is the most conservative and has become the focus of HCV molecular diagnostic research.
  • HCV antibody detection is currently the most commonly used method for hospitals and blood stations to determine and screen patients for HCV infection, but its fatal disadvantage is the existence of a "window period", that is, there is a 40-70 days between HCV infection and HCV antibody production. Blood donors have been infected and are infectious, but cannot be detected with current antibody detection reagents. This stage is called the Preseroconversion Window Phase (PWP) after infection. The existence of the window period is the main cause of blood transfusion infection.
  • PWP Preseroconversion Window Phase
  • HCV nucleic acid detection is the most reliable of the three detection methods. It can be detected at the initial stage of infection and can also reflect the content of the virus. It is mainly used for the selection of antiviral treatment and the monitoring of efficacy.
  • RNA is aseptically extracted, which is easy to operate and equipment. Factors such as the environment and the environment cause errors, resulting in false positives or false negatives, which is not conducive to promotion in ordinary hospitals and has a relatively small market share.
  • HCV antigen-antibody joint test can simultaneously detect HCV antigen and antibody in the sample.
  • HCV antigen and antibody joint detection method a large number of antigens and antibodies must be screened and tested to avoid overlapping antigen and antibody epitopes and the interaction between monoclonal antibodies against hepatitis C virus antigens and hepatitis C virus recombinant antigens. The problem of cross-reactivity.
  • the selected antigen region must also have high immunogenicity, which is conducive to preparing antibodies and capturing the antigen in the sample.
  • CN105228649A discloses a mutant core protein antigen containing deletions of amino acids 34 and 48 and amino acids 115-121 for joint inspection; it also discloses deletion of 5 amino acids (C11-9 binding regions 32, 33 and 34 and core C11-14 binding Amino acid residues 47 and 48) to avoid the problem of reaction between the core antigen used to capture the core antibody and the detection antibody used to detect the core antigen.
  • the prior art CN1489692A discloses an HCV antigen-antibody combination for HCV detection, which teaches HCV core antigens such as amino acids 10-53 and 120-130 and NS3 antibodies for combined detection.
  • Such a joint inspection method usually requires a large number of cross-experiments and screenings for the epitopes of the antibodies used to capture the HCV antigens of the patients and the capture of the HCV antibodies of the patients, and requires high manpower, equipment, and reagent costs.
  • the inventor After a lot of theoretical research and experimental exploration, the inventor fully considered the entire process of HCV infection, analyzed and studied the antibodies used in various HCV antigens of the patients to be captured and captured the HCV antibodies of the patients by using the antigen interval, and through a large number of cross experiments and screening, obtained A combination of hepatitis C virus core antigen regions for hepatitis C virus detection.
  • the inventors have proved that the selected epitope region has excellent immunogenicity, and the antibodies prepared therefrom can unexpectedly be combined with each other for high activity detection of HCV core antigen.
  • the inventor also surprisingly discovered that the monoclonal antibody prepared from the antigen can be combined with the HCV antigen to complement each other's deficiencies in the detection of HCV antigen and antibody, thereby reducing the risk of missed detection and shortening the window period.
  • the present invention provides a hepatitis C virus detection kit and preparation method.
  • the kit of the invention has improved sensitivity and stability, shortened reaction time, simple operation, and is suitable for popularization and application.
  • the kit of the invention particularly shortens the window period and reaction time, and can be used for the rapid diagnosis of early acute hepatitis C.
  • the present invention provides a hepatitis C virus detection kit, which includes a first antibody and a second antibody for detecting hepatitis C virus core antigen, wherein the first antibody is directed against the hepatitis C virus core antigen No. 95-117 The second antibody is directed against the epitope in the 55-72 amino acid sequence of the hepatitis C virus core antigen.
  • the first antibody specifically binds to the 95-117th amino acid sequence of the hepatitis C virus core antigen.
  • the second antibody specifically binds to the 55-72 amino acid sequence of the hepatitis C virus core antigen.
  • the first antibody and/or the second antibody may be monoclonal antibodies.
  • the antibodies of the invention are prepared by methods known in the art, such as the first antibody and/or the second antibody.
  • the antibody of the present invention, such as the first antibody and/or the second antibody can be prepared by immunizing an animal with an antigen comprising the 55-72 amino acid sequence and/or an antigen comprising the 95-117 amino acid sequence.
  • the 55-72 amino acid sequence and/or the 95-117 amino acid sequence may be used as an antigen to immunize an animal to prepare the antibody of the present invention, such as the first antibody and/or the second antibody.
  • "specifically binds” can mean that the antibody selectively or preferentially binds to the amino acid sequence. Standard measurement methods such as surface plasmon resonance techniques (e.g. ) Determine the binding affinity.
  • the first antibody binds to the same epitope as an antibody that specifically binds the 95-117th amino acid sequence of the hepatitis C virus core antigen.
  • the second antibody binds to the same epitope as the antibody that specifically binds the 55-72 amino acid sequence of the hepatitis C virus core antigen.
  • the "antibody that binds to the same epitope" as the reference antibody means that, for example, it blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay; or the reference antibody blocks more than 50% of the reference antibody in a competition assay. The binding of an antibody to its antigen.
  • any suitable in vitro assays, cell-based assays, in vivo assays, animal models, etc. can be used to test the effects of the antibodies of the present invention, such as binding activity and/or cross-reactivity.
  • the assay may include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, and the like.
  • the EC50 value of the antibody (or antigen-binding fragment thereof) of the present invention binding to the antigen may be, for example, 1 ⁇ M-1 pM, such as 1 nM-1 pM, such as 100 pM-1 pM.
  • the first antibody (for amino acids 95-117 of the epitope) and the second antibody (for amino acids 55-72 of the epitope) can be used as capture antibodies (or called coating antibodies) and labeled antibodies,
  • the first antibody is a capture antibody and the second antibody is a labeled antibody, or the first antibody is a labeled antibody and the second antibody is a capture antibody.
  • the first antibody is a capture antibody (or called a coating antibody)
  • the second antibody is a labeled antibody.
  • alternative antibodies can also be used as coating antibodies or labeled antibodies.
  • an antibody against amino acids 17-35 of the epitope can be used as a capture antibody (or called a coating antibody).
  • the capture antibody is bound to a solid phase.
  • the capture antibody can be used to coat a solid support.
  • the solid support is not particularly limited, and it may be, for example, magnetic particles such as magnetic beads, latex particles, and microtiter plates.
  • the labeled antibody is labeled with a detectable label, for example, with a fluorescent label such as acridinium ester, for example, by a linker such as biotin-avidin and a fluorescent label such as acridinium ester.
  • antibody herein is used in the broadest sense, which can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antigen-binding fragments of antibodies, as long as they Display the required biological activity, such as specific binding to HCV antigen.
  • Antibody fragment includes a portion of a full-length antibody, preferably its antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies or domain antibodies.
  • the kit further includes a first antigen and a second antigen for detecting hepatitis C virus antibodies in a subject sample.
  • the first antigen and the second antigen may be, for example, hepatitis C virus core antigen, E1, E2, NS2, NS3, NS4 [Mimms et al., Lancet 336:1590 (1990); Bresters et al. al., Vox Sang 62:213 (1992)] and NS5.
  • the first antigen and the second antigen are from different locations on the same antigen.
  • the first antigen and the second antigen may be selected from the antigens shown in SEQ ID NO: 1 and SEQ ID NO: 2 or immunogenic fragments thereof.
  • the first antigen and the second antigen may be a sequence of amino acids from positions 1 to 56 of the HCV core antigen, amino acids from positions 1201 to 1490 of NS3, and amino acids from positions 1883 to 1925 of NS4; HCV core antigen Sequences of amino acids 1 to 35, amino acids 1223 to 1426 of NS3, and amino acids 1890 to 1923 of NS4.
  • the first antigen and the second antigen can be used as capture antigens and labeled antigens.
  • the first antigen is a capture antigen and the second antigen is a labeled antigen, or the first antigen is a labeled antigen and the second antigen is a capture antigen. antigen.
  • the first antigen is a capture antigen and the second antigen is a labeled antigen.
  • the capture antigen is bound to a solid phase.
  • the capture antigen can be used to coat a solid support.
  • the solid support is not particularly limited, and it may be, for example, magnetic particles such as magnetic beads, latex particles, and microtiter plates.
  • the labeled antigen is labeled with a detectable label, such as a fluorescent label such as an acridinium ester, for example, by a linker such as biotin-avidin and a fluorescent label such as an acridinium ester.
  • the detectable label used to label the antigen or antibody is not particularly limited.
  • the labels may include, but are not limited to, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels, as well as indirect labels such as enzymes or ligands, for example, through enzymatic reactions or Indirect detection of molecular interactions.
  • exemplary labels include, but are not limited to, radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ - Galactosidase, glucoamylase, lysozyme, sugar oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin label , Phage markers and so on.
  • HRP horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ - Galactosidase glucoamylase
  • lysozyme sugar oxidase
  • sugar oxidase for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
  • kits of the invention include reagents suitable for performing immunoassays.
  • the kit of the present invention can be used to perform immunoassays, such as ELISA, indirect immunofluorescence assay IFA, radioimmunoassay RIA, and other non-enzyme-linked antibody binding assays or methods.
  • HCV antibody can be coated with a solid phase such as magnetic beads, the HCV antigen in the sample can be captured, and then the labeled antibody can be combined with the antigen bound on the reaction plate again. Read the result after coloring.
  • the HCV antibody of the present invention can be used to coat solid phases such as magnetic beads or as a labeled secondary antibody.
  • the antibody or antigen-binding fragment thereof is immobilized on a surface, such as a solid support, such as plastic, membrane such as nitrocellulose membrane, glass, magnetic beads or metal support.
  • a sample from a subject is contacted with the solid support, and then contacted with an antibody indicator with a detectable label for color development.
  • blocking agents such as bovine serum albumin, milk powder solution, gelatin, PVP, Superblock can be used to block non-specific sites, thereby reducing the background caused by non-specific binding.
  • a diluent such as the application of BSA and phosphate buffered saline (PBS)/Tween to dilute the antiserum, can be used to help reduce non-specific background.
  • a sample from a subject may include biological tissues, cells, or body fluids in a healthy or pathological state, such as blood samples, such as plasma, serum, blood products, such as semen or vaginal secretions.
  • biological tissues, cells, or body fluids in a healthy or pathological state such as blood samples, such as plasma, serum, blood products, such as semen or vaginal secretions.
  • the kit also includes a virus lysis solution.
  • the virus lysate may contain, for example, denaturants, surfactants, protective proteins, ammonium sulfate, and absolute ethanol.
  • the virus lysate may be a buffer, such as a phosphate buffer.
  • the lysis solution does not need to dissociate the antigen and antibody. By adjusting the mild lysis solution, the sensitivity of the antibody is not affected, which is conducive to the combination of antigen and antibody, and can release the core antigen in the virus, thereby Realize the efficient reaction of antigen and antibody and increase the detection rate of virus.
  • the present invention provides the use of the first antibody and the second antibody for detecting hepatitis C virus core antigen in preparing a kit for detecting hepatitis C virus.
  • the present invention provides a method of detecting hepatitis C virus, the method comprising contacting a sample from a subject with a first antibody and a second antibody.
  • the first antibody is directed against the epitope in the 95-117 amino acid sequence of the hepatitis C virus core antigen
  • the second antibody is directed against the epitope in the 55-72 amino acid sequence of the hepatitis C virus core antigen.
  • the present invention provides a combination of an immunogenic polypeptide comprising amino acids 95-117 of the hepatitis C virus core antigen and an immunogenic polypeptide comprising amino acids 55-72 of the hepatitis C virus core antigen for preparation Use of antibodies to detect hepatitis C virus core antigen.
  • the present invention provides a method for preparing antibodies for detecting hepatitis C virus core antigen, the method comprising combining an immunogenic polypeptide comprising amino acids 95-117 of the hepatitis C virus core antigen and The immunogenic polypeptides of the 55-72 amino acids of the virus core antigen are respectively immunized to animals, thereby preparing antibodies such as monoclonal antibodies for detecting the hepatitis C virus core antigen.
  • the immunogenic polypeptide includes amino acids 95-117 of the hepatitis C virus core antigen and/or adjuvant and amino acids 55-72 of the hepatitis C virus core antigen and/or adjuvant.
  • the core antigen epitope regions identified in the present invention such as the 95-117th amino acids of the hepatitis C virus core antigen and the 55-72 amino acids of the hepatitis C virus core antigen (which can be artificially synthesized by, for example, chemical methods) )
  • an appropriate carrier protein to prepare antibodies such as monoclonal antibodies for immunizing animals.
  • the appropriate carrier protein is known to those skilled in the art, and it can be, for example, KLH, BSA, and the like.
  • the kit of the present invention may include the above-mentioned first antibody and second antibody, and further include the hepatitis C virus first antigen and/or second antigen.
  • the method for detecting hepatitis C virus of the present invention may further include contacting a sample from a subject with a first antigen and/or a second antigen from hepatitis C virus.
  • the first antigen and/or the second antigen may be, for example, hepatitis C virus core antigen, E1, E2, NS2, NS3, NS4, and NS5, for example, the first antigen and/or the second antigen are derived from Different positions of the same hepatitis C virus antigen, for example, from the 7-48 amino acid sequence of the hepatitis C virus core antigen, for example, from the 7-21 amino acid sequence of the hepatitis C virus core antigen, and/or 29-48
  • the amino acid sequence may include any of the following amino acid fragments or combinations thereof: amino acids from positions 1 to 56 of the HCV core antigen, amino acids from positions 1201 to 1490 of NS3, and amino acids from positions 1883 to 1925 of NS4
  • the present invention provides a method for preparing a reagent or kit for detecting hepatitis C virus, wherein the first core antigen of hepatitis C virus and the second core antigen of hepatitis C virus are used to prepare antibodies.
  • the present invention provides the use of the first core antigen of hepatitis C virus and the second core antigen of hepatitis C virus in preparing reagents or kits for detecting hepatitis C virus.
  • the hepatitis C virus first core antigen may comprise or consist of amino acids 55-72 of the hepatitis C virus core antigen
  • the hepatitis C virus second core antigen may comprise the hepatitis C virus core antigen No. 95-117 amino acids or consist of them.
  • the kit of the present invention may include the first core antigen of hepatitis C virus and the second core antigen of hepatitis C virus, and may also include other locations of hepatitis C virus (ie, different from the first core antigen). The position of the antigen and the second core antigen), such as monoclonal antibodies.
  • one or two antigens in other locations of the hepatitis C virus may be, for example, hepatitis C virus core antigens, E1, E2, NS2, NS3, NS4, and NS5, for example from the same hepatitis C virus
  • the different positions of the antigen for example, from the 7-48th amino acid sequence of the hepatitis C virus core antigen, for example from the 7-21th amino acid sequence and/or the 29-48th amino acid sequence of the hepatitis C virus core antigen, for example, may be Including any of the following amino acid fragments or combinations thereof: HCV core antigen 1 to 56 amino acids, NS3 1201 to 1490 amino acids, NS4 1883 to 1925 amino acid sequence; HCV core antigen 1 to 35 Amino acids, the sequence of amino acids from 1223 to 1426 of NS3, and amino acids from 1890 to 1923 of NS4, for example from the amino acid sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the first core antigen of hepatitis C virus and the second core antigen of hepatitis C virus can be used to prepare antibodies, such as monoclonal antibodies.
  • the kit of the present invention may include antibodies prepared by the above-mentioned hepatitis C virus first core antigen and hepatitis C virus second core antigen, such as monoclonal antibodies, and optionally may also include hepatitis C One or two antigens elsewhere in the virus.
  • one or two antigens in other locations of the hepatitis C virus may be, for example, hepatitis C virus core antigens, E1, E2, NS2, NS3, NS4, and NS5, for example from the same hepatitis C virus
  • the different positions of the antigen for example, from the 7-48th amino acid sequence of the hepatitis C virus core antigen, for example from the 7-21th amino acid sequence and/or the 29-48th amino acid sequence of the hepatitis C virus core antigen, for example, may be Including any of the following amino acid fragments or combinations thereof: HCV core antigen 1 to 56 amino acids, NS3 1201 to 1490 amino acids, NS4 1883 to 1925 amino acid sequence; HCV core antigen 1 to 35 Amino acids, the sequence of amino acids from 1223 to 1426 of NS3, and amino acids from 1890 to 1923 of NS4, for example from the amino acid sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the present invention provides methods, uses, and related kits for simultaneously detecting at least one HCV antigen and at least one HCV antibody.
  • the method may include contacting the sample with at least one HCV antigen or fragment thereof coated on a solid phase to form an immune complex, and simultaneously with at least one HCV antibody coated on the solid phase. Or a fragment thereof is contacted to form an immune complex; the presence of the complex is detected to determine the presence of HCV antigen and/or antibody in the sample.
  • the method may include contacting the sample with at least one HCV antigen or fragment thereof coated on a solid phase to form an immune complex, and simultaneously with at least one HCV antibody coated on the solid phase.
  • the present invention provides a kit for the method, which comprises 1) a container containing at least one HCV antigen coated on a solid phase, and 2) containing at least one HCV antigen coated on a solid phase A container for HCV antibodies; or a container containing at least one HCV antigen coated on a solid phase and at least one HCV antibody coated on a solid phase.
  • the kit further comprises a second antigen and/or a second HCV antibody linked to a detectable label.
  • the at least one HCV antibody coated on the solid phase does not cross-react with the at least one HCV antigen coated on the solid phase.
  • the at least one HCV antibody is a monoclonal antibody to the HCV core antigen.
  • the at least one HCV antigen is an HCV core antigen, such as a recombinant antigen.
  • the HCV core antigen does not include an epitope bound by the antibody, for example, does not include an epitope in the 95-117th amino acid sequence of the core antigen and an epitope in the 55-72 amino acid sequence of the core antigen Bit.
  • the method and/or use of the present invention does not require the dissociation of antigen and antibody.
  • the antibody in the method and/or use of the present invention, can be reacted in the first step to facilitate the preferential binding of the antigen and antibody, and then the lysis solution can be added in the second step to release the core antigen in the virus. So as to achieve an efficient antigen-antibody reaction and increase the virus detection rate.
  • the first antigen and the second antigen can be used as capture antigens and labeled antigens.
  • the first antigen is a capture antigen and the second antigen is a labeled antigen, or the first antigen is a labeled antigen and the second antigen is a capture antigen. antigen.
  • the first antigen is a capture antigen and the second antigen is a labeled antigen.
  • the present invention provides a magnetic bead antigen antibody combined detection kit.
  • the kits of the present invention may include reagents suitable for mechanical energy chemiluminescence detection.
  • the kit of the present invention can use a chemiluminescence automatic instrument to detect the antigen and antibody in HCV quickly and accurately with high throughput, shorten the detection time, and quickly display the test result.
  • the present invention provides a magnetic bead antigen-antibody joint detection kit, which includes antigens and antibodies labeled on magnetic beads.
  • the kit of the present invention can use magnetic beads as a solid phase to directly label antigens and antibodies on the magnetic beads, and use the principle of double antigen sandwich method and double antibody sandwich method to detect antigens and antibodies in HCV. The detection rate shortens the window period.
  • the kit of the present invention detects the HCV core antigen, significantly shortening the window period, about 50 days in advance on average, and shortening the risk of HCV infection in the window period.
  • the combined detection of HCV core antigen and HCV antibody of the present invention can overcome the shortcomings of HCV antigen detection or HCV antibody detection alone, that is, significantly shorten the window period, reduce the risk of missed detection, reduce workload, and reduce The cost of manpower, equipment, and reagents for the two methods of separate testing.
  • the present invention provides a hepatitis C virus magnetic bead antigen antibody combined detection kit and preparation method, which solves the problems existing in the prior art, such as low sensitivity, poor stability, and long reaction time. And/or technical problems such as cumbersome operation.
  • the present invention shortens the window period, reduces the reaction time, and can be used for the rapid diagnosis of early acute hepatitis C.
  • the present invention may use magnetic beads as a carrier for detecting antigen and antibody.
  • the present invention can quickly and accurately detect antigen and antibody in HCV through a chemiluminescence automatic instrument.
  • the double antibody sandwich principle can be used to prepare the kit.
  • the antibodies in the sample are first labeled with the hepatitis C virus recombinant antigen AgI (HCV-AgI) and the biotinylated hepatitis C virus recombinant antigen AgII (HCV-AgII-BIO) on magnetic beads. ) The captured form a double antigen sandwich state.
  • the core antigen is labeled with the hepatitis C virus antigen monoclonal antibody AbI (HCV-AbI) and biotinylation on the magnetic beads.
  • HCV-AbI hepatitis C virus antigen monoclonal antibody AbII
  • HCV-AbII-BIO hepatitis C virus antigen monoclonal antibody AbII
  • the avidinized marker SA-AE can be added to form the monoclonal antibody AbI-hepatitis C virus antigen-biotinylated monoclonal antibody AbII-avidinated marker SA-AE With recombinant antigen AgI-hepatitis C virus antibody-biotinylated recombinant antigen II-avidinated marker SA-AE.
  • the plate is washed and the excitation solution is added, and the luminescence value is measured with a chemiluminescence automatic instrument. The luminescence value is positively correlated with the total concentration of antigen and antibody in the sample, and compared with the critical value to determine negative or positive.
  • the detection antigen antibody of the present invention is obtained by analyzing the sequence of hepatitis C virus.
  • Two strains of anti-hepatitis C virus antigen monoclonal antibodies (AbI, AbII), two hepatitis C virus recombinant antigens (AgI , AgII), and there is no cross-reaction between the two anti-hepatitis C virus antigen monoclonal antibodies and the two hepatitis C virus recombinant antigens.
  • the anti-hepatitis C virus antigen monoclonal antibody AbI and the hepatitis C virus recombinant antigen AgI can be used as the magnetic bead coating materials, and the anti-hepatitis C virus antigen monoclonal antibody AbII, type C Hepatitis virus recombinant antigen AgII is used as the raw material for biotinylation.
  • the double antigen sandwich method is used to detect hepatitis C virus antigen
  • the double antibody sandwich method is used to detect hepatitis C virus antibodies.
  • the invention discloses the sequences of HCV-AgI and HCV-AgII antigens.
  • HCV-AgI antigen sequence SEQ ID NO:1(W135)
  • HCV-AgII antigen sequence SEQ ID NO: 2 (W102)
  • the invention discloses a method for preparing HCV-core antigen McAb.
  • the present invention can also be performed in the mode of directly labeling HCV-AbII antibodies with acridinium esters.
  • the present invention may use magnetic beads as a solid phase carrier.
  • the present invention provides an in vitro labeling method for biotinylated hepatitis C virus antigen antibodies.
  • the present invention provides an in vitro labeling method of biotinylated hepatitis C virus antigen antibody using magnetic beads as a carrier.
  • the present invention provides a lysate for the detection of HCV antigen-antibody joint test. It does not require the dissociation of the antigen and antibody. By adjusting the mild lysate, the sensitivity of the antibody is not affected, which is conducive to the combination of antigen and antibody and can remove the virus. The core antigens in the media are released, thereby realizing an efficient reaction of antigens and antibodies and increasing the virus detection rate.
  • the present invention may use avidinylated or biotinylated SA-AE for labeling.
  • the antibodies of the invention are directed against epitopes 95-117aa of the core antigen or specifically bind to the sequence.
  • the epitope targeted by the labeled antibody is preferably between 55-72aa or specifically binds to the sequence.
  • the epitope that the coating antibody preferably targets is 95-117aa or specifically binds to the sequence.
  • there is no cross-reaction between the coated antigen and the antibody and there is no overlap of epitopes, which avoids the difficulty of staggering epitopes for core antigen antibodies and the inability to prepare antigen-antibody joint test kits in the past.
  • the method of mutating core epitopes to avoid cross-reaction with antibodies also has the problem of loss of activity after mutation.
  • the antigen and antibody epitopes of the present invention have no crossover and overlap, and do not affect the activity.
  • the present invention uses a labeled antibody and a coating antibody to target paired epitopes for HCV antigen-antibody joint detection, which advantageously avoids the problems of cross-reactivity and loss of detection activity.
  • the HCV antigen I and HCV antigen II of the present invention can be any suitable HCV antigen, such as core antigen, E1, E2, NS2, NS3, NS4, and NS5.
  • the HCV genotype detected in the present invention is not particularly limited. For example, it may be type I/1a, II/1b, III/2a, IV/2b, V/3a and then VI/3b.
  • the HCV genotype detected by the present invention is HCV 1b.
  • gene fragments of different genotypes or a combination of gene fragments of several genotypes can be used for the distribution of genotypes in different regions, but the selected amino acid segments remain unchanged.
  • the present invention analyzes and screens the core segment, and determines that the core epitope segment is 7-21aa and 29-48aa of the core antigen.
  • the core antigen segment of the present invention is preferably selected from 7-48a.
  • the HCV core antigen and HCV-core antibody of the present invention can be used for joint testing at the same time, avoiding cross-reaction between the core antigen and the core antibody.
  • the method can also overcome the shortcomings of HCV antigen detection or HCV antibody detection methods alone, that is, significantly shorten the window period, reduce the risk of missed detection, reduce workload, and reduce the cost of manpower, equipment, and reagents for separate detection of the two methodologies.
  • the antibody can be reacted in the first step to facilitate the preferential binding of antigen and antibody, and then the lysis solution will be added in the second step. It can release the core antigen in the virus, so as to achieve an efficient antigen-antibody reaction and increase the virus detection rate.
  • the combined detection method provided by the present invention can simultaneously detect HCV antigens and antibodies, which solves the need for two kits for the independent detection of core antigens and HCV antibodies on the market.
  • Figure 1 Principle diagram of antigen-antibody joint detection in a non-limiting embodiment of the present invention.
  • Figure 2 A: PCR amplification product map, 1. DNA marker (DL2000); 2. PCR amplification product; B: PKO-C175C recombinant plasmid PCR identification map, 1. PCR amplification product; 2. PKO-C175 colony PCR amplification product.
  • Figure 3 Analysis of induced expression and purity of recombinant fusion protein.
  • A 1. Protein marker (KD); 2. BL21 (DE3) without PKO-C175 expression plasmid; 3. BL21 (DE3) with PKO-C175 expression plasmid without IPTG induction; 4. BL21 (DE3) with PKO-C175 The expression plasmid IPTG was induced for 4h.
  • B 1. Protein marker (KD); 2. BL21 (DE3) IPTG containing PKO-C175 expression plasmid for 4 hours; 3. Affinity purification of C175-A samples containing fusion protein; 4. Enterokinase digestion to remove the fusion protein The sample C175-B.
  • HCV-AgI coating antigen Using genetic engineering techniques, through a large number of molecular biology analysis software, the dominant epitope segments of HCV NS3, NS4, and core antigens are selected.
  • the sequence is SEQIDNo.1 (named For W135), optimize the codon sequence to synthesize the gene, and design primers (W135-F: CGCGGATCCATGTCTACCAACCCGAAACCG; W135-R: CCGGAATTCACGAGAAGCGAAAGCGATCA) to amplify the DNA segment corresponding to W135.
  • the upstream primers have BamHI restriction sites and the downstream primers There are EcoRI restriction sites.
  • the PCR fragments were recovered by the kit (purchased from Shanghai Huashun Biological Engineering Co., Ltd.) and digested with BamHI and EcoRI (various molecular biology enzymes used in the present invention were purchased from Dalian Bao Biological Engineering Co., Ltd.), Connected to the expression vector pET-24a (Novagen company, catalog number: 69864-3) digested with BamHI and EcoRI to obtain the recombinant plasmid pET-24a-W135.
  • BamHI and EcoRI variant molecular biology enzymes used in the present invention were purchased from Dalian Bao Biological Engineering Co., Ltd.
  • the cells per liter of bacterial solution were resuspended in 20ml lysis buffer (50mM Tirs-HCl, pH8.0, 1mM EDTA, 100mM NaCl), sonicated, and centrifuged at 12000g for 20 minutes at 4°C After identification by SDS-PAGE electrophoresis, most of the target protein was distributed in the supernatant of the lysis buffer.
  • 20ml lysis buffer 50mM Tirs-HCl, pH8.0, 1mM EDTA, 100mM NaCl
  • Ni-NTA affinity column Qiagen, Catalog No. 30210
  • add protein sample wash off the unbound protein with 10 times the medium volume of the equilibration buffer, and then use 5 times Volume elution buffer (20mM Na2HPO4, 300mM NaCl, 250mM imidazole, pH 8.0) to elute the target protein, dialyze off the imidazole, determine the protein concentration, and store at -20°C for later use.
  • HCV-AgII labeled antigen Using genetic engineering techniques, through a large number of molecular biology analysis software, the dominant epitope segments of HCV NS3, NS4, and core antigens are selected.
  • the sequence is SEQIDNo.2 (named W102), optimize the codon sequence to synthesize the gene, design primers (W102-F: CGCGGATCCATGTCTACCAACCCGAAACCG; W102-R: CCGGAATTCAGCGATCAGACGGTTCATCCAC) to amplify the DNA segment corresponding to W102, the upstream primers respectively have BamHI restriction sites, and the downstream primers have EcoRI restriction site.
  • the PCR fragments were recovered by the kit (purchased from Shanghai Huashun Biological Engineering Co., Ltd.) and digested with BamHI and EcoRI (various molecular biology enzymes used in the present invention were purchased from Dalian Bao Biological Engineering Co., Ltd.), Connected to the expression vector pGEX-6P-1 (Phamacia Company, Item No.: 27-4597-01) digested with BamHI and EcoRI to obtain the recombinant plasmid pGEX-6P-1-W102, which is the recombinant plasmid of the labeled antigen of the present invention , Hereinafter referred to as 6P-W102.
  • the cells were collected by centrifugation at 5000g for 20 minutes at 4°C.
  • the cells per liter of bacterial solution were resuspended in 20ml lysis buffer (50mM Tirs-HCl, pH8.0, 1mM EDTA, 100mM NaCl), sonicated, and centrifuged at 12000g for 20 minutes at 4°C After identification by SDS-PAGE electrophoresis, 80% of the target protein was distributed in the supernatant of the lysate.
  • 20ml lysis buffer 50mM Tirs-HCl, pH8.0, 1mM EDTA, 100mM NaCl
  • HCV-AgI-1 and HCV-AgII-1 where the sequence of HCV-Ag I-1 is the amino acid sequence from position 1201 to 1490 of NS3, and the sequence of HCV-AgII-1 is from position 1890 to 1923 of NS4 The sequence of the amino acid at position.
  • the specific band of about 500 bp is consistent with the expected size of 525 bp, and the target band is cut and recovered.
  • the fragments digested with BamHI and EcoRI were ligated to the PET32a vector digested with BamHI and EcoRI, and the BL21(DE3) strain was transformed and identified by PCR.
  • the recombinant plasmid PET32a-C175 was sequenced to confirm that the C175 gene was correctly inserted into the vector , Without any base or amino acid mutation, keep the correct reading frame.
  • the recombinant genetically engineered bacteria containing the PET32a-C175 plasmid was induced with IPTG at a final concentration of 0.25 mM for 4 hours.
  • the result showed that an induced expression band of about 33 KD appeared in the induced sample.
  • the fusion protein is mainly expressed in a soluble form, and its relative molecular mass is consistent with the theoretical molecular mass. After the remaining cells were disrupted by ultrasound, the supernatant was collected by centrifugation and purified by nickel affinity chromatography to obtain the fusion protein.
  • a part of the obtained fusion protein is stored for later use, and a part is digested with enterokinase to remove the N-terminal fusion protein, and the non-fusion target protein is obtained by reverse affinity, which is stored for later use.
  • the protein sample was analyzed by SDS-PAGE gel.
  • the fusion protein sample was named C175-A about 33KD, and the non-fusion protein sample was named C175-B about 20KD.
  • the purity of the purified target protein was above 90%.
  • C175-A and C175-B antigens have good reactivity to HCV antibody-positive serum.
  • the overall reactivity of C175-B is higher than that of C175-A, so C175-B was chosen to immunize mice.
  • BALB/c mouse peritoneal macrophages were used as feeder cells.
  • BALB/c rats were put to death by pulling their necks, immersed in 75% alcohol, in a clean bench, cut the abdominal skin with scissors under aseptic operation, exposed the peritoneum, and injected 5 mL of RPMI 1640 basic culture medium into the abdominal cavity with a syringe. Wash repeatedly, collect the washing solution, centrifuge at 1000 rpm for 5 minutes, leave the pellet, resuspend with RPMI 1640 screening medium (in RPMI 1640 complete medium containing HAT), adjust the cell concentration to 1 ⁇ 10 5 cells/mL, and add to 96 wells Plate, 150 ⁇ L/well, 37°C, 5% CO2 culture overnight.
  • the spleen was taken out under aseptic conditions, placed in a petri dish, washed once with RPMI1640 basic culture medium, and placed on a nylon mesh in a small beaker to grind and filter to prepare a cell suspension. Centrifuge, discard the supernatant, resuspend the RPMI 1640 basic culture medium, repeat this three times, and count.
  • Mouse myeloma cells Sp2/0 (preserved by Feipeng Biological Co., Ltd.) were screened by 8-azaguanine and cultured to the logarithmic growth phase. Two large flasks were taken to prepare cell suspension, centrifuged, and the supernatant was discarded. Use RPMI Resuspend the 1640 basic medium, repeat these three times, and count.
  • the myeloma cells and immune spleen cells were mixed in a ratio of 1:10, washed once with RPMI 1640 basic culture medium in a 50 mL plastic centrifuge tube, centrifuged at 1200 rpm for 8 minutes. Discard the supernatant, mix the cells, and slowly add 1 mL of 50% PEG1500 for fusion. After 1 minute of fusion, add 15 mL of RPMI 1640 basal medium to stop the cell fusion. Centrifuge at 1000 rpm for 5 minutes.
  • RPMI 1640 screening medium Discard the supernatant, gently suspend it with 50 mL of RPMI 1640 screening medium, and divide it equally into 10 96-well plates with feeder cells, and incubate at 50 ⁇ L/well at 37°C and 5% CO2. Culture to the sixth day, change the HT medium (RPMI 1640 complete medium containing HT) twice.
  • a total of 12 cell lines that stably secrete monoclonal antibodies against the HCV core antigen 175B recombinant protein were obtained by fusion three times, and their titers were all between 10 5 and 10 7 .
  • Monoclonal antibodies against HCV core antigen were classified and identified by ELISA method.
  • the six monoclonal antibodies of 3C-28, 11C-13, 14C-1, 1D-9, 8H-53, and 5G-28 were IgG1 type.
  • 14C-77, 4G-19, 5B-36, 8D-73, 3G-42, 2H-49 six monoclonal antibodies are IgG2 type.
  • a total of 21 cell lines that stably secrete monoclonal antibodies against the HCV core antigen 175B recombinant protein were obtained by fusion three times, and their titers were all between 10 5 and 10 7 .
  • Monoclonal antibodies against HCV core antigen were classified and identified by ELISA method.
  • the six monoclonal antibodies of 3C-28, 11C-13, 14C-1, 1D-9-10, 8H-53 and 5G-28 were IgG1 type, 14C-77, 3F-41, 5B-36, 8D-73, 7C-14-9, 2H-49, 12F-19 six monoclonal antibodies are IgG2a type, 4D-19, 3C-7, 2D -32, 5G-12, 6F-78, 6G-5-1, 15D-8 strains of monoclonal antibodies are IgG2b type.
  • HCV short titanium antigens A1 ⁇ A8 were used to coat the microwells.
  • PBS+20% NBS was used as the diluent.
  • the diluted monoclonal antibody was used as the primary antibody to a concentration of 1ug/ml.
  • the goat anti-mouse IgG-HRP was used as the secondary antibody.
  • the response of each monoclonal antibody to different antigens is used to determine the monoclonal antibody epitope.
  • the monoclonal antibody prepared from the C175-B antigen can recognize 5 epitopes, the non-recognizing monoclonal antibodies of C70-100 and C120-C175, among which C17-35, C55-72 and C95-117 epitopes are recognized There are the most monoclonal antibodies, with 5 strains for C17-35, 6 strains for C60-72 (C55-72 verified by further analysis), and 7 strains for C100-120aa (C95-117 verified by further analysis). From the above epitope recognition, the main epitopes of the antigen recognized by antibodies are distributed in the three segments C17-35, C55-72, and C95-117, and the reactivity to the antigen is the best.
  • the laboratory collected 78 samples with positive PCR and antibody positives, and used the hepatitis C virus core antigen detection kit purchased by Shandong Labbiotech Co., Ltd. to detect the positive status of the core antigen in 78 sera.
  • the content in the serum is very low, and part of the antigen may be neutralized by the antibody. Therefore, a more active antibody pairing is required to improve the detection sensitivity of the core antigen.
  • These core antigen-positive samples are subsequently used to screen for natural core antigens. Use paired monoclonal antibodies with high reactivity for positive samples.
  • the antibodies 3C-28, 14C-1, 11C-13, 15D-8, and 3G-42 were respectively coated with magnetic beads with 14C-77 , 1D-9-10, 2H-49, 8D-73 labeled AE, 20 groups of monoclonal antibody compatibility, selected 10 RNA-positive 5 copies with S/CO greater than 5, and 5 copies with S/CO greater than 1-4 Response to natural positive specimens: the detection rate of cross-paired monoclonal antibody combinations on core positive specimens.
  • the monoclonal antibody paired with C95-117aa coated with C55-72aa label combination has the highest detection rate.
  • 11C-13 with 14C-77-AE can detect all 10 sera, which is better than other groups. Pairing, followed by the combination of C15-35aa and C55-72aa, followed by the combination of C95-117aa and C95-117aa and C17-35aa and C95-117aa with the lowest detection rate.
  • HCV-Core-McAb23 and HCV-Core-McAb19 monoclonal antibodies that specifically bind to the 95-117 region sequence and the 55-72 region sequence
  • HCV-Core-McAb23 monoclonal antibody specifically binds to HCV core antigen 95-117 region sequence
  • HCV-Core-McAb19 monoclonal antibody specifically binds to HCV core antigen 55-72 region sequence
  • antibodies 2C-18, 3D-10, 5G-22, HCV-Core-McAb23 against epitopes in regions 95-117 and 6G-15, 7H-3, HCV-Core-McAb19 against epitopes in regions 55-72 were found All combinations were detected on all 10 natural specimens, with high reactivity to core antigen, high detection rate on natural HCV-core antigen positive specimens, and good negative serum background. The above results also indicate that the detection sensitivity and specificity of antibody combinations against 95-117aa and 55-72aa epitope amino acids are better than those against other epitope antibody pairs.
  • the antibody 11C-13 was selected as the coating/14C-77-AE pair for experiment. Name 11C-13 as HCV-AbI and 14C-77-AE as HCV-AbII for joint inspection pairing.
  • AE acridinate
  • Biotin solution 2.2mg sulfo-NHS-LC-biotin is dissolved in 0.4ml ultrapure water, and 143ul of biotin is added to the above dialyzed antigen.
  • reaction solution was dialyzed in PB (100mM PB, pH7.2) buffer containing 0.05% SDS to remove free biotin;
  • Biotin solution 2.2mg sulfo-NHS-LC-biotin is dissolved in 0.4ml ultrapure water, and 53ul of biotin is added to the above dialyzed antibody.
  • SDS concentration is 0.5% to 1%, preferably 0.8%.
  • NP-40 0.5%-1%, preferably 0.5%.
  • TRITONX-100 and TWEEN-20 are added 0.5% to 1%, preferably 0.5%.
  • BSA protective protein the concentration is 0.5% to 1%, preferably 1%.
  • the concentration of ammonium sulfate is 1% to 2.5%, preferably 1%.
  • Anhydrous ethanol the concentration can be 0.1%-10%, preferably 1%.
  • the above concentration ratio is the mass volume ratio, 1% means 1g/100ml.
  • Magnetic beads working solution preparation of HCV-AbI and HCV-AgI mixed solution labeled with magnetic beads:
  • Biotin working solution preparation of biotinylated HCV-AbII-BIO and HCV-AgII-BIO mixture
  • HCV-AgII-BIO Dilute the labeled HCV-AgII-BIO with biotin diluent (containing 20mMPB+150mMNacl+0.%Casein-2Na+0.1%P300+0.1% mercaptoethanol) to 0.2mg/ml
  • HCV-AgII-BIO uses HCV Biotin diluent (containing 20mMPB+150mMNacl+0.%Casein-2Na+0.1%P300+0.1% mercaptoethanol) is diluted to 0.2mg/ml, and then mixed 1:2 (biotin diluent contains 1:1000 mercapto Ethanol), wherein the reducing agent can be added DTT, mercaptoethanol, etc., preferably mercaptoethanol.
  • kit 2 was prepared by replacing HCV-AgI and HCV-AgII in the kit with HCV-AgI-1 and HCV-AgII-1.
  • Table 5 Comparison of data between antigen-antibody joint test and single test: result judgment: greater than 1 is positive, less than 1 is negative.
  • the epitope of the monoclonal antibody against hepatitis C virus antigen of the present invention has no overlap, which avoids the problem that the core antigen antibody is difficult to stagger the epitopes for joint inspection.
  • the anti-hepatitis C virus antigen monoclonal antibody and the hepatitis C virus recombinant antigen of the present invention do not cross-react with each other, do not affect the activity, and advantageously avoid the problems of cross-reaction and loss of detection activity.
  • the method and the kit of the present invention significantly shorten the window period, reduce the risk of missed detection, reduce the workload, reduce the manpower, instrument, and reagent costs for separate detection of the two methodologies, and improve the virus detection rate and sensitivity.

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Abstract

一种丙型肝炎病毒检测试剂盒、检测丙型肝炎病毒的方法以及制备检测丙型肝炎病毒的试剂或试剂盒的方法，该试剂盒包括检测丙型肝炎病毒核心抗原的第一抗体和第二抗体，其中第一抗体针对丙型肝炎病毒核心抗原第95-117位氨基酸序列中的表位或特异性结合丙型肝炎病毒核心抗原第95-117位氨基酸序列，第二抗体针对丙型肝炎病毒核心抗原第55-72位氨基酸序列中的表位或特异性结合丙型肝炎病毒核心抗原第55-72位氨基酸序列。该试剂盒灵敏度高，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

一种丙型肝炎病毒检测试剂盒
相关申请的交叉引用
本申请要求于2019年4月30日提交中国专利局的申请号为201910367283.6、名称为“一种丙型肝炎病毒检测试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及病毒检测领域。具体而言，本发明涉及丙型肝炎病毒的检测方法和试剂盒。
背景技术
丙型肝炎是严重威胁人类健康的传染病之一,目前还没有有效的疫苗防止其传播。直接抗病毒药物(DAAs)的出现大大提高了丙型肝炎治疗效果，但目前DAAs药物尚未在中国上市。我国现阶段HCV感染者接受抗病毒治疗的主要方案仍然是基于干扰素的治疗方案。在临床表现上与乙型肝炎相比,丙型肝炎症状较轻、发展慢、重症化少见,不易引起临床医师和患者的重视。HCV主要是通过输血及输入血液制品传播,丙肝病毒可引起急性或慢性感染。急性丙肝病毒感染通常没有症状，且仅在十分罕见情况下才会导致危及生命的疾病。约有15％-45％的感染者不经任何治疗即可在感染6个月之内自行清除病毒。其余60％–80％的感染者会发生慢性丙肝病毒感染。在这些慢性丙肝病毒感染者中，20年内出现肝硬化的危险为15％–30％。丙型肝炎治疗难度大、疗程长、疗效欠佳、费用高,所以选择理想的检测方法及早检出HCV非常重要。
HCV为有包膜呈球形的正单链RNA病毒，属于黄病毒家族，全长约9500碱基。HCV基因组两侧分别为5’和3’非编码区，中间为开放读码框(ORF)，分为结构区和非结构区。结构区包括核心蛋白区(C)和两个包膜蛋白区(E1、E2)，分别编码核心蛋白和包膜蛋白。非结构蛋白区包括NS2、NS3、NS4和NS5区，编码功能蛋白，如蛋白酶(NS2、NS3和NS4A区)，螺旋酶(NS3)以及依赖RNA的RNA多聚酶(NS5B区)。丙肝病毒核心蛋 白约含190aa，在病毒复制中起到非常重要的作用。通常采用基因工程表达上述结构和非结构蛋白作为包被抗原，来建立抗-HCV检测的ELISA方法。HCV基因组具有显著的异质性，同一基因组不同区段变异程度有显著差别。5’非编码区最为保守，已成为HCV分子诊断研究的焦点。
目前HCV的检测方法主要有三种：HCV抗体检测、HCV核心抗原检测，HCV核酸检测丙型肝炎病毒RNA。HCV抗体检测是目前医院及血站判断及筛选患者是否感染HCV病毒最常用的方法，但其致命缺点是存在“窗口期”，即HCV感染后至HCV抗体产生中间有40-70天的时间，献血员已经被感染并具有传染性，但是用目前的抗体检测试剂没法检出，该阶段称为感染后血清阳转前的窗口期(Preseroconversion Window Phase,PWP)。窗口期的存在是输血感染的主要原因。目前丙型肝炎的输血后感染占肝炎病例的70％，而在丙型肝炎病毒感染者中更有80-90％是属于输血后感染。在HCV核酸出现后的1-2天内，感染者体内就会出现HCV核心抗原，而且与HCV核酸的水平具有一定的相关性，可以作为HCV检测的标志物。HCV核酸检测(NAT)是三种检测中方法最可信的一种，在感染的初期就能检测出来，同时还能反应病毒的含量，主要用于抗病毒治疗的选择与疗效监测，但它需严格按照PCR操作规程操作，检测人员需经过专业培训并取得相应资质，且样本的质量控制要求高，必须是采血后2小时内低温条件送检，无菌抽提RNA，容易因为操作、设备及环境等因素造成误差，从而产生假阳性或假阴性，不利于在普通医院推广，市场份额比较小。
HCV抗原抗体联检可以同时检测样品中的HCV抗原和抗体。然而，在HCV抗原、抗体联检方法中，必须对抗原和抗体进行大量筛选和实验，以避免抗原抗体表位重叠和抗丙型肝炎病毒抗原单克隆抗体和丙型肝炎病毒重组抗原相互之间的交叉反应的问题。在HCV抗原、抗体联检方法中，所选抗原区域还必须具备高免疫原性，有利于制备抗体和捕获样品中的抗原。现有技术中通常需要对核心抗原中单克隆抗体已知表位结合区进行突变或缺失，使得用于HCV核心抗原检测的单克隆抗体不会结合这些突变或缺失的核心抗原，但是仍然结合来自样品的完整核心抗原。例如CN105228649A公开了用于联检的包含氨基酸34和48和氨基酸115-121缺失的突变体核心蛋白抗原；还公开了缺失5个氨基酸(C11-9结合区32、 33和34和核心C11-14结合区氨基酸残基47和48)来避免用于捕获核心抗体的核心抗原与检测核心抗原的检测抗体反应的问题。但是，这样的构建体的核心抗体检测效果可能较差，因为缺失的残基在患者中是高免疫原性的(参见CN105228649A)。因此，有利的是所选择的抗原区域不被检测抗体检测到，但是同时保留或增强对于抗核心抗体的样品的检测。现有技术CN1489692A公开了用于HCV检测的HCV抗原抗体组合，其中教导了HCV核心抗原如氨基酸10-53和120-130等和NS3抗体进行组合检测。这样的联检方法通常需要对各种待捕获患者HCV抗原所用抗体的表位和捕获患者HCV抗体采用抗原区间进行大量交叉实验和筛选，需要较高的人力、仪器、试剂成本。
从目前市场上的抗原抗体联检项目检测比较局限于ELISA和板式发光、以及时间分辨上，这些方法学都存在反应时间长，而且耗费减少人力物力，增加成本。
发明内容
发明人经过大量理论研究和实验摸索，充分考虑HCV感染整个过程，对各种待捕获患者HCV抗原所用抗体和捕获患者HCV抗体采用抗原区间进行分析研究，并通过大量交叉实验和筛选，获得了能够用于丙型肝炎病毒检测的丙型肝炎病毒核心抗原区域组合。发明人已经证明所选抗原表位区域具有优异的免疫原性，由其制备的抗体出乎意料的能够相互组合用于HCV核心抗原的高活性检测。发明人还令人吃惊地发现所述抗原制备的单克隆抗体能够与HCV抗原组合，相互补充HCV抗原、抗体各自检测的不足，降低漏检风险和缩短窗口期。
因此，在一些实施方案中，本发明提供了一种丙型肝炎病毒检测试剂盒以及制备方法。本发明的试剂盒具备改善的灵敏度和稳定性以及缩短的反应时间，操作简单，适合推广应用。本发明的试剂盒尤其缩短了窗口期和反应时间，可用于早期急性丙型肝炎的快速诊断。在一些实施方案中，本发明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和第二抗体，其中第一抗体针对丙型肝炎病毒核心抗原第95-117位氨基酸序列中的表位，第二抗体针对丙型肝炎病毒核心抗原第55-72位氨基酸序列中的表位。在一些实施方案中，第一抗体特异性结合 丙型肝炎病毒核心抗原第95-117位氨基酸序列。在一些实施方案中，第二抗体特异性结合丙型肝炎病毒核心抗原第55-72位氨基酸序列。在一些实施方案中，第一抗体和/或第二抗体可以是单克隆抗体。在一些实施方案中，本领域已知的方法制备本发明的抗体，例如第一抗体和/或第二抗体。在一些实施方案中，可以以包含第55-72位氨基酸序列的抗原和/或包含第95-117位氨基酸序列的抗原免疫动物制备本发明的抗体，例如第一抗体和/或第二抗体。在一些实施方案中，可以以第55-72位氨基酸序列和/或第95-117位氨基酸序列为抗原免疫动物制备本发明的抗体，例如第一抗体和/或第二抗体。在一些实施方案中，可以通过当用于本文中时，"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。可以使用标准测定法如表面等离子共振技术(例如
Figure PCTCN2020086437-appb-000001
)确定结合亲和力。在一些实施方案中，第一抗体与特异性结合丙型肝炎病毒核心抗原第95-117位氨基酸序列的抗体结合相同表位。在一些实施方案中，第二抗体与特异性结合丙型肝炎病毒核心抗原第55-72位氨基酸序列的抗体结合相同表位。与参比抗体“结合相同表位的抗体”是指例如在竞争测定中阻断50％以上的所述参比抗体与其抗原的结合；或者参比抗体在竞争测定中阻断50％以上的该抗体与其抗原的结合。
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、动物模型等检测本发明抗体的效果如结合活性和/或交叉反应性。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中本发明的抗体(或其抗原结合片段)与抗原结合的EC50值可以为例如1μM-1pM,例如1nM-1pM，例如100pM-1pM。
在一些实施方案中，试剂盒中的第一抗体和第二抗体不存在交叉反应。在一些实施方案中，第一抗体(针对表位95-117位氨基酸)和第二抗体(针对表位55-72位氨基酸)可以用作捕获抗体(或称为包被抗体)和标记抗体，例如第一抗体为捕获抗体，第二抗体为标记抗体，或者第一抗体为标记抗体，第二抗体为捕获抗体。优选地，在一些实施方案中，第一抗体为捕获抗体(或称为包被抗体)，第二抗体为标记抗体。在一些实施方案中，还可以使用备选抗体作为包被抗体或标记抗体。例如，在一些实施方案中，可以使用针对表位17-35位氨基酸的抗体作为捕获抗体(或称 为包被抗体)。
在一些实施方案中，所述捕获抗体结合至固相。在一些实施方案中，所述捕获抗体可以用于包被固相支持物。在一些实施方案中，固相支持物没有特别限制，其可以是例如磁性颗粒如磁珠，胶乳粒子和微量滴定板。在一些实施方案中，所述标记抗体用可检测标记物标记，例如用荧光标记物如吖啶酯标记，例如通过接头如生物素-亲和素与荧光标记物如吖啶酯标记。
在一些实施方案中，本文中的术语“抗体”在最广义上使用，其可以包括全长单克隆抗体，双特异性或多特异性抗体，嵌合抗体，以及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。“抗体片段”包括全长抗体的部分，优选地其抗原结合区或可变区。抗体片段的实例包括Fab，Fab'，F(ab') 2，Fd，Fv，dAb，互补决定区(CDR)片段，单链抗体(例如，scFv)，双价抗体或结构域抗体。
在一些实施方案中，试剂盒还包括用于检测受试者样品中丙型肝炎病毒抗体的第一抗原和第二抗原。在一些实施方案中，所述第一抗原和第二抗原可以是例如丙型肝炎病毒核心抗原，E1、E2、NS2、NS3、NS4[Mimms et al.,Lancet 336:1590(1990)；Bresters et al.,Vox Sang 62:213(1992)]和NS5。在一些实施方案中，第一抗原和第二抗原来自同一抗原的不同位置。在一些实施方案中，第一抗原和第二抗原可以选自SEQ ID NO:1和SEQ ID NO:2所示的抗原或其免疫原性片段。例如在一些实施方案中，第一抗原和第二抗原可以是HCV核心抗原第1位至56位氨基酸，NS3第1201位至1490位氨基酸，NS4的1883位至1925位氨基酸的序列；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位氨基酸的序列。
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，第一抗原为捕获抗原，第二抗原为标记抗原。
在一些实施方案中，所述捕获抗原结合至固相。在一些实施方案中，所述捕获抗原可以用于包被固相支持物。在一些实施方案中，固相支持物没有特别限制，其可以是例如磁性颗粒如磁珠，胶乳粒子和微量滴定板。 在一些实施方案中，所述标记抗原用可检测标记物标记，例如用荧光标记物如吖啶酯标记，例如通过接头如生物素-亲和素与荧光标记物如吖啶酯标记。在一些实施方案中，用于标记抗原或抗体的可检测标记没有特别限制。在一些实施方案中，所述标记可以包括但不限于荧光标记，发色团标记，电子致密标记，化学发光标记，和放射性标记，以及间接标记如酶或配体，例如，通过酶促反应或分子相互作用来间接检测。在一些实施方案中，示例性标记包括但不限于放射性同位素，荧光团，罗丹明及其衍生物，荧光素酶，荧光素，辣根过氧化物酶(HRP)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记，噬菌体标记等等。
在一些实施方案中，本发明的试剂盒包括适合进行免疫测定的试剂。在一些实施方案中，本发明的试剂盒可以用于进行免疫测定，例如ELISA，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HCV抗体包被固相如磁珠，捕获样品中的HCV抗原，然后用标记的抗体与结合在反应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，本发明的HCV抗体可以用于包被固相如磁珠或用作标记的第二抗体。在一些实施方案中，抗体或其抗原结合片段固定到一个表面上，例如固相支持物上，例如塑料、膜如硝酸纤维素膜、玻璃、磁珠或金属支持物。在一些实施方案中，来自受试者的样品与所述固相支持物接触，然后接触带有可检测标记的抗体指示剂显色。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，因此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)/吐温稀释抗血清，有助于减少非特异性背景。
在本文中，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，试剂盒还包括病毒裂解液。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，不影响抗体的灵敏度，有利于抗原抗体的结合，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，本发明提供用于检测丙型肝炎病毒核心抗原的第一抗体和第二抗体在制备检测丙型肝炎病毒的试剂盒中的用途。在一些实施方案中，本发明提供检测丙型肝炎病毒的方法，所述方法包括使来自受试者样品与第一抗体和第二抗体接触。在一些实施方案中，第一抗体针对丙型肝炎病毒核心抗原第95-117位氨基酸序列中的表位，第二抗体针对丙型肝炎病毒核心抗原第55-72位氨基酸序列中的表位。在一些实施方案中，本发明提供包含丙型肝炎病毒核心抗原第95-117位氨基酸的免疫原性多肽和包含丙型肝炎病毒核心抗原第55-72位氨基酸的免疫原性多肽组合用于制备检测丙型肝炎病毒核心抗原的抗体的用途。在一些实施方案中，本发明提供制备检测丙型肝炎病毒核心抗原的抗体的方法，所述方法包括将包含丙型肝炎病毒核心抗原第95-117位氨基酸的免疫原性多肽和包含丙型肝炎病毒核心抗原第55-72位氨基酸的免疫原性多肽分别免疫动物，从而制备检测丙型肝炎病毒核心抗原的抗体如单克隆抗体。在一些实施方案中，所述免疫原性多肽包括丙型肝炎病毒核心抗原第95-117位氨基酸和/或佐剂以及丙型肝炎病毒核心抗原第55-72位氨基酸和/或佐剂。在一些实施方案中，本发明鉴定的核心抗原表位区域如丙型肝炎病毒核心抗原第95-117位氨基酸和丙型肝炎病毒核心抗原第55-72位氨基酸(其可以通过例如化学法人工合成)可以与适当的载体蛋白连接，用于免疫动物制备抗体如单克隆抗体。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，本发明的试剂盒可以包含上述第一抗体和第二抗体，并进一步包含丙型肝炎病毒第一抗原和/或第二抗原。在一些实施方案中，本发明的检测丙型肝炎病毒的方法还可以包括使来自受试者样品与来自丙型肝炎病毒的第一抗原和/或第二抗原接触。在一些实施方案中，所述第一抗原和/或第二抗原可以是例如丙型肝炎病毒核心抗原，E1、E2、NS2、NS3、NS4和NS5，例如第一抗原和/或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，例如可以是包括以下任一氨基酸片段或其组合：HCV核心抗原第1位至56位氨基酸，NS3第1201位至1490位氨基酸，NS4的1883位至1925位氨基酸的序列；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位氨基酸的序列，例如来自SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列。
在一些实施方案中，本发明提供制备检测丙型肝炎病毒的试剂或试剂盒的方法，其中使用丙型肝炎病毒第一核心抗原和丙型肝炎病毒第二核心抗原制备抗体。在一些实施方案中，本发明提供丙型肝炎病毒第一核心抗原和丙型肝炎病毒第二核心抗原在制备检测丙型肝炎病毒的试剂或试剂盒中的用途。在一些实施方案中，丙型肝炎病毒第一核心抗原可以包含丙型肝炎病毒核心抗原第55-72位氨基酸或由其组成，丙型肝炎病毒第二核心抗原可以包含丙型肝炎病毒核心抗原第95-117位氨基酸或由其组成。在一些实施方案中，本发明的试剂盒可以包含上述丙型肝炎病毒第一核心抗原和丙型肝炎病毒第二核心抗原，还可以包含针对丙型肝炎病毒其它位置(即不同于上述第一核心抗原和第二核心抗原的位置)的一种或两种抗原的抗体，如单克隆抗体。在一些实施方案中，所述丙型肝炎病毒其它位置的一种或两种抗原可以是例如丙型肝炎病毒核心抗原，E1、E2、NS2、NS3、NS4和NS5，例如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，例如可以是包括以下任一氨基酸片段或其组合：HCV核心抗原第1位至56位氨基酸，NS3第1201位至1490位氨基酸，NS4的1883位至1925位氨基酸的序列；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位氨基酸的序列，例如来自SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列。在一些实施方案中，所述丙型肝炎病毒第一核心抗原和丙型肝炎病毒第二核心抗原可以用于制备抗体，如单克隆抗体。在一些实施方案中，本发明的试剂盒可以包含通过上述丙型肝炎病毒第一核心抗原和丙型肝炎病毒第二核心抗原制备的抗体，如单克隆抗体，任选地还可以包含丙型肝炎病毒其它位置的一种或两种抗原。在一些实施方案 中，所述丙型肝炎病毒其它位置的一种或两种抗原可以是例如丙型肝炎病毒核心抗原，E1、E2、NS2、NS3、NS4和NS5，例如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，例如可以是包括以下任一氨基酸片段或其组合：HCV核心抗原第1位至56位氨基酸，NS3第1201位至1490位氨基酸，NS4的1883位至1925位氨基酸的序列；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位氨基酸的序列，例如来自SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列。
在一些实施方案中，本发明提供同时检测至少一种HCV抗原和至少一种HCV抗体的方法、用途以及相关试剂盒。在一些实施方案中，所述方法可以包括将样品与包被于固相上的至少一种HCV抗原或其片段接触形成免疫复合物，和同时与包被于固相上的至少一种HCV抗体或其片段接触形成免疫复合物；检测复合物的存在，以确定所述样品中HCV抗原和/或抗体的存在。在一些实施方案中，所述方法可以包括将样品与包被于固相上的至少一种HCV抗原或其片段接触形成免疫复合物，和同时与包被于固相上的至少一种HCV抗体或其片段接触形成免疫复合物；向产生的复合物中添加与可检测标记连接的第二HCV抗原，和与可检测标记连接的第二HCV抗体；和检测产生的信号，以确定所述样品中HCV抗原和/或抗体的存在。在一些实施方案中，本发明提供用于所述方法的试剂盒，其包含1)含有包被于固相上的至少一种HCV抗原的容器，2)包含包被于固相上的至少一种HCV抗体的容器；或者含有包被于固相上的至少一种HCV抗原和包被于固相上的至少一种HCV抗体的容器。在一些实施方案中，所述试剂盒进一步包含与可检测标记连接的第二抗原和/或第二HCV抗体。在一些实施方案中，所述包被于固相上的至少一种HCV抗体与包被于固相上的至少一种HCV抗原不发生交叉反应。在一些实施方案中，所述至少一种HCV抗体为HCV核心抗原的单克隆抗体。在一些实施方案中，所述至少一种HCV抗原为HCV核心抗原，例如重组抗原。在一些实施方案中，所述HCV核心抗原不包含所述抗体结合的表位，例如不包含核心抗原第95-117位氨基酸序列中的表位和核心抗原第55-72位氨基酸序列中的表位。
在一些实施方案中，本发明的方法和/或用途中不需要进行抗原抗体的解离。在一些实施方案中，本发明的方法和/或用途中，可以第一步先让抗体反应，有利于抗原抗体的优先结合，再第二步再加入裂解液，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，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，第一抗原为捕获抗原，第二抗原为标记抗原。
在一些实施方案中，本发明提供了一种磁珠抗原抗体联检试剂盒。在一些实施方案中，本发明的试剂盒可以包括适合机械能化学发光检测的试剂。在一些实施方案中，本发明的试剂盒可以利用化学发光全自动仪器高通量、快速准确地检测HCV中的抗原抗体，缩短检测时间，快速的出检验结果。
在一些实施方案中，本发明提供了一种磁珠抗原抗体联检试剂盒，其包括标记在磁珠上的抗原和抗体。在一些实施方案中，本发明的试剂盒可以利用磁珠为固相，将抗原和抗体直接标记在磁珠上，采用双抗原夹心法和双抗体夹心法原理检测HCV中的抗原和抗体，提高检出率，缩短窗口期。
在一些实施方案中，本发明的试剂盒检测HCV核心抗原，显著的缩短窗口期，平均提前50天左右，缩短窗口期HCV感染的风险。
在一些实施方案中，本发明结合HCV核心抗原及HCV抗体联合检测,能够克服单独HCV抗原检测或HCV抗体检测方法的不足，即显著的缩短窗口期，降低漏检的风险，减少工作量，降低两种方法学单独检测的人力、仪器、试剂成本。
在一些实施方案中，本发明提供一种丙型肝炎病毒磁珠抗原抗体联合检测试剂盒以及制备方法，解决了现有技术存在的问题，所述问题包括例如灵敏度低、稳定性差、反应时间长和/或操作繁琐等技术问题。在一些实施方案中，本发明缩短了窗口期，降低了反应时间，可用于早期急性丙型肝炎的快速诊断。
在一些实施方案中，本发明可以采用磁珠为检测抗原抗体的载体。在 一些实施方案中，本发明可以通过化学发光全自动仪器快速准确地检测HCV中的抗原抗体。在一些实施方案中，可以利用双抗夹心原理制备试剂盒。例如，在一些实施方案中，样品中的抗体首先被标记在磁珠上的丙型肝炎病毒重组抗原AgI(HCV-AgI)和生物素化的丙型肝炎病毒重组抗原AgII(HCV-AgII-BIO)所捕获形成双抗原夹心状态。在一些实施方案中，在加入裂解液对样本中丙型肝炎病毒进行裂解获得核心抗原后核心抗原被标记在磁珠上的丙型肝炎病毒抗原单克隆抗体AbI(HCV-AbI)和生物素化的丙型肝炎病毒抗原单克隆抗体AbII(HCV-AbII-BIO)所捕获形成双抗体夹心状态，然后洗掉样品的其他成份。在一些实施方案中，可以再加入亲和素化标记物SA-AE，就可以形成单克隆抗体AbI-丙型肝炎病毒抗原-生物素化单克隆抗体AbII-亲和素化标记物SA-AE与重组抗原AgI-丙型肝炎病毒抗体-生物素化重组抗原II-亲和素化标记物SA-AE。在一些实施方案中，洗板加入激发液，用化学发光全自动仪器测量发光值，发光值与样品中的抗原抗体总浓度呈正相关，与临界值相比较，从而判断阴阳性。
在一些实施方案中，本发明的检测抗原抗体是通过分析丙型肝炎病毒序列而得到的2株抗丙型肝炎病毒抗原单克隆抗体(AbI、AbII)，2种丙型肝炎病毒重组抗原(AgI、AgII)，且2株抗丙型肝炎病毒抗原单克隆抗体与2种丙型肝炎病毒重组抗原两两之间不发生交叉反应。在一些实施方案中，可以使用其中的抗丙型肝炎病毒抗原单克隆抗体AbI、丙型肝炎病毒重组抗原AgI作为磁珠包被原料，其中的抗丙型肝炎病毒抗原单克隆抗体AbII、丙型肝炎病毒重组抗原AgII作为生物素化原料，用双抗原夹心法检测丙型肝炎病毒抗原，用双抗体夹心法检测丙型肝炎病毒抗体。
本发明公开了HCV-AgI和HCV-AgII抗原的序列。
HCV-AgI抗原序列：SEQ ID NO:1(W135)
Figure PCTCN2020086437-appb-000002
Figure PCTCN2020086437-appb-000003
HCV-AgII抗原序列：SEQ ID NO:2(W102)
Figure PCTCN2020086437-appb-000004
C175序列(SEQ ID NO:3)
Figure PCTCN2020086437-appb-000005
本发明公开了制备HCV-核心抗原McAb的方法。
在一些实施方案中，本发明还可以将HCV-AbII抗体直接标记吖啶酯模式进行。
在一些实施方案中，本发明可以采用磁珠作为固相载体。
在一些实施方案中，本发明提供了生物素化丙型肝炎病毒抗原抗体的体外标记方法。
在一些实施方案中，本发明提供了采用磁珠作为载体的生物素化丙型肝炎病毒抗原抗体的体外标记方法。
在一些实施方案中，本发明提供检测HCV抗原抗体联检的裂解液，不需要进行抗原抗体的解离，通过调整温和的裂解液，不影响抗体的灵敏度，有利于抗原抗体的结合，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，本发明可以采用亲和素化或生物素化SA-AE进行标记。
在一些实施方案中，本发明的抗体针对核心抗原的表位95-117aa或特异性结合所述序列。在一些实施方案中，标记抗体优选针对的表位在 55-72aa或特异性结合所述序列。在一些实施方案中，包被抗体优选针对的表位在95-117aa或特异性结合所述序列。在一些实施方案中，包被抗原与抗体不存在交叉反应，表位无重叠，避免了以往核心抗原抗体难以错开表位，无法制备抗原抗体联检试剂盒困局。另外突变核心抗原表位来避开与抗体交叉反应的方法也存在突变后的活性损失问题。本发明的抗原和抗体表位上无交叉重叠，对活性不造成影响。在一些实施方案中，本发明采用标记抗体和包被抗体针对配对的表位，用于进行HCV抗原抗体联检，有利地避免了交叉反应和检测活性损失的问题。
在一些实施方案中，本发明的HCV抗原I与HCV抗原II可以是任何适当的HCV抗原，例如核心抗原、E1、E2、NS2、NS3、NS4和NS5。在一些实施方案中，本发明中检测的HCV基因型没有特别限制，例如可以是I/1a，II/1b，III/2a，IV/2b，V/3a进而VI/3b型。在一些实施方案中，本发明检测的HCV基因型为HCV 1b。在一些实施方案中，针对不同地区的基因型分布情况可以采用不同基因型的基因片段或几种基因型基因片段的组合，但选用的氨基酸区段不变。在一些实施方案中，本发明对核心区段进行分析和筛选，确定了其中的核心表位区段为核心抗原的7-21aa、29-48aa。在一些实施方案中，本次发明优选核心抗原区段选取7-48a。
本发明有利地具有以下一种或多种优点：
1、在一些实施方案中，本发明的HCV核心抗原和HCV-核心抗体可以同时用于联检，避免了核心抗原和核心抗体相互交叉反应。该方法还能够克服单独HCV抗原检测或HCV抗体检测方法的不足，即显著的缩短窗口期，降低漏检的风险，减少工作量，降低两种方法学单独检测的人力、仪器、试剂成本。
2、不需要进行抗原抗体的解离，通过调整温和的裂解液，不影响抗体的灵敏度，可以第一步先让抗体反应，有利于抗原抗体的优先结合，再第二步再加入裂解液，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。
3、在一些实施方案中，本发明提供的联检方法可以同时检出HCV抗原和抗体，解决市场上单独检测核心抗原和HCV抗体需要两种试剂盒。
附图说明
图1：本发明非限制性实施方案中抗原抗体联检原理图。
图2：A:PCR扩增产物图谱，1.DNA marker(DL2000)；2.PCR扩增产物；B:PKO-C175C重组质粒PCR鉴定图谱，1.PCR扩增产物；2.PKO-C175菌落PCR扩增产物。
图3：重组融合蛋白诱导表达分析及纯度分析。A：1.protein marker(KD)；2.BL21(DE3)不含PKO-C175表达质粒；3.BL21(DE3)含PKO-C175表达质粒非IPTG诱导；4.BL21(DE3)含PKO-C175表达质粒IPTG诱导4h。B：1.protein marker(KD)；2.BL21(DE3)含PKO-C175表达质粒IPTG诱导4h；3.亲和纯化含融合蛋白的C175-A样品；4.肠激酶酶切去除融合蛋白后的样品C175-B。
具体实施方式
下面主要结合附图及具体实施例对丙型肝炎病毒磁珠抗原抗体联合检测试剂盒以及制备方法作进一步详细的说明。
试剂和材料：
实施列1 HCV抗原制备
(1)HCV-AgI包被抗原的制备：采用基因工程技术手段,通过大量的分子生物学分析软件分析筛选出HCV NS3,NS4,核心抗原的优势表位区段，序列为SEQIDNo.1(命名为W135),优化密码子序列合成基因，设计引物(W135-F:CGCGGATCCATGTCTACCAACCCGAAACCG；W135-R:CCGGAATTCACGAGAAGCGAAAGCGATCA)扩增W135所对应的DNA区段,上游引物分别带有BamHI酶切位点，下游引物带有EcoRI酶切位点。PCR的片段经试剂盒(购自上海华舜生物工程有限公司)回收后，用BamHI和EcoRI酶切(本发明所采用的各种分子生物学用酶均购自大连宝生物工程有限公司)，连接到用BamHI和EcoRI酶切之后的表达载体pET-24a(Novagen公司，货号：69864-3)中，得到重组质粒pET-24a-W135。
上述阳性克隆用含100ug/ml硫酸卡那霉素(上海生工生物工程技术服务有限公司，以下简称生工，货号KB0286)的500ml LB培养基37℃振荡培养至OD600＝1.0左右，用终浓度为0.5mM的IPTG(生工，货号IB0168)37℃诱导4小时。4℃5000g离心20分钟收集菌体，每升菌液的菌体用20ml裂解缓冲液(50mM Tirs-HCl，pH8.0，1mM EDTA，100mM NaCl) 重悬，超声破碎，4℃12000g离心20分钟，经SDS-PAGE电泳鉴定后，大部分目的蛋白分布在裂解液上清中。收集上清，逐滴缓慢加入饱和硫酸铵溶液(广东光华化学试剂公司，货号：7783-20-2，调pH7.4)至硫酸铵终浓度为25％，4℃静置30min，4℃12000g离心20分钟，收集上清，继续逐滴缓慢加入饱和硫酸铵溶液至硫酸铵终浓度为40％，4℃静置30min，4℃12000g离心20分钟，收集沉淀，用5ml平衡缓冲液(10mM Na2HPO4，1.8mM KH2PO4，140mM NaCl，2.7mM KCl，25mM咪唑(美国Sigma-Aldrich公司，货号I5513)，pH8.0)溶解。用10倍柱床体积的平衡缓冲液平衡Ni-NTA亲和柱(Qiagen公司，货号30210)之后，加入蛋白样，用10倍介质体积的平衡缓冲液洗去未结合的蛋白，再用5倍体积洗脱缓冲液(20mM Na2HPO4，300mM NaCl，250mM咪唑，pH8.0)，洗脱目的蛋白，透析掉咪唑，测定蛋白浓度，-20℃保存备用。
(2)HCV-AgII标记抗原的制备：采用基因工程技术手段,通过大量的分子生物学分析软件分析筛选出HCV NS3,NS4,核心抗原的优势表位区段，序列为SEQIDNo.2(命名为W102),优化密码子序列合成基因，设计引物(W102-F:CGCGGATCCATGTCTACCAACCCGAAACCG；W102-R:CCGGAATTCAGCGATCAGACGGTTCATCCAC)扩增W102所对应的DNA区段,上游引物分别带有BamHI酶切位点，下游引物带有EcoRI酶切位点。PCR的片段经试剂盒(购自上海华舜生物工程有限公司)回收后，用BamHI和EcoRI酶切(本发明所采用的各种分子生物学用酶均购自大连宝生物工程有限公司)，连接到用BamHI和EcoRI酶切之后的表达载体pGEX-6P-1(Phamacia公司，货号：27-4597-01)中得到重组质粒pGEX-6P-1-W102，即本发明的标记抗原的重组质粒，以下简称6P-W102。
上述阳性克隆，接种于含100ug/ml氨苄西林钠(生工，货号A0339)的500ml LB培养基37℃振荡培养至OD600＝1.0左右，用终浓度为0.5mM的IPTG 37℃诱导4小时。4℃5000g离心20分钟收集菌体，每升菌液的菌体用20ml裂解缓冲液(50mM Tirs-HCl，pH8.0，1mM EDTA，100mM NaCl)重悬，超声破碎，4℃12000g离心20分钟，经SDS-PAGE电泳鉴定后，80％目的蛋白分布在裂解液上清中。收集上清，逐滴缓慢加入饱和硫酸铵溶液至硫酸铵终浓度为15％，4℃静置30min，4℃12000g离心20 分钟，收集上清，继续逐滴缓慢加入饱和硫酸铵溶液至硫酸铵终浓度为45％，4℃静置30min，4℃12000g离心20分钟，收集沉淀，用10ml平衡缓冲液(pH 7.3PBS，140mMNaCl，2.7mMKCl，10mM Na2HPO4，1.8mM NaH2PO4)溶解。用10倍柱床体积的平衡缓冲液平衡GSTrap亲和柱(Amersham公司，货号：17-5130-02)之后，加入蛋白样，用10倍介质体积的平衡缓冲液洗去未结合的蛋白，再用5倍体积洗脱缓冲液(50mMTris-HCl，10mM还原型谷胱甘肽(Amresco公司，货号：0399)，pH8.0)，洗脱目的蛋白，测定蛋白浓度，-20℃保存备用。
类似地，制备HCV-AgI-1，HCV-AgII-1，其中HCV-Ag I-1的序列为NS3第1201位至1490位氨基酸序列，HCV-AgII-1的序列为NS4的1890位至1923位氨基酸的序列。
实施列2HCV-单克隆细胞制备过程以及抗体筛选
2.1 HCV核心抗原蛋白C175基因片段的获得及PET32a-C175克隆构建采用基因工程技术手段,通过大量的分子生物学分析软件分析筛选出核心抗原的优势表位区段，序列为SEQIDNo.3(命名为C175),优化密码子序列合成基因，设计引物(C175-F:CGCGGATCCATGTCTACCAACCCGAAACCG；C175-R:CCGGAATTCAGAGAAAGAGCAACCCGGCA)扩增C175所对应的DNA区段,取PCR产物于1.5％的琼脂糖凝胶进行鉴定，可见约500bp左右的特异性条带，与预期525bp大小相符，切下目的条带回收。将经BamHI、EcoRI双酶切的片段连接于经BamHI、EcoRI双酶切的PET32a载体上，转化BL21(DE3)菌株并进行PCR鉴定，重组质粒PET32a-C175经测序，证实C175基因正确插入载体中，无任何碱基或者氨基酸突变，保持正确的读码框。
2.2融合蛋白C175的原核表达和纯化
按照上述方法，将含PET32a-C175质粒重组基因工程菌以终浓度0.25mM的IPTG进行诱导4h，结果表明诱导样品中出现约33KD的诱导表达带。融合蛋白主要以可溶性形式进行表达，且其相对分子质量与理论分子质量相符合。剩余菌体经超声破碎后，离心收集上清经镍亲和层析纯化获得融合蛋白。获得的融合蛋白一部分保存备用，一部分经肠激酶酶切 去除N端融合蛋白，反向亲和得到非融合目的蛋白，保存备用。蛋白样品经SDS-PAGE胶分析，融合蛋白样品命名为C175-A约33KD，非融合蛋白样品命名为C175-B约20KD，纯化后的目的蛋白纯度均在90％以上。
2.3 HCV核心抗原C175-A、C175-B的抗原性
用纯化的目的蛋白C175-A、C175-B分别包被酶标板，间接ELISA法检测HCV阳性质控血清，结果表明8份质控血清对两个蛋白均有较好反应性，8份质控血清均呈阳性反应，C175-A均值为1.090，C175-B均值为1.219；平行比较的8份非HCV阳性的临床血清均呈阴性反应，C175-A均值为0.025，C175-B均值为0.014；C175-A和C175-B两个抗原相比，反应性上C175-B要略好。此结果我们可以这样认为：通过PET32a融合蛋白的协助表达、低温诱导等使得HCV核心抗原蛋白具有更好的抗原性。
如下表：重组HCV核心抗原C175-A、C175-B抗原的反应性。
Figure PCTCN2020086437-appb-000006
通过对比，C175-A和C175-B抗原对HCV抗体阳性血清具有较好的反应性，C175-B整体的反应性要高于C175-A，故选C175-B来进行免疫小鼠。
3重组抗原免疫小鼠
取1ml C175-B抗原与等量与弗氏完全佐剂混合，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。
3.1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1×10 5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，RPMI1640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。
3.2抗HCV核心抗原蛋白C175的抗体的筛选
以核心抗原C175-A及C175-B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 pH7.2PBS，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。
3.3抗HCV核心抗原蛋白单克隆抗体细胞株的建立
融合三次共获得12株稳定分泌抗HCV核心抗原175B重组蛋白单克隆抗体的细胞株，其效价均在10 5～10 7之间。抗HCV核心抗原单克隆抗 体经ELISA方法进行单克隆抗体分型鉴定，其中3C-28、11C-13、14C-1、1D-9、8H-53、5G-28六株单克隆抗体为IgG1型，14C-77、4G-19、5B-36、8D-73、3G-42、2H-49六株单克隆抗体为IgG2型。
3.4抗HCV核心抗原McAB表位鉴定
融合三次共获得21株稳定分泌抗HCV核心抗原175B重组蛋白单克隆抗体的细胞株，其效价均在10 5～10 7之间。抗HCV核心抗原单克隆抗体经ELISA方法进行单克隆抗体分型鉴定，其中3C-28、11C-13、14C-1、1D-9-10、8H-53、5G-28六株单克隆抗体为IgG1型，14C-77、3F-41、5B-36、8D-73、7C-14-9、2H-49、12F-19六株单克隆抗体为IgG2a型，4D-19、3C-7、2D-32、5G-12、6F-78、6G-5-1、15D-8株单克隆抗体为IgG2b型。
3.4抗HCV核心抗原McAB表位鉴定
采用8种HCV短钛抗原A1～A8分别包被微孔，以PBS+20％NBS为稀释液，稀释单抗为一抗浓度到1ug/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。
结果如下表所示：C175-B抗原制备的单抗能识别5个表位，C70～100和C120～C175的无识别单抗，其中识别C17～35、C55～72和C95～117表位的单抗最多，C17～35有5株，C60～72有6株(经进一步分析验证为C55～72)，C100-120aa有7株(经进一步分析验证为C95～117)。从以上表位识别看，抗体识别抗原的主要表位分布在C17～35，C55-72，C95～117三个区段，而且对抗原的反应性最好。
HCV-核心单克隆抗体表位鉴定
Figure PCTCN2020086437-appb-000007
3.5抗体配对筛选：
21株单抗通过ELISA夹心法正交实验，检测稀释度以C175B core抗原来筛选检测灵敏度高的配伍单抗，3C-28、14C-1、6F-78、11C-13、15D-8、3G-42包被磁珠配14C-77、1D-9-10、2H-49、8D-73标记AE 20组单抗配伍的效果好，能检测到97pg/ml，其中14C-1、6F-78、11C-13配1D-9-10、14C-77、2H-49、8D-73标记AE配伍4组单抗能检测到48.5ng/ml，为灵敏度最高。而且还发现14-77用于标记端和其他抗体配对亲和力高，而且能和20株单抗都有反应，配对成功率最高。
见下表
Figure PCTCN2020086437-appb-000008
备注：/表示配对没有反应，*表示C175B抗原对97ng/ml和48.5ng/ml的两组抗原浓度有较好的反应性。
通过配对结果说明：C17～35表位和C95～117表位用于包被活性优于其他的表位，C55-72表位用于标记活性优于其他表位，C95～117表位与C55-72表位组合效果在灵敏度上好于C17～35配C55-72的组合。
3.6核心抗原天然阳性标本的筛选。
本实验室收集PCR阳性，抗体阳性标本78份，用山东莱博生物技术有限公司采购的丙型肝炎病毒核心抗原检测试剂盒进行检测78份血清中的核心抗原的阳性情况，结果78份血清中，S/CO大于临界值的标本有47份，其中大部分血清的反应性都比较低，只有10份S/CO大于5的，其他都分布在S/CO 1.0～5之间，说明核心抗原在血清中的含量很低，另外可能有一部分抗原被抗体中和掉，所以需要活性更加高的抗体配对来提高核心抗原的检出灵敏度，后续用这些核心抗原阳性标本用于筛选对天然核心抗原阳性标本反应性高的配对单抗使用。
3.7通过双抗体夹心法磁酶免平台来筛选对天然core抗原阳性的标本检测率高的单抗配伍。
由于我们无法确定筛选到的配对对核心抗原阳性标本的检出率如何，所以将抗体3C-28、14C-1、11C-13、15D-8、3G-42分别包被磁珠配14C-77、1D-9-10、2H-49、8D-73标记AE，20组单抗配伍，挑选了10份RNA阳性S/CO大于5的5份，S/CO大于1-4的5份来看对天然阳性标本的反应情况：交叉配对单抗组合对核心阳性标本检出率。
Figure PCTCN2020086437-appb-000009
通过上述筛选，可以看出C95-117aa做包被配C55-72aa标记组合的单抗配对检出率高最高，11C-13配14C-77-AE对10份血清都检出，优于其他组配对，其次是C15-35aa配C55-72aa组合，再次是C95-117aa包被配C95-117aa标记与C17-35aa配C95-117aa检出率最低。说明对核心抗原检 出的优势表位配对主要集中在C95-117aa做包被配C55-72aa标记和C15-35aa包被配C55-72aa标记组合上。选两组优势表位组合进行阳性和临床血清的放大。
3.8两组优势表位组合进行阳性和临床阴性血清的放大对比
Figure PCTCN2020086437-appb-000010
结论：通过对比筛选到与山东莱博试剂盒检出率一致的配对11C-13包被配14C-77标记的组合，而且特异性也满足要求。而其他配对均出现部分低值标本漏检的问题，从而体现出C95-117aa包被配C55-72aa标记组合为最佳，其中11C-13包被配14C-77标记配对在灵敏度和特异性均优于其他配对，故选此配对进行联检组合，通过错开表位方式实现联检试剂盒的制备。
另外，商购获得特异性结合特异性结合95-117区序列和特异性结合55-72区序列的单克隆抗体(HCV-Core-McAb23和HCV-Core-McAb19，购自菲鹏生物股份有限公司，HCV-Core-McAb23单抗特异性结合HCV核心抗原95-117区序列，HCV-Core-McAb19单抗特异性结合HCV核心抗原55-72区序列)。测定上述抗体对C175-B检出灵敏度最高的以及对HCV-核心抗原阳性的10份天然标本检出反应性。结果发现针对95-117区表位的抗体2C-18、3D-10、5G-22、HCV-Core-McAb23和针对55-72区表位的6G-15、7H-3、HCV-Core-McAb19所有组合对10份天然标本全部检出，对核心抗原的反应性高，对天然HCV-核心抗原阳性标本检出率高，阴性血清本底好。上述结果也表明针对95-117aa和55-72aa表位氨基酸的抗体组合的检测灵敏度和特异性优于针对其他表位抗体的配对。在下述实验中，选择抗体11C-13做包被/14C-77-AE配对进行实验。将11C-13命名为 HCV-AbI，14C-77-AE命名为HCV-AbII，用于联检配对。
实施例3磁珠标记抗原和抗体制备
1)取10mg羧基磁珠(Merk EM1-100/40羧基磁珠)用活化缓冲液(100mM MES pH5.5)洗涤4次，每次10mL，最后加8mL活化缓冲液，超声分散。称取约NHS(10mg)和EDC(5mg)，(NHS(N-hydroxysuccinimide购于Thermo，型号是：24510)和EDC购于Thermo，型号是：22891)分别溶解至10mg/mL和1mg/mL，加入1mL NHS溶液和1mL EDC溶液，充分混匀，室温旋转(30rpm)反应10分钟。
2)磁分离，弃去上清，不要洗涤，直接加9mL交联缓冲液(同活化缓冲液：100mM MES pH5.5)，超声分散；将活化好的磁珠分成两份，取一份4.5ml活化后的磁珠加入HCV-AbI 0.5mL(4.0mg/mL)，另一份4.5ml磁珠加入HCV-AgI 0.5ml(加入前先过Zeba的脱盐柱，购买于Thermo，型号为：89891)，室温旋转(30rpm)反应4小时。
3)用洗涤液洗2次，每次10mL；加10mL封闭液(含0.5％BSA)，室温旋转(30rpm)反应4小时。
4)用洗涤液洗3次，每次10mL；最后分别加5mL磁珠保存液(25mM MES+150mM NaCl+0.2％(w/v)Casein+1mM EDTA+5％(v/v)NBS+0.2％Proclin-300)重悬，终浓度10mg/mL固体含量。+2～+8℃保存。
实施例4亲和素(SA)与吖啶酯(AE)偶联制备
实施例：取已纯化好的SA，纯度大于90％，装入透析袋中用20m M PB(pH 7.4)透析4个小时，按照比例加入AE(吖啶酯)进行偶联(AE购买于赫利森NSP-SA-NHS，型号：199293-83-9)标记10min，后继续透析4小时，从透析袋中吸出，加入50％甘油保存在-20度中待用。
实施例5 HCV体外生物素化抗原制备
1)在此我们以sulfo-NHS-LC-生物素标记HCV抗原氨基进行说明，其标记过程如下：
2)取HCV-AgII抗原1mg在缓冲液中(100mM PB+150mM NaCl， pH7.2)透析过夜；
3)生物素溶液：2.2mg sulfo-NHS-LC-生物素溶解在0.4ml超纯水，取143ul的生物素加入到上述透析好的抗原中，
4)将蛋白溶液和生物素溶液以1:50摩尔比混合，0-4℃交联2小时；
5)将反应液在含有0.05％SDS的PB(100mM PB，pH7.2)缓冲液中透析，除去游离生物素；
6)加入终浓度50％的甘油后储存于-20℃备用。
实施例6 HCV体外生物素化抗体制备
1)取HCV-AbII 4mg在缓冲液中(100mM PB+150mM NaCl，pH7.2)透析过夜；
2)生物素溶液：2.2mg sulfo-NHS-LC-生物素溶解在0.4ml超纯水，取53ul的生物素加入到上述透析好的抗体中，
3)将蛋白溶液和生物素溶液以1:20摩尔比进行混合，2～8℃交联2小时；在含有0.05％SDS的PB(100mM PB，pH7.2)缓冲液透析，除去游离生物素；
4)加入终浓度50％的甘油后储存于-20℃备用。
实施例7检测抗原抗体联检的裂解液
1)选取10-100mM，优选20mM PB磷酸盐缓冲液，
2)变性剂：SDS浓度为0.5％～1％，优选0.8％。
3)表面活性剂：NP-40浓度为0.5％～1％，优选0.5％。TRITONX-100、TWEEN-20加入0.5％～1％，优选0.5％。
4)BSA保护蛋白：浓度为0.5％～1％，优选1％。
5)硫酸铵浓度为1％～2.5％，优选1％。
6)无水乙醇：浓度可以为0.1％～10％，优选1％。
注：以上浓度比例为质量体积比，1％表示1g/100ml。
实施例8试剂盒内试剂的制备
1、磁珠工作液(磁珠标记的HCV-AbI和HCV-AgI混合液的制备)：
将制备好的HCV-AgI磁珠10mg/ml用保存液稀释成0.2mg/ml， HCV-AbI磁珠10mg/ml用保存液稀释成0.2mg/ml，将前面两组液体按照1：2的体积比合备用。
2、生物素工作液(生物素化的HCV-AbII-BIO和HCV-AgII-BIO混合液的制备)：
将标记好的HCV-AgII-BIO用生物素稀释液(含20mMPB+150mMNacl+0.％Casein-2Na+0.1％P300+0.1％巯基乙醇)稀释成0.2mg/ml，HCV-AgII-BIO用HCV生物素稀释液(含20mMPB+150mMNacl+0.％Casein-2Na+0.1％P300+0.1％巯基乙醇)稀释成0.2mg/ml，然后按1：2混合(生物素稀释液中含1：1000巯基乙醇)，其中还原剂可以加入DTT、巯基乙醇等，优选巯基乙醇。
3、亲和素化标记物SA-AE的制备：
将SA-AE用稀释液200mM HEPES+0.5％BSA+0.1％叠氮钠稀释成0.5μg/ml备用，加入阻断剂0.5μg/ml。
4、裂解液的制备。
5、20X洗涤液的制备，稀释成1X后备用。
6、阴阳性质控品的制备。
7、激发液的制备。
通过上述步骤制备试剂盒1。
类似地，通过HCV-AgI-1和HCV-AgII-1替代试剂盒中的HCV-AgI和HCV-AgII，制备试剂盒2。
实施例9测操作过程
1、检测试剂的准备；
2、每孔加入50μl磁珠工作液(抗原抗体磁珠混合液)+100μl样本+50μl生物素工作液(抗原抗体标记生物素混合物)37℃恒温箱反应15min，加入50μl裂解液37℃恒温箱反应15min，洗涤4次；
3、每孔加入200μl亲和素化SA-AE 37℃恒温箱反应10min，洗涤4次
4、每孔加入100μl激发液A和100μl激发液B，用化学发光全自动仪器测量发光值，与临界值相比较，从而判断阴阳性。
表5：抗原抗体联检与单检的数据对比：结果判断：大于1为阳性， 小于1为阴性。
试剂盒1检测结果
Figure PCTCN2020086437-appb-000011
Figure PCTCN2020086437-appb-000012
Figure PCTCN2020086437-appb-000013
Figure PCTCN2020086437-appb-000014
裂解液对比分析数据：
Figure PCTCN2020086437-appb-000015
裂解液效果对比数据分析表：(S/CO)结果判断：大于1为阳性，小于1为阴性。
结论：从以上结果看，调整的抗原抗体联检裂解液对抗体检出没有影响，同时对抗原检出起到裂解的作用，提高了抗原的检出率。
因此，本发明的抗丙型肝炎病毒抗原单克隆抗体表位无重叠，避免了核心抗原抗体难以错开表位进行联检的问题。本发明的抗丙型肝炎病毒抗原单克隆抗体和丙型肝炎病毒重组抗原相互之间不发生交叉反应，对活性不造成影响，有利地避免了交叉反应和检测活性损失的问题。本发明的方法和试剂盒显著的缩短窗口期，降低漏检的风险，减少工作量，降低两种方法学单独检测的人力、仪器、试剂成本，提高了病毒检出率和灵敏性。

Claims (15)

  1. 丙型肝炎病毒检测试剂盒，其包括用于检测来自受试者样品中丙型肝炎病毒核心抗原的第一抗体和第二抗体，其中第一抗体针对丙型肝炎病毒核心抗原第95-117位氨基酸序列中的表位或特异性结合丙型肝炎病毒核心抗原第95-117位氨基酸序列，第二抗体针对丙型肝炎病毒核心抗原第55-72位氨基酸序列中的表位或特异性结合丙型肝炎病毒核心抗原第55-72位氨基酸序列。
  2. 权利要求1所述的试剂盒，其中第一抗体为捕获抗体，第二抗体为标记抗体，或者第一抗体为标记抗体，第二抗体为捕获抗体，优选第一抗体为捕获抗体，第二抗体为标记抗体。
  3. 权利要求2所述的试剂盒，其中所述捕获抗体结合至固相，所述标记抗体用可检测标记物标记，例如荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，酶标记，例如放射性同位素，荧光团，罗丹明及其衍生物，荧光素酶，荧光素，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，生物素/抗生物素蛋白，自旋标记，噬菌体标记，例如吖啶酯标记，例如通过接头如生物素-亲和素添加荧光标记如吖啶酯标记。
  4. 权利要求1所述的试剂盒，其还包括用于检测来自受试者样品中丙型肝炎病毒抗体的第一抗原和/或第二抗原，所述第一抗原和/或第二抗原可以是例如丙型肝炎病毒核心抗原，E1、E2、NS2、NS3、NS4和NS5，例如第一抗原和/或第二抗原来自同一丙型肝炎病毒抗原的不同位置，例如丙型肝炎病毒核心抗原第1位至56位氨基酸，例如丙型肝炎病毒核心抗原第1位至35位氨基酸，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，第29-48位氨基酸序列，例如NS3第1201位至1490位氨基酸，NS4的1883位至1925位氨基酸的序列；NS3第1223位至1426位氨基酸，NS4的1890位至1923位氨基酸的序列；例如第一抗原包括以下任一氨基酸片段或其组 合：HCV核心抗原第1位至56位氨基酸，NS3第1201位至1490位氨基酸，NS4的1883位至1925位氨基酸的序列；第二抗原包括以下任一氨基酸序列或其组合：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位氨基酸的序列；例如来自SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列。
  5. 权利要求4所述的试剂盒，其中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。
  6. 权利要求5所述的试剂盒，其中所述捕获抗原结合至固相，所述标记抗原用可检测标记物标记，例如荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，酶标记，例如放射性同位素，荧光团，罗丹明及其衍生物，荧光素酶，荧光素，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，生物素/抗生物素蛋白，自旋标记，噬菌体标记，例如吖啶酯标记，例如通过接头如生物素-亲和素添加荧光标记如吖啶酯标记。
  7. 权利要求3所述的试剂盒，其中所述固相包括磁性颗粒，胶乳粒子和微量滴定板。
  8. 权利要求1所述的试剂盒，其包括病毒裂解液，例如磷酸盐缓冲液，例如包含变性剂，表面活性剂，保护蛋白，硫酸铵和无水乙醇的缓冲液。
  9. 权利要求8所述的试剂盒，其中病毒裂解液包含10-100mM磷酸盐缓冲液，0.5％～1％(m/v)的变性剂，如SDS，0.5％～1％(m/v)的表面活性剂，如NP-40，TRITONX-100和/或TWEEN-20，0.5％～1％(m/v)的保护蛋白，如BSA，1％～2.5％(m/v)的硫酸铵，0.1％～10％(m/v)的无水乙醇。
  10. 权利要求1所述的试剂盒，其中所述样品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，所述丙型肝炎病毒包括HCV基因型I/1a，II/1b，III/2a，IV/2b，V/3a和VI/3b型。
  11. 检测丙型肝炎病毒的方法，所述方法包括使来自受试者样品与第一抗体和第二抗体接触，其中第一抗体针对丙型肝炎病毒核心抗原第 95-117位氨基酸序列中的表位或特异性结合丙型肝炎病毒核心抗原第95-117位氨基酸序列，第二抗体针对丙型肝炎病毒核心抗原第55-72位氨基酸序列中的表位或特异性结合丙型肝炎病毒核心抗原第55-72位氨基酸序列。
  12. 权利要求11所述的方法，其中所述方法还包括使来自受试者样品与来自丙型肝炎病毒的第一抗原和/或第二抗原接触，所述第一抗原和/或第二抗原可以是例如丙型肝炎病毒核心抗原，E1、E2、NS2、NS3、NS4和NS5，例如第一抗原和/或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，例如第一抗原和/或第二抗原包括以下任一氨基酸片段或其组合：HCV核心抗原第1位至56位氨基酸，NS3第1201位至1490位氨基酸，NS4的1883位至1925位氨基酸的序列；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位氨基酸的序列，例如来自SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列。
  13. 制备检测丙型肝炎病毒的试剂或试剂盒的方法，其中使用丙型肝炎病毒第一核心抗原和丙型肝炎病毒第二核心抗原制备抗体，所述丙型肝炎病毒第一核心抗原包含丙型肝炎病毒核心抗原第55-72位氨基酸或由其组成，所述丙型肝炎病毒第二核心抗原包含丙型肝炎病毒核心抗原第95-117位氨基酸或由其组成。
  14. 权利要求13所述的方法，其中所述抗体为单克隆抗体。
  15. 权利要求13所述的方法，其中所述方法包括制备检测丙型肝炎病毒的试剂盒，所述试剂盒包含所述抗体，还包含丙型肝炎病毒的一种或两种抗原，所述一种或两种抗原可以是例如丙型肝炎病毒核心抗原，E1、E2、NS2、NS3、NS4和NS5，例如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，一种或两种抗原包括以下任一氨基酸序列或其嵌合片段：HCV核心抗原第1位至56位氨基酸，NS3第1201位至1490位氨基酸，NS4的1883位至1925位氨基酸的序列；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位氨基酸的序列，例如来自SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列。
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