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WO2008003236A1 - Procédé de détection conjointe de l'antigène pres1 et de l'antigène de noyau de vhb, coffret d'expérimentation, substrat solide et solution de lyse de virus - Google Patents

Procédé de détection conjointe de l'antigène pres1 et de l'antigène de noyau de vhb, coffret d'expérimentation, substrat solide et solution de lyse de virus Download PDF

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Publication number
WO2008003236A1
WO2008003236A1 PCT/CN2007/002000 CN2007002000W WO2008003236A1 WO 2008003236 A1 WO2008003236 A1 WO 2008003236A1 CN 2007002000 W CN2007002000 W CN 2007002000W WO 2008003236 A1 WO2008003236 A1 WO 2008003236A1
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WIPO (PCT)
Prior art keywords
antibody
antigen
virus
hepatitis
buffer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2007/002000
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English (en)
Chinese (zh)
Inventor
Shengxiang Ge
Quan Yuan
Yu Zhao
Wenxin Luo
Jun Zhang
Ningshao Xia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xiamen University
Beijing WanTai Biological Pharmacy Enterprise Co Ltd
Original Assignee
Xiamen University
Beijing WanTai Biological Pharmacy Enterprise Co Ltd
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Filing date
Publication date
Application filed by Xiamen University, Beijing WanTai Biological Pharmacy Enterprise Co Ltd filed Critical Xiamen University
Publication of WO2008003236A1 publication Critical patent/WO2008003236A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—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
    • G01N33/5761—Hepatitis B
    • G01N33/5762—Hepatitis B core antigen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • G01N33/5764—Hepatitis B surface antigen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus

Definitions

  • the present invention relates to the field of hepatitis B virus detection, and more particularly to a method for diagnosing hepatitis B virus infection by detecting hepatitis B virus-associated pre-S1 antigen (HBV PreSl) and core antigen (HBcAg).
  • HBV PreSl hepatitis B virus-associated pre-S1 antigen
  • HBcAg core antigen
  • the invention also discloses a kit for detecting combined pre-S1 antigen and core antigen of hepatitis B virus (especially enzyme-linked immunosorbent assay and chemiluminescence method).
  • the present invention also relates to a method for detecting a pre-S1 antigen, a method for lysing a virus, and a related kit. Background of the invention
  • Hepatitis B virus (Hepa t i t i s B vi rus ) infection is one of the most important public health problems in the world.
  • HBV serum markers are widely used as routine testing standards for HBV infection and previous HBV infection.
  • HBV serum markers conventional such as: HBsAg, HBsAb, HBeAg, HBeAb, HBcAb, commonly known as "two-and-a-half" test
  • HBsAg, HBsAb, HBeAg, HBeAb, HBcAb commonly known as "two-and-a-half” test
  • HBeAg HBeAg
  • HBeAb HBeAb
  • HBcAb HBcAb
  • HBV DNA is a direct indicator of HBV replication
  • its dot blot hybridization assay (or PCR assay) is the gold standard for infection and infectivity judgment in patients with hepatitis B and HBV carriers. Both PCR and dot blot hybridization assays can be used as direct indicators of HBV infection and infectivity, but are not suitable for large-scale census or routine use.
  • HBcAg Serum marker antigen (HBV PreSl, which is highly correlated with HBV DNA, HBcAg, HBxAg, DNAP, HBV PreS2, etc.): HBcAg—is directly considered to be an antigen directly related to HBV DNA.
  • the detection is of particular diagnostic significance in HBcAg in HBV-infected and HBsAg-negative HBV-infected and HB-negative patients.
  • the current developed or reported HBcAg immunological diagnostic reagents usually pre-treat the sample before treatment (cracking the virus, rupturing the membrane and inactivating the HBcAb) or using the bypass detection to detect the HBcAg-HBcAb immune complex.
  • HBV PreSl also has a good correlation with HBV DNA (D. Buff el lo, 2000, 96%; Kui jpers, 1989, 88%; Thei lraan, 1986).
  • HBV DNA D. Buff el lo, 2000, 96%; Kui jpers, 1989, 88%; Thei lraan, 1986.
  • the diagnostic method of HBV PreSl antigen the current developed or reported PreSl immunological diagnostic reagents consider that the absolute concentration of PreSl is lower than that of HBsAg.
  • the conventional double PreSl antibody sandwich method is difficult to achieve the desired sensitivity, often using the whole detection.
  • LHBs ie, HBsAg-mediated detection on LHBs, specific antibodies against HBsAg as capture or reporter antibodies, and paired with specific antibodies against PreSl
  • this detection mode effectively amplifies the detection of PreSl Sensitivity, but the detection of PreSl is completely dependent on HBsAg, does not form an independent detection system, may lead to the false negative of PreSl due to HBsAg mutation, can not accurately reflect the PreSl dynamics of mutant virus or different subtype virus infection.
  • An object of the present invention is to establish a simple, effective HBcAg detection method without sample preparation process and a highly sensitive double Presl antibody sandwich method for detecting PreSl, and to compensate for possible HBcAg or PreSl mutations by jointly detecting HBcAg and PreSl modes. Or missed detection caused the disadvantage of ⁇ negative, construct a combination of HBVAg and PreSl detection modes, and apply this model to enzyme-linked immunosorbent assay, chemiluminescence, time-resolved, colloidal gold, Qualitative or quantitative immunological diagnosis such as enzyme-linked immunofiltration.
  • the present invention relates to an immunological diagnostic method highly correlated with a nucleic acid detection method established by jointly detecting a pre-S1 antigen/core antigen of hepatitis B virus.
  • the present invention relates to a kit for the simultaneous detection of pre-S1 antigen/core antigen of hepatitis B virus and a preparation method thereof.
  • the present invention relates to an enzyme-linked immunosorbent kit and a chemiluminescent kit for jointly detecting a pre-S1 antigen/core antigen of hepatitis B virus and a preparation method thereof.
  • the combined detection mode of the present invention not only effectively reflects the complementary advantages of the combined detection of the two antigens, but also the new index formed by the combined detection results - "nucleic acid-related antigen" reflects a high correlation with the hepatitis B virus nucleic acid index.
  • the present invention discloses for the first time a method for sandwich detection of pre-S1 antigen with a high sensitivity double pre-S1 antibody, which is different from other published methods for detecting pre-S1 antigen.
  • the present invention is the first to use a series of buffer solutions containing different doses of different types of surfactants for lysing the virus, greatly improving the combination of the pre-S1 antigen and the core antigen and the pre-S1 antigen. Sensitivity of detection of individual indicators. DRAWINGS
  • Figure 1 shows the recombinant plasmid of the triple tandem cascade of the 21-47AA fragment of the preSl region.
  • Figure 2 shows the results of SDS-PAGE electrophoresis of the expressed and purified recombinant protein GST-2147*6. The results showed that the purity of the target protein after purification was over 85%.
  • Lane 1 Protein marker
  • Lane 2 Expression of pGEX-2147 * 6
  • Lane 3 Purified pGEX-2147 *6 D
  • Figure 3 shows the correlation between chemiluminescence detection and real-time PCR. As described in Example 14, the logistic correlation analysis was performed after taking the logarithm of the virus content and the luminescence intensity of each specimen. Detailed description of the invention
  • the present invention relates to an immunological diagnostic method highly correlated with a nucleic acid detection method established by jointly detecting a pre-S1 antigen/core antigen of hepatitis B virus.
  • a nucleic acid detection method established by jointly detecting a pre-S1 antigen/core antigen of hepatitis B virus.
  • the core antigen and the combined detection of the pre-S1 antigen/core antigen enzyme-linked immunosorbent assay
  • the combined detection of the pre-S1 antigen/core antigen is compared with the pre-S1 detection alone.
  • Core antigens have distinct advantages.
  • the pre-S1 antigen/core antigen (enzyme-linked immunosorbent assay) combined with detection of hepatitis B virus is compared with conventional two-and-a-half detection of hepatitis B, and the detection method can accurately determine a part of the virus-containing specimen. Qualitative, and two-and-a-half tests are not deterministic for such specimens.
  • the method for detecting the pre-S1 antigen/core antigen of the combined detection of hepatitis B virus is applied to the chemiluminescence diagnosis, and the detection result and the virus content of the pre-S1 antigen/core antigen of the hepatitis B virus are jointly detected. Have better Linear correlation.
  • the detection method of the present invention is carried out by an enzyme-linked immunosorbent assay. Accordingly, the present invention provides a method of preparing a related enzyme-linked immunosorbent kit and a kit prepared thereby.
  • the detection method of the present invention is carried out by a chemiluminescence method. Accordingly, the present invention provides a method of preparing a related chemiluminescent kit and a kit prepared thereby. The above aspects and other aspects are explained in more detail below.
  • the invention relates to a method for detecting hepatitis B virus in a sample or detecting hepatitis B virus infection, including joint detection of hepatitis B virus Pre-S1 antigen and core antigen.
  • combined detection includes the detection of the pre-S1 antigen and the detection of the core antigen, either separately or in combination, and simultaneously and sequentially in any order, as long as the detection of the pre-S1 antigen and The signal processing of both the detection of the core antigen can be performed in combination.
  • the combined signal processing means that when the sample is judged to be positive or negative for hepatitis B virus or hepatitis B virus infection, the signal obtained by detecting the pre-S1 antigen and the signal detected by the core antigen are combined to form a single indicator.
  • the signals detected by the two antigens can be simply added and compared with a predetermined value.
  • the signals detected by the two antigens can be judged by appropriate processing or calculation (for example, weighting one of the signals or weighting both), combining them into a new single index, and comparing with a predetermined value. This can improve sensitivity, detection rate, and the like. This may be due to avoiding missed detection, for example due to a single antigen ⁇ negative.
  • detection of the pre-S1 antigen and detection of the core antigen can be independent of each other, that is, they can occur in the same or different time, the same or different assays.
  • detection of the pre-S1 antigen and detection of the core antigen can be carried out by the same method or by different methods; for example, detection of an antigen is carried out by one of the following methods, and the other Detection of antigens is carried out using different (or the same) methods: enzyme-linked immunosorbent assay, immunodiafiltration, immunochromatography, chemiluminescence, time-resolved.
  • both antigens are detected using the same method.
  • detection of the pre-S1 antigen can occur before, after, or at the same time as the core antigen is detected.
  • the two antigens are detected by the same method, they can be carried out in the same or different test batches, in the same or different reaction vessels (e.g., reaction wells), on the same or different solid phase supports. Both can use the same or different signal generators.
  • both antigens are detected simultaneously and in combination.
  • detection of both antigens is performed simultaneously in the same reaction vessel (e.g., reaction well). This can be carried out, for example, by immobilizing a capture antibody that captures two antigens on the same vector, and simultaneously capturing the two antigens in the same medium using the same virus lysate, optionally simultaneously in the same medium. Binding and detection of the two antibodies.
  • the sample is an isolated biological sample from the individual to be tested, preferably the sample is serum.
  • the method is for determining hepatitis B disease in serum The presence or absence of toxic nucleic acids.
  • the combined detection of the hepatitis B virus pre-S1 antigen and the core antigen is achieved by one or more methods selected from the group consisting of: enzyme-linked immunosorbent assay, immunodiafiltration, immunization Chromatography, chemiluminescence, time resolution; preferably enzyme-linked immunosorbent assay or chemiluminescence.
  • the pre-S1 antigen and the core antigen are simultaneously detected in the same reaction in the same reaction vessel (e.g., reaction well).
  • the pre-S1 antigen and the core antigen are detected using a double antibody sandwich method.
  • no pretreatment of the sample, such as serum is required. For example, there is no need to denature the virus in advance.
  • cleavage of the hepatitis B virus and capture of the pre-S1 antigen and the core antigen are carried out simultaneously in the same medium in the same reaction vessel (e.g., reaction well).
  • the lysate of the present invention for joint detection is used as the medium.
  • the joint detection method of the present invention comprises the following steps:
  • the vector is ELISA plate;
  • the sample to be tested contacting the sample to be tested with the first antibody-carrier conjugate such that the hepatitis B virus pre-S1 antigen and core antigen in the sample, if present, are captured, shaped Forming an antigen-first antibody-carrier conjugate;
  • the sample may be pre-treated or not pretreated; preferably, the sample is not subjected to prior viral lysis, and the sample to be tested is bound to the first antibody-vector
  • the contacting is carried out under conditions allowing for viral lysis and antigen capture; more preferably, the sample to be tested is contacted with the ⁇ -antibody-carrier conjugate in the lysate for combined detection according to the present invention;
  • step c contacting a second anti-HBV PreSl antibody and a second anti-HBc antibody (both collectively referred to as a second antibody) with the product (vehicle) of step c), which allows for the binding of the genomic antibody to the captured antigen. get on;
  • the antigen-first antibody-carrier conjugate of step c) comprises a hepatitis B virus pre-S1 antigen-first anti-HBV PreSl antibody-carrier conjugate and a core antigen-first anti-HBc antibody-carrier conjugate.
  • step d) if a hepatitis B virus pre-S1 antigen and a core antigen are present, a second antibody-antigen-first antibody-carrier conjugate is formed, including a sputum secondary antibody HBV PreSl antibody-hepatitis B virus pre-S1 Antigen- ⁇ primary antibody HBV PreSl antibody-carrier conjugate, and second anti-HBc antibody-core antigen-first anti-HBc antibody-carrier conjugate
  • the first anti-HBV PreSl antibody and the second anti-HBV PreSl antibody may be the same or different and may be monoclonal antibodies or polyclonal antibodies, preferably different monoclonal antibodies.
  • the first anti-HBc antibody and the second anti-HBc antibody may be the same or different and may be monoclonal antibodies or polyclonal antibodies, preferably different monoclonal antibodies.
  • Step e) detecting the amount of the bound second antibody can be carried out, for example, by providing a first signal producer capable of efficiently binding to the second anti-HBV PreS1 antibody and capable of being effective against the second anti-HBc antibody.
  • the combined ⁇ 2 signal generator, the signal intensity generated by the ⁇ 1 and ⁇ 2 signal generators are only related to the amount of the second anti-HBV PreSl antibody and the second anti-HBc antibody, respectively; the first and second signals are The generating body is in contact with the second antibody; the signal generated by the signal generating body is detected.
  • the first signal generator and the second signal generator may be the same or different.
  • the first signal generator and the second signal generator are the same, or the signals generated by the first signal generator and the second signal generator are the same, and can be detected by the same method; more preferably, the detection The sum of the signals generated by the first and second signal generators is used as an indicator of the judgment result.
  • Step e) Detecting the amount of the bound second antibody can also be carried out, for example, by the following method:
  • the second antibody is directly labeled with a signal generating body, and the signal generated by the signal generating body is detected.
  • the second anti-HBV PreSl antibody and the second antibody anti-HBc antibody can be labeled with the same signal generator, or different signal generators.
  • the second anti-HBV PreSl antibody and the second anti-HBc antibody labeled signal generator are identical, or they produce the same signal and can be detected by the same method; more preferably, the second anti-HBV PreSl is detected
  • the sum of the signals generated by the antibody and the second anti-HBc antibody-labeled signal generator is used as an indicator of the judgment result.
  • the signal generating body may be any substance which can be detected by an appropriate method, and may be, for example, an enzyme, a fluorescein, a chemiluminescent substance, an isotope, or a rare earth element.
  • immunodetection is carried out using a monoclonal antibody against hepatitis B virus pre-S1 antigen and anti-core antigen.
  • the monoclonal antibody against hepatitis B virus pre-S1 antigen is selected from the group consisting of 3H5, 7H11, 2A7, 4D11, 6F1, 13G2 and 16F5; preferably, 7H11 mAb is used as the first anti-HBV PreSl antibody HBV PreSl was detected by pairing with 4D11-HRP as a second anti-HBV PreSl antibody.
  • the method further comprises the step of: lysing the virus in the sample using the lysate before contacting the sample to be tested with the ⁇ -antibody-carrier conjugate, wherein the lysate is preferably used in the present invention Combined detection of virus lysates.
  • steps e.g. between steps b) and c), and/or c) and d), and/or d) and e
  • steps may also contain steps of washing, drying, etc. .
  • the invention also relates to a viral lysate useful for the combined detection of hepatitis B virus pre-S1 antigen and core antigen, comprising a surfactant and a suitable buffer selected from the group consisting of: Chaps, sulfobetain 8-18 (SB8) , SB10, SB12, SB14, SB16, SB18), Bridger 35, Tween series and/or HP- ⁇ -CD, preferably selected from SB14, SB16, SB18, HP- ⁇ -CD, TWEEN20, TWEEN40> TWEEN60, TWEEN80 .
  • a surfactant selected from the group consisting of: Chaps, sulfobetain 8-18 (SB8) , SB10, SB12, SB14, SB16, SB18), Bridger 35, Tween series and/or HP- ⁇ -CD, preferably selected from SB14, SB16, SB18, HP- ⁇ -CD, TWEEN20, TWEEN40> TWEEN60, TW
  • the surfactant may be a combination of one or more of the above surfactants (2, 3, 4, 5), such as one of HP, TWEEN series and SB series. Or a combination of two or three.
  • HP + TWEEN series, TWEEN series + SB series, HP + SB series or HP + TWEEN series + SB series preferably HP+TWEEN40, HP + TWEEN40 + SB14 HP + TWEEN40 + SB16, HP + TWEEN40 + SB18, HP + TWEEN40 + SB18, HP + TWEEN20 + SB14, HP + TWEEN60 + SB14, HP + T EEN80 + SB14
  • the surfactant may be HP: 0.5%-4; Tween series: 0.
  • the surfactant is selected from the group consisting of lysisl - lysis 24 shown in Table 4, more preferably lysisl, lysislO, lysisl2, lysisl3, lysisl4, lysisl5, lysisl6, lysisl7, lysisl8, lysisl9, lysis20, lysis21, lysis22, lysis23, and lysis24 More preferably, the tables described in lysis10, lysisl3, lysisl6, lysisl7, lysisl8, lysisl9, lysis20, lysis21, and lysis24, most preferably lysis10, 13, 16, 18 Surfactant.
  • the buffer is selected from the group consisting of citrate buffer, phosphate buffer, Tris buffer, amino acid buffer, acetate buffer, HEPES buffer, carbonate buffer.
  • the buffer may be one or a combination of the above buffers (2, 3, 4, 5).
  • the buffering agent is preferably a citrate buffer or a phosphate buffer.
  • the pH range and concentration range of the buffer may be: phosphate buffer: 10- 50 mM pH 6.0-7.0 preferably 20 mM 11 value 7.4; citrate buffer: 10-50 mM pH 3.0-6.6 preferably 10 mM pH 6.6; Tris buffer: 10-50 mM pH 7.0-9.0 preferably 20 mM pH 7.0; acetate buffer: 10-50 mM pH 3.8-6.0 preferably 10 mM pH 6.0.
  • the lysate may also contain one or more (eg 1, 2, 3, 4 , 5, 6-10) other ingredients, such as reducing agents (eg DTT, DTE, ⁇ -mercaptoethanol), chelate Mixture (eg EDTA), osmotic pressure regulator (eg salt such as NaCl; sucrose).
  • reducing agents eg DTT, DTE, ⁇ -mercaptoethanol
  • chelate Mixture eg EDTA
  • osmotic pressure regulator eg salt such as NaCl; sucrose.
  • the lysate may also contain one or a combination of the following components: ⁇ -mercaptoethanol: 0.01% to 1%, preferably 0.1% to 0.5%, more preferably 0.4%; DTT: 1 to 30 mM, preferably 5 to 10 mM, More preferably 5 mM; DTE: l-30 mM preferably 5- lOmM, more preferably 5 mM; EDTA: 5 mM-20 mM preferably 5 mM; NaCl: 50 mM-300 mM more preferably: 50-150 mM, more preferably 100 mM; sucrose: 2%-20% 5% to 10% is more preferably 10%.
  • ⁇ -mercaptoethanol 0.01% to 1%, preferably 0.1% to 0.5%, more preferably 0.4%
  • DTT 1 to 30 mM, preferably 5 to 10 mM, More preferably 5 mM
  • DTE l-30 mM preferably 5- lOmM, more preferably 5 mM
  • the lysate is selected from the lysis of lysis 24 shown in Table 4, and more preferably, the lysate is selected from the group consisting of lysis1, lysis10, lysisll, lysisl2, lysisl3, lysisl4, lysisl5, lysisl6, lysisl7, lysisl8, lysisl9, lysis20, lysis21 , lysis22, lysis23, and lysis24, more preferably lysis10, lysisl3, lysisl6, lysisl7, lysisl8, lysisl9, lysis20, lysis21, and lysis24, most preferably, the lysate is selected from the group consisting of: lysislO - pH 6.6 citrate buffer Liquid, 2% SB14, 5mM DTE, lOOmM NaC 5raM EDTA; lys i sl 3 - pH 6.
  • the virus lysate of the present invention for the combined detection of the pre-S 1 antigen and core antigen of hepatitis B virus can also be used to detect the core antigen alone or to detect the pre-S1 antigen alone. Accordingly, the present invention also provides the above virus lysate for detecting a core antigen. The present invention also provides the above virus lysate for detecting the pre-S1 antigen. The present invention also provides the use of the above lysate for detecting a core antigen, and the use of the above lysate for detecting the pre-S1 antigen.
  • NP4Q is generally used for the virus-breaking envelope, but these known lysates are not suitable for the combined detection of pre-S1 antigen and core antigen, such as CN1352392A.
  • virus lysate of the present invention can be used as a medium for capturing viral antigens.
  • the virus lysate of the present invention is characterized by sufficient lysis of the virus but mild enough to not substantially inactivate the antibody coated on the carrier.
  • the use of the virus lysate of the present invention allows for efficient antigen capture while lysing the virus.
  • the invention is not limited by this mechanism of action.
  • the virus lysate for combined detection of the present invention is used to simultaneously lyse the virus and capture the antigen (pre-S1 antigen and core antigen) in the same reaction medium without The virus is pre-cleaved prior to antigen capture. This is also an important advantage of the present invention.
  • the present invention also relates to a kit for joint detection comprising an agent for jointly detecting a hepatitis B virus pre-S1 antigen and a core antigen according to the above-described joint detection method of the present invention.
  • the combined detection kit of the present invention comprises an anti-HBV PreSl antibody (preferably comprising two different anti-HBV PreSl antibodies, particularly a monoclonal antibody) and an anti-HBc antibody (preferably comprising two different Anti-HBc antibodies, particularly monoclonal antibodies), and/or viral lysates of the invention for use in combination assays.
  • the anti-HBV PreSl antibodies and at least one anti-HBc antibody in the antibody is labeled by a signal generator or is operably associated with a signal generator.
  • the anti-HBV PreSl antibody and the anti-HBc antibody-labeled signal generator or the combined signal-generating body may be the same or different, and preferably may be detected by the same detection method.
  • the antibody is preferably a monoclonal antibody.
  • the anti-HBV PreSl antibody is selected from the group consisting of: 3H5, 7H11, 2A7, 4D11, 6F1, 13G2 and 16F5, especially 7H11 and 4D11.
  • the kit further comprises a reagent (e.g., a detection reagent for detecting a second antibody) suitable for detecting the antigen-antibody reaction.
  • a reagent e.g., a detection reagent for detecting a second antibody
  • the combined detection kit of the present invention comprises: (1) a solid phase carrier immobilized with a first anti-HBV PreSl antibody and a first anti-HBc antibody; and/or 2) a virus lysate for co-detection of the present invention; and (3) a second anti-HBV PreSl antibody and a second anti-HBc antibody which are optionally labeled or unlabeled by a signal generator; and (4)
  • the detection reagent for detecting the ruthenium antibody may be optionally included; and (5) the buffer solution, the washing solution, and the instruction manual (the method for joint detection of the present invention is described in the preferred instruction manual).
  • the solid phase carrier in the kit of the present invention is a porous reaction plate (for example, an ELISA plate), and the first anti-HBV PreSl antibody and the primary antibody HBc are immobilized in the same or different wells of the porous reaction plate. antibody.
  • the present invention also relates to a solid phase support to which an anti-HBV PreSl antibody and an anti-HBc antibody are immobilized.
  • the solid phase carrier is a porous reaction plate (eg, ELISA plate, chemiluminescence) Plate), an anti-HBV PreSl antibody and an anti-HBc antibody are immobilized in the same or different wells of the porous reaction plate.
  • the antibody is preferably a monoclonal antibody, particularly a specific antibody disclosed herein.
  • the invention further relates to the use of a reagent for the combined detection of a hepatitis B virus pre-S1 antigen and a core antigen for the preparation of a kit for the detection of hepatitis B virus infection.
  • the reagent is a combined detection reagent as described herein.
  • the combined detection reagents described in the present invention are anti-HBV PreSl antibodies (preferably comprising two different anti-HBV PreSl antibodies, particularly monoclonal antibodies) and anti-HBc antibodies (preferably comprising two different anti-HBc antibodies, In particular, monoclonal antibodies), and/or viral lysates of the invention for use in combination detection.
  • the present invention relates to a method for detecting hepatitis B virus pre-S1 antigen by a double antibody sandwich method, characterized in that both antibodies (capture antibody and reporter antibody) used are specific to hepatitis B virus pre-S1 antigen. Binding of anti-HBV PreSl antibodies.
  • the sample is not required to be pretreated.
  • cleavage of the hepatitis B virus and capture of the pre-S1 antigen are carried out simultaneously in the same medium in the same reaction vessel (e.g., reaction wells).
  • the lysate for detecting the pre-S1 antigen described in the present invention is used as the medium.
  • the method of the invention comprises:
  • the vector is an ELISA plate
  • step c) contacting the second anti-HBV PreSl antibody with the product of step c), which allows binding of the second anti-HBV PreSl antibody to the captured antigen to form a second anti-HBV PreSl antibody-antigen-first anti-HBV PreSl antibody- Carrying out under the conditions of the carrier conjugate;
  • step e) detecting the amount of bound second antibody is carried out by: providing a signal generator capable of efficiently binding to said purine secondary antibody HBV PreS1 antibody, produced by the signal generator The signal intensity is only related to the amount of the second anti-HBV PreSl antibody; the signal generator is contacted with the second antibody; and the signal generated by the signal generator is detected.
  • step e) detecting the amount of bound genomic antibody is carried out by the following method: The second antibody is directly labeled with a signal generating body, and the signal generated by the signal generating body is detected.
  • the signal generator is an enzyme, a fluorescein, a chemiluminescent substance, an isotope, Rare earth elements.
  • the detection of the hepatitis B virus pre-S1 antigen is achieved by one or more of the following methods: enzyme-linked immunosorbent assay, immunodiafiltration, immunochromatography, chemistry Luminescence method, time resolution method; enzyme-linked immunosorbent assay or chemiluminescence method is preferred.
  • between the various steps of the invention may also contain washing, and / or dry and other steps.
  • the invention also relates to a viral lysate useful for detecting pre-S1 antigen, comprising a surfactant and a suitable buffer selected from the group consisting of: Chaps, sulfobetain 8-18, Bridger 35, Triton X100, Tween series And/or ⁇ - ⁇ -CD, preferably selected from the group consisting of SB8, SB12, SB14, SB16, SB18, HP- ⁇ -CD, TWEEN20, TWEEN40, T-cut, T-wave N80, BRJ35, more preferably SB12, SB14, SB16, SB18, TWEEN80 o
  • the surfactant may be a combination of one or more of the above surfactants (2, 3, 4, 5), preferably SB16 + SB14, SB18 + SB14.
  • the concentration of the surfactant may range from SB14%: 1% to 10%; SB16: 0.5% to 1%; SB18: 0.5% to 1%; more preferably: SB14: 2% to 8%; SB16: 1 %; SB18: 1%; such as: l%SB14+0.5%SB16 or 1 Jane 4+0.5%SB18 D
  • the surfactant is selected from the group consisting of A-P lysate shown in Table 2, more preferably CM lysate and P lysate, more preferably E, F, G, H, J lysate, most preferably G lysate, H
  • the buffer is selected from the group consisting of citrate buffer, phosphate buffer, Tris buffer, amino acid buffer, acetate buffer, HEPES buffer, carbonate buffer.
  • the buffering agent may be one or more of the above buffers ( 2 , 3, 4 , A combination of 5).
  • the buffering agent is preferably a citrate buffer or a phosphate buffer.
  • the pH range and concentration range of the buffer may be, phosphate buffer: 10- 50 niM pH 6. 0-7. 0 preferably 20 mM pH 7.4; citrate buffer: 10- 50 mM pH 3 0-6. 6
  • Tri is buffer: 10-50 mM pH 7. 0-9. 0 preferably 20 mM pH ⁇ . 0; acetate buffer: 10-50 mM 0 ⁇ The pH value of 3. 0-6. 0.
  • the lysate may also contain one or more (eg 1, 2, 3, 4, 5, 6 - 10) other ingredients, such as reducing agents (eg DTT, DTE, ⁇ -mercaptoethanol), chelating Mixtures (eg EDTA), osmotic pressure regulators (eg salts such as NaCl; sucrose).
  • reducing agents eg DTT, DTE, ⁇ -mercaptoethanol
  • chelating Mixtures eg EDTA
  • osmotic pressure regulators eg salts such as NaCl; sucrose
  • the lysate may further comprise one or more components selected from the group consisting of: ⁇ -mercaptoethanol: 0. 01%-1%, preferably 0. 1%-0.
  • DTT l-30 mM, preferably 5- l QmM, more preferably 5 mM
  • DTE l-30 mM, preferably 5-10 mM, more preferably 5 mM
  • EDTA 5 mM-2 QraM is preferably 5 raM.
  • the lysate is selected from the group consisting of A-P lysates shown in Table 2, more preferably C-M lysate and P lysate, more preferably E, F, G, H, J lysates, most preferably G lysates, H lysate; or selected from the ⁇ lysate, hydrazine lysate, ⁇ lysate, hydrazine lysate shown in Table 6.
  • the invention also relates to a kit for detecting a hepatitis B virus pre-S1 antigen, comprising an agent for detecting a hepatitis B virus pre-S1 antigen according to the method of the present invention.
  • the kit of the present invention comprises: a first anti-HBV PreSl antibody and a second anti-HBV PreSl antibody that specifically bind to the hepatitis B virus pre-S1 antigen (the two antibodies may be the same or different and may be Monoclonal or polyclonal antibodies) and/or lysates of the invention for detecting pre-S1 antigen of hepatitis B virus.
  • the kit further comprises a reagent (for example, a detection reagent for detecting a quinone antibody) suitable for detecting the antigen-antibody reaction.
  • a reagent for example, a detection reagent for detecting a quinone antibody
  • at least the antibody An anti-HBV PreSl antibody is labeled by a signal generator or can bind efficiently to a signal generator.
  • the antibody is preferably a monoclonal antibody.
  • the anti-HBV PreSl antibody is selected from the group consisting of: 3H5, 7H11, 2A7, 4D1 6F1, 13G2 and 16F5, in particular 7H11 and 4D1 L
  • the kit comprises: (1) a solid phase support on which the first anti-HBV PreSl antibody is immobilized; and/or (2) the invention is used to detect hepatitis B virus a lysate of the pre-S1 antigen; and (3) a second anti-HBV PreSl antibody that is optionally labeled or unlabeled; and (4) a detectable reagent for detecting the ruthenium antibody And (5) optional buffers, washing solutions, and instructions for use (preferred instructions for use of the present invention for detecting the pre-S1 antigen of hepatitis B virus).
  • the solid phase carrier is a porous reaction plate (e.g., an enzyme plate, a chemiluminescent plate), and an anti-HBV PreSl antibody is immobilized in the well of the porous reaction plate.
  • a porous reaction plate e.g., an enzyme plate, a chemiluminescent plate
  • an anti-HBV PreSl antibody is immobilized in the well of the porous reaction plate.
  • the reagent is a reagent for detecting a pre-S1 antigen of hepatitis B virus described in the present invention .
  • the reagent comprises: a first anti-HBV PreSl antibody and a second antibody anti-HBV PreSl antibody that can specifically bind to the hepatitis B virus pre-S1 antigen (the two antibodies may be the same or different, and may be a monoclonal antibody or a polyclonal antibody) And/or the lysate of the present invention for detecting the pre-S1 antigen of hepatitis B virus, an optional reagent suitable for detecting the antigen-antibody reaction (for example, a detection reagent for detecting a second antibody); in particular: (1) a solid phase carrier on which a sputum anti-HBV PreSl antibody is immobilized; and/or (2) a lysate for detecting a pre-S1 antigen of hepatitis B virus; and (3) dispensable a second anti-HBV PreSl antibody labeled or unlabeled by the signal generator; and (4) a detectable reagent for detecting the second antibody; and
  • Lysis of the virus under mild conditions for example, without substantially inactivating the antibody, such as the pre-S1 antigen or the core antigen antibody
  • the virus is preferably a hepatitis B virus.
  • substantially means at least 70%, such as at least 80%, at least 90%, at least 95% of things happen.
  • not substantially denatured protein or “not substantially inactivated antibody” means that at least 70%, such as at least 80%, preferably at least 90%, or at least 95% of the protein or antibody is not denatured or inactivated.
  • the invention further relates to a mild viral lysate capable of not substantially denatured a protein (e.g., does not substantially inactivate an antibody, such as a pre-S1 antigen or a core antigen antibody), and lyses the virus.
  • a mild viral lysate capable of not substantially denatured a protein (e.g., does not substantially inactivate an antibody, such as a pre-S1 antigen or a core antigen antibody), and lyses the virus.
  • the invention also relates to a method of detecting a pre-S1 antigen and/or a core antigen of hepatitis B virus, comprising: under mild conditions that do not substantially denature a protein (eg, an antibody that does not substantially inactivate an antibody, such as a pre-S1 antigen or a core antigen antibody)
  • the virus is cleaved, preferably by using the lysate of the present invention for detecting the pre-S1 antigen of hepatitis B virus or the lysate of the combined detection of the present invention; and detecting the pre-S1 antigen and/or the core antigen of hepatitis B virus.
  • the detection of the hepatitis B virus pre-S1 antigen and the core antigen can be carried out by a known method or the method disclosed in the present invention.
  • the present invention relates to a method for detecting a pre-S1 antigen of hepatitis B virus, comprising: lysing a virus under mild conditions that do not substantially inactivate an anti-pre-S1 antigen antibody, preferably before detecting hepatitis B virus by the present invention
  • the lysate of the S1 antigen or the lysate of the combined detection of the present invention cleaves the virus; the hepatitis B virus pre-S1 antigen is detected.
  • the invention also relates to a method for detecting a hepatitis B virus core antigen, comprising: The virus is lysed under mild conditions in which the anti-core antigen antibody is inactivated, and the lysate which is combined with the detection of the present invention or the lysate used for detecting the core antigen in the embodiment is preferably used to lyse the virus; and the hepatitis B virus core antigen is detected.
  • the invention relates to a method of lysing a virus in a sample comprising cleavage of the virus using the viral lysate of the invention.
  • the present invention also provides a method of detecting a core antigen, which comprises lysing a virus in a sample using the above lysate.
  • the core antigen can then be detected using known methods or methods disclosed herein.
  • the invention also provides a method of detecting a pre-S1 antigen, the method comprising lysing a virus in a sample using the lysate described above.
  • the pre-S1 antigen can then be detected using known methods or methods disclosed herein.
  • the virus is preferably a hepatitis B virus.
  • the invention in another specific embodiment, relates to a method of lysing a virus, comprising lysing a virus using a lysate for detecting a pre-S1 antigen of hepatitis B virus of the invention; preferably the virus is hepatitis B virus.
  • the present invention also provides a method of detecting a pre-si antigen, which comprises lysing a virus in a sample using the above lysate.
  • the pre-S1 antigen can then be detected using known methods or methods disclosed herein.
  • Template preparation HBV DNA was extracted from a serum sample of chronic hepatitis B patients (No. 263) stored internally using the proteinase K-phenol: chloroform method. Multiple primers were designed, and the HBV gene was isolated by PCR and cloned into the PMD18-T vector (Takara NO. CK2401) for sequencing.
  • the assembled full-length HBV sequence has the GenBank accession number of AF233236, and the HBV strain has a genome length of 3068 bp, which is the adw2 subtype.
  • the cloned plasmid pT-PreS contains the full-length PreS1 and PreS2 genes. The sequence is as follows:
  • Atgcagtggaactccacaacattccaccaagctctgctagatcccagagtga ggggcctatattttcctgctggtggctccagttccggaacagtaaaccctgttcc aac ( PreS2 ).
  • the upstream primer 2147F1 (ATG AGA TCT CCT CTG GGA TTC TTT CC ) was designed, and the downstream primer 2147R1 (TTA GGA TCC ACC TCC ACC CGG ATT AAA GTC CCA ATC TGG ATT GTT ) was cloned with the plasmid pT- PreS as a template.
  • the gene sequence of the 21-47AA segment in the preS1 region was reverse-inserted into the pMD-18T plasmid pT-2147*1 as a starting plasmid, and the 21-47 fragment double tandem plasmid pT-2147*2 was first obtained.
  • the above six-fold tandem recombinant plasmid pT-2147*6 was digested with Bgl II and Eco I, and the gel recovery kit (Shanghai Huashen Bioengineering Co., Ltd. NO. W5212) was used to recover the target fragment 2147*6.
  • the expression plasmid PGEX-20T was digested with Ban I and Eco I, and the vector was recovered by ethanol precipitation.
  • the target fragment was ligated with PGEX-2QT by DNA ligase, and after digestion with the enzyme, a GST-2147*6 recombinant expression plasmid was obtained.
  • the constructed GST-2147*6 recombinant plasmid was transformed into the expression strain E. coli ER2566 for expression.
  • the expression results were analyzed by SDS-PAGE electrophoresis.
  • GST-2147*6 is present in a soluble form.
  • mice Six to eight weeks old female Balb/c mice were immunized, and each mouse was injected intramuscularly with 5 g of GST-2147*6 recombinant antigen (total volume 50 ⁇ l) emulsified with Freund's complete adjuvant. A secondary basal immunization was performed 15 days later by emulsification and intramuscular injection of the same amount of antigen with Freund's incomplete adjuvant. After 30 days, the tail vein was intensively injected with 5 ⁇ 8 of adjuvant-free antigen, and 72 hours after booster immunization, the mice were sacrificed, blood was collected, and spleen cell suspension was prepared by spleen (suspended in RPMI 16 40 medium). , cell counting plate cell count.
  • the cultured SP2/0 mouse myeloma cells were taken at a ratio of 1/6 to the spleen cells, mixed, centrifuged, and spleen cells were fused with mouse myeloma cells SP2/G using polyethylene glycol (PEG 1500).
  • Cell suspension with equal volume of feeder cells (Balb/c Mouse macrophages and thymocytes were mixed and placed in a 96-well cell culture plate (200 ⁇ l/well) in a 5% carbon dioxide incubator (ESPEC BNA-311) at 37 °C.
  • HT medium hyperxanthn, H
  • T + thymidine
  • RPMI 1640 GPI 1640
  • Semi-retained liquid change After 7 days, the peptide-coated plates were synthesized with HBV PreSl 21-47, and the resulting hybridoma cell supernatant culture medium in a 96-well cell culture plate was detected by the following enzyme-linked immunosorbent assay (ELISA). For cell clones that were positive for ELISA, cloning was performed using the limiting dilution method.
  • ELISA enzyme-linked immunosorbent assay
  • Coating buffer 0.05 mol/L carbonate buffer pH 9.6 (20.02 g Na 2 C0 3 , 2.52 g NaHC0 3 plus deionized water to 1 liter).
  • 100 ⁇ l of the coating solution was added to each well of a 96-well microtiter plate, coated at 37 ° C for 2 hours, and then transferred to 2 to 8 ° C for 16 hours.
  • PBST wash (20 mM PB 7.4, 150 mM NaCl, 0.1% Tween 20). Then add 200 ⁇ l of blocking solution (20 mM Na 2 HP0 4 /NaH 2 P0 4 buffer solution containing 20% calf serum and 1% casein, pH 7.4) to each well, and place at 37 ° C. 2 hours; remove the blocking solution. After drying, put it in an aluminum foil bag and store it at 2 ⁇ 8 °C.
  • Dilute solution (20 mM Na 2 HPO 4 /NaH 2 P0 4 buffer solution containing 20% calf serum and 1% casein with a pH value of 7.4) 1: 1000 dilution of horseradish peroxidase-labeled goat anti-mouse immunization Globulin (HRP-GAM Ig, DAKO), bath at 37 °C for 30 minutes; wash with PBST (20 mM PB7.4, 150raM NaCl, 0.1% Tween20) for 5 times, pat dry and add A and B Color liquid (provided by Beijing Wantai Biopharmaceutical Co., Ltd.) 50 ⁇ 1, 37 each.
  • HRP-GAM Ig, DAKO horseradish peroxidase-labeled goat anti-mouse immunization Globulin
  • the cloned hybridoma cells were collected, centrifuged to remove the supernatant, and serum-free medium (1640HT) was added to adjust the cell density to 2 ⁇ 10 5 - 2 10 6 /1111, 0.5 injection per mouse. Ml. After 7 to 10 days, the abdomen of the mice increased and ascites began to collect. After centrifugation at 3000 rpm for 15 minutes, the liquid in the middle clarified portion was aspirated, and the microporous membrane of 0 ⁇ 4 5 ⁇ was filtered and sterilized, and stored at - 20 ° C after dispensing.
  • serum-free medium (1640HT) was added to adjust the cell density to 2 ⁇ 10 5 - 2 10 6 /1111, 0.5 injection per mouse. Ml. After 7 to 10 days, the abdomen of the mice increased and ascites began to collect. After centrifugation at 3000 rpm for 15 minutes, the liquid in the middle clarified portion was aspirated, and the microporous membrane of 0 ⁇ 4 5 ⁇ was
  • the treated ascites was diluted with 0.02 mol/L PBS (81 ml 0.2 mol/L Na 2 HP0 4 , 19 ml 0.2 mol/L NaH 2 P0 4 , 20 ml 3 mol/L NaCl, added with water to 100 ml).
  • Saturated ammonium sulfate (concentration reached 50% saturation) was slowly added dropwise with stirring, at 2-8 ° C overnight. Centrifuge at 12000 rpm for 15 minutes at 4 ° C, discard the supernatant, and sink The lake was dissolved in PBS twice the volume of ascites. Saturated ammonium sulfate was slowly added dropwise with stirring to bring the ammonium sulfate concentration to 33 ° /.
  • the monoclonal antibody after completion of dialysis was purified by a DE52 column (Whatman No. 4057050) under FPLC, and a breakthrough peak was collected. At this time, the purity of the monoclonal antibody can reach more than 90%.
  • the purified monoclonal antibody was stored at - 20 °C.
  • HBV PreSl monoclonal antibody of the present invention Preliminary identification
  • a modified sodium periodate method is employed. Take 20mg 3H5 monoclonal antibody as an example: Accurately weigh 40mg of HRP, add 2ml of 0.2M pH 5.6 acetate buffer, dissolve and add 2ml of 0.06M NaI0 4 solution, react at room temperature for 20 minutes; add 0.16M ethylene glycol- 2ml of 10% NaCl solution, react at room temperature for 20 minutes, put the enzyme solution into the dialysis bag, dialyze overnight against 0.001M pH4.0 acetate buffer; add 2M pH9.6 carbonate buffer solution 0.8ml, add 3H5 mAb 20mg, stir reaction at 4 °C for 2 hours; add freshly prepared NaBH 4 solution (5mg / ral) 0.4ml, stir the reaction at 4 °C for 2 hours; add saturated (NH 4 ) 2 S0 4 The solution was 9.2 ml, and the reaction was stirred at 4 ° C for 30 minutes and centrifuged at 3500 ° C for 20 minutes.
  • the 3H5, 7H11, 2A7, 4D11, 6F1, 13G2 and 16F5 monoclonal antibodies were HRP-labeled by the above methods.
  • the HBV PreSl monoclonal antibody was diluted with H7.4 in 20 mM PB buffer (Na 2 HP0 4 /3 ⁇ 41 ⁇ 0 4 buffer, final concentration 20 mM, pH 7.4) to a final concentration of 5 ⁇ g/ml.
  • 100 ⁇ l of the coating solution was added to each well of a 96-well microtiter plate, and coated at 2 to 8 ° C for 6 to 24 hours.
  • PBST wash (20 mM PB 7.4, 150 mM NaCl, 0.1% Tween 20). Then, 200 ⁇ l of blocking solution (20 mM Na 2 HP0 4 /NaH 2 P0 4 buffer solution containing 20% calf serum and 1% casein, pH 7.4) was added to each well, and placed at 37 ° C to block 2 Hour; remove the blocking solution. After drying, it is placed in an aluminum foil bag and stored at 2 ⁇ 8 °C.
  • HBV virus positive (PCR detection) clinical serum samples HBV virus negative clinical serum samples.
  • the plate after the reaction was washed 5 times with PBST washing solution (20 raM PB7.4, 150 mM NaCl, 0.1% Tween 20), and the enzyme dilution (containing 20% calf serum and 1% casein) was added to each well.
  • the plate after the reaction was washed 5 times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), 50 ⁇ l of each of A and B coloring agents was added to each well, and placed in a 37 ° C incubator reaction. 15 minutes.
  • Stop solution 2 MH 2 S0 4 50 ⁇ l was added to each well of the reaction plate, and the ⁇ 450/630 value of each well was measured on a microplate reader.
  • the HBV PreSl monoclonal antibody 7H11 was diluted with ⁇ 4 in 20 ⁇ M MPB buffer to a final concentration of 5 ⁇ g/ml.
  • 100 ⁇ l of the coating solution was added to each well of the 96-well microtiter plate, and coated at 2 to 8 ° C for 16 to 24 hours.
  • HP ⁇ - ⁇ -CD, hydroxypropyl beta-cyclodextrin (Hydroxypropyl- ⁇
  • HBV virus positive Clinical serum samples, normal human serum samples.
  • the plate after the reaction was washed 5 times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), and the enzyme dilution (containing 20% calf serum and 1% casein) was added to each well.
  • PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), and the enzyme dilution (containing 20% calf serum and 1% casein) was added to each well.
  • the plate after the reaction was washed 5 times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), 50 ⁇ l of each of A and B coloring agents was added to each well, and placed in a 37 ° C incubator reaction. 15 minutes.
  • Stop solution (2M H 2 S0 4) 50 ⁇ 1 was added to each well of the reaction plate, and the ⁇ 45 of each well was detected on a microplate reader. /63 . value.
  • the positive sample detection titer refers to the dilution of the positive specimens until they are not detected as positive (the positive judgment is the negative sample A 45Q/63 corresponding to the same lysis condition.
  • x 2.1 is Cutoff, greater than or equal to Cutoff Positive, and vice versa is negative, the maximum dilution factor that can be detected positive is the detection titer of the positive specimen.
  • DTAC dodecyltrimethylammonium chloride
  • DTT dithiothreitol
  • DTE dithioerythritol
  • the 7H11 monoclonal antibody, anti-HBc monoclonal antibody G8 was diluted with buffer at pH7.4 20mMPB final concentration of the other points is 1 J 5 ⁇ g / ral and 4 ⁇ g /ml.
  • 100 ⁇ l of the coating solution was added to each well of the 96-well microtiter plate, and coated at 2 to 8 ° C for 16 to 24 hours.
  • PBST wash (20 mM PB 7.4, 150 raM NaCl, 0.1% Tween 20). Then, 200 ⁇ l of blocking solution (20 mM Na 2 HP0 4 /NaH 2 P0 4 buffer solution containing 20% calf serum and 1% casein with a pH value of 7.4) was added to each well, and placed at 37 ° C to block 2 Hour; remove the blocking solution. After drying, it is placed in an aluminum foil bag and stored at 2 ⁇ 8 °C.
  • HBV virus positive (PCR detection) clinical serum samples HBV virus negative clinical serum samples.
  • the plate after the reaction was washed 5 times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), and the enzyme dilution (containing 20% calf serum and 1% casein) was added to each well.
  • the value of 7.4 is ⁇ 2 0mM Na 2 HP0 4 / NaH 2 P0 4 buffer solution) 1: 1000 dilution 4D11 enzyme (HRP) labeled monoclonal antibody and an enzyme (HRP) labeled anti HBc monoclonal antibody CZ (Xiamen Bosheng Biotechnology Co., Ltd., NO.04042901) was placed in a 37 °C incubator for 30 minutes.
  • the plate after the reaction was washed 5 times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), 50 ⁇ l of each of the AB developers was added to each well, and placed in a 37 ° C incubator for 15 minutes. .
  • Stop solution (2M H 2 S0 4 ) 50 ⁇ 1 was added to each well of the reaction plate, and the ⁇ 45 of each well was detected on a microplate reader. /63 . value.
  • Lysisl 2.426 0.031 78 lysis2 1.446 0.044 33 lys is 3 1.574 0.046 34 lysis4 1.521 0.059 26 lysis5 0.071 22 lysis6 0.037 41 lys is7 2.765 0.286 10 lys is8 1.534 0.053 29 lysis9 1.409 0.031 45 lysislO 2.428 0.011 221 lys isll 0.019 70 lysisl2 1.468 0.016 92 Lysisl3 3.468 0.013 267 lysisl4 0.018 87 lys isl5 1.344 0.02 67 lysisl6 2.441 0.012 203 Lysisl7 2.444 0.015 163 lysisl8 2.422 0.
  • lysislO, lysisl3, lysisl6, lysisl8 are the best lysate formulations.
  • Example 6 Analysis of the effect of adding a lysis system to the detection of a conventional hepatitis B virus pre-S1 antigen
  • the traditional hepatitis B virus pre-S1 antigen detection mode is also generally detected by double antibody sandwich ELISA, but anti-HBsAg (signal-labeled anti-HBsAg) is used as a capture antibody (4 antibody) and paired with signal-labeled anti -PreSl (anti-PreSl) as the corresponding reporter antibody (capture antibody), and the detection sensitivity of the pre-S1 antigen is improved by the high abundance of HBsAg on the hepatitis B virus particles.
  • anti-HBsAg signal-labeled anti-HBsAg
  • anti-PreSl anti-PreSl
  • HBV PreSl monoclonal antibody 4D11 was diluted with 20mMPB buffer at pH 7.4 to a final concentration of 5 g/ml; the purchased anti-HBsAg (Xiamen Bosheng Biotechnology Co., Ltd.) was diluted with 20mMPB buffer at pH 7.4, and finally The concentration is 3 g/ml.
  • the coated A and B plates were washed once with PBST washing solution (0.1% Tween 20 in PBS). Then, 200 ⁇ l of blocking solution (20 mM Na 2 HP0 4 /NaH 2 P0 4 buffer solution containing 20% calf serum and 1% casein) was added to each well, and placed in 37 ° C is blocked for 2 hours; the blocking solution is removed. After drying, put it in an aluminum foil bag and store it at 2 ⁇ 8 °C.
  • the alpha lysate is: 2 % Chaps+5raMEDTA+20raMPB7. 4
  • the ⁇ lysate is: 2 HP+20mMPB7. 4
  • the y lysate is: 2 % SB8 + 5mMEDTA+20mMPB7. 4
  • the ⁇ lysate is: 2 % SB12 + 5raMEDTA+20mMPB7. 4
  • the ⁇ lysate is: 2 % SB14 + 5mMEDTA+20mMPB7. 4
  • the sputum lysate is: 1 % SB16 + 5raMEDTA+20raMPB7. 4+0. 5 SB14 ⁇ lysate: 1 % SB18 + 5mMEDTA+20raMPB7. 4+0. 5% SB14 ⁇ Lysate: 2 % SB14+5mMEDTA+ 20mMPB7. 4
  • I lysate is: 2 % Tw80 + 5mMEDTA + 20raMPB7. 4
  • the ⁇ lysate is: 2 % Tw60+5mMEDTA+20raMPB7. 4
  • the lambda lysate is: 2 % Tw40 + 5mM EDTA + 20mMPB7. 4
  • the ⁇ lysate is: 2 % Tw20+5raMEDTA+20mMPB7. 4
  • V lysate is: 2% TX100+5mMEDTA+20mMPB7. 4
  • the sputum lysate is: 2 % BRJ 35+5mMEDTA+20mMPB7. 4
  • HBV virus positive (PCR detection) clinical serum samples normal human serum samples.
  • the A plate and the B plate after the reaction were washed 5 times with a PBST washing solution, and the A plate was added with an enzyme dilution (a 20 mM Na containing 20% calf serum and 1% casein) having a pH value of 7.4.
  • an enzyme dilution (a 20 mM Na containing 20% calf serum and 1% casein) having a pH value of 7.4.
  • HRP-labeled ant i-HBsAg 1: 10000
  • B plate per well added ⁇ ⁇ ⁇ enzyme diluted solution (containing 20% calf serum and 1% casein)
  • the ⁇ value of 7.4 is 20 mM a 2 HP0 4 /NaH 2 P0 4 buffer solution)
  • HRP-labeled 4D11 (1 : 1000 ) was placed in a 37 ° C incubator for 30 minutes.
  • the reacted A plate and B plate were washed 5 times with PBST washing solution, 50 ⁇ l each of A and B color developing agents were added to each well, and placed in a 37 C incubator for 15 minutes.
  • Stop solution 50 ⁇ l was added to each well of the reacted A plate and B plate, and A 45 of each well was detected on a microplate reader. /63Q value.
  • Positive specimens detect titer means that the positive specimens are diluted by dilution until they are not detected as positive (positive judgments are negative samples corresponding to the same lysis condition ⁇ 45. /63 ⁇ 2. 1 is Cutoff, greater than or equal to Cutoff If it is judged to be positive, otherwise it is judged to be negative, the maximum dilution factor at which the specimen can be detected positive is the detection titer of the positive specimen.
  • ⁇ lysate, sputum lysate, ⁇ lysate, and sputum lysate are very helpful for improving the sensitivity of the traditional hepatitis B virus pre-S1 antigen detection system.
  • Example 7 Preparation of a diagnostic kit for detecting hepatitis B virus pre-S1 antigen (HBV PreSl) and its detection method (enzyme-linked immunosorbent assay).
  • Hepatitis B virus pre-S1 antigen diagnostic kit for HBV PreSl monoclonal antibody 7H11, 4D11 (other anti-pre-S1 antigen antibody can also be used) The composition and preparation are as follows:
  • a suitable concentration of the coating solution was prepared as in Example 3 and coated on the ELISA plate.
  • the HRP-labeled 4D11 was formulated into a suitable concentration (1:1000) of the enzyme-labeled reagent, and the dilution was 20% Na 2 HPO 4 /NaH 2 P0 4 having a pH of 7.4 with 20% calf serum and 1% casein. Buffer solution.
  • the diagnostic kit for detecting hepatitis B virus pre-S1 antigen (HBV PreSl) is as follows:
  • Lysis Pipette 50 ⁇ l of sample lysate (as in Example 4, F or G or ⁇ lysate) using a pipette.
  • washing After incubation, remove the sealing film, blot the liquid in the well, wash it with washing solution (lx) for 5 times, soak for 30-60 seconds each time.
  • Washing After incubation, remove the sealing film, blot the liquid in the well, wash it with washing solution (lx) for 5 times, soak for 30-60 seconds each time.
  • Add enzyme Add 100 ⁇ l of enzyme labeling reagent to the corresponding wells, except for blank wells.
  • washing After incubation, remove the sealing film, blot the liquid in the well, wash it with washing liquid (lx) for 5 times, soak for 30 ⁇ 60 seconds each time.
  • Color development add color developer A and B solution to each well 50 ⁇ 1, gently shake and mix, seal the plate with sealing film and place 37 soil at 1°C for 15 minutes.
  • Termination Add stop solution (2M H 2 S0 4 ) 50 ⁇ 1 to each well and mix gently by shaking.
  • Measurement Set the microplate reader wavelength to 450/630nm and measure the 0D value of each well.
  • Negative result (S/C.0. ⁇ 1): The absorbance value of the sample is less than the Cut Off value, indicating that HBV PreSl antigen was not detected in the sample.
  • composition and preparation of the hepatitis B virus core antigen diagnostic kit encompassed by the present invention are as follows:
  • Anti-HBc monoclonal antibody G8 (Xiamen Bosheng Biotechnology Co., Ltd. N0.050824) Dilute with 20 mM PB buffer, pH 7.4, to a final concentration of 4 ⁇ g/ml.
  • the purchased HRP-labeled anti-HBc monoclonal antibody CZ (Xiamen Bosheng Biotechnology Co., Ltd., ⁇ 0 ⁇ 04042901) was formulated into a suitable concentration (1:1 1000 dilution) of the enzyme standard reagent, and the dilution was 20% calf serum and 1 A 20 mM Na 2 HP0 4 /NaH 2 P0 4 buffer solution having a pH of 7.4 of casein.
  • Lysis Pipette 50 ⁇ l sample into the corresponding wells (see lysislO, lysisl3, lysisl6, lysisl8 in Example 5).
  • washing After the incubation, the sealing film is peeled off, the liquid in the hole is sucked out, and washed with washing liquid for 5 times, soaking for 30 to 60 seconds each time.
  • Color development Add 50 ⁇ l of each of the developer A and B solution, gently shake and mix. Seal the plate with a sealing film and set it at 37 ⁇ 1 °C for 15 minutes.
  • Termination Add stop solution (2M H 2 S0 4 ) 50 ⁇ 1 to each well and mix gently by shaking.
  • Measurement Set the microplate reader wavelength to 450/630nm and measure the 0D value of each well.
  • Negative result (S/C.0. ⁇ 1): The absorbance value of the sample is less than the Cut Off value, which means that HBcAg is not detected in the sample.
  • composition and preparation of the kit for the simultaneous detection of hepatitis B virus pre-S1 and core antigens (enzyme-linked immunosorbent assay) included in the present invention are as follows:
  • 7H11 other anti-pre-S1 antigen antibodies can also be used
  • purchased anti-HBc monoclonal antibody G8 Xiamen Bosheng Biotechnology Co., Ltd. No. 050824
  • HBc antibody is formulated into a suitable concentration (1:1000 dilution) of the enzyme standard reagent. The dilution is 20raM Na 2 HP0 4 /NaH 2 P0 4 with a pH of 7.4 containing 20% calf serum and 1% casein. Flush solution.
  • hepatitis B virus pre-S1 antigen and core antigen HBcAg
  • washing After the incubation, the sealing film is peeled off, the liquid in the hole is sucked out, and washed with washing liquid for 5 times, soaking for 30 to 60 seconds each time.
  • Color development Add 50 ⁇ l of each of the developer A and B, and mix gently by shaking. After sealing with a sealing film, place 37 °C at 1 °C for 15 minutes.
  • Termination Add stop solution (2M H 2 S0 4 ) 50 ⁇ 1 to each well and mix gently by shaking.
  • Measurement Set the microplate reader wavelength to 450/630nm and measure the 0D value of each well.
  • Negative result (S/C.0. ⁇ 1): The absorbance value of the sample is less than the Cut Off value, indicating that HBV PreSl and HBcAg were not detected in the sample.
  • Example 10 Combined detection of pre-hepatitis B virus S1 antigen and core antigen (nucleic acid-associated antigen) and hepatitis B virus nucleic acid detection, and similar pre-S1 antigen detection parallel comparison
  • Hepatitis B virus pre-S1 antigen diagnostic kit (enzyme-linked immunosorbent assay) was prepared as in Example 7; Hepatitis B virus core antigen diagnostic kit preparation (enzyme-linked immunosorbent assay) as in Example 8; combined detection of hepatitis B Pre-S1 antigen and core antigen diagnosis
  • the preparation of the break kit (enzyme-linked immunosorbent assay) was as in Example 9.
  • the commercial hepatitis B virus pre-S1 antigen diagnostic kit was purchased from Shanghai Fosun Long March Medicine Technology Co., Ltd. (NO. 3150480) and Shanghai Alpha Biotechnology Company.
  • Hepatitis B virus nucleic acid quantitative PCR detection reagent purchased from Shenzhen Piki Bioengineering Co., Ltd. ( ⁇ 0 ⁇ 06010121), Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Daan Gene Co., Ltd. (NO. #DA-B051) .
  • Hepatitis B virus "two-and-a-half" enzyme-linked immunosorbent assay was purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd. (WB2196), Yingke Xinchuang (Xiamen) Technology Co., Ltd. (IPT2100K IPT21201, IPT2130K IPT2140U IPT21501) and Shanghai Industry Branch Hua Biotechnology (NO. 267 ⁇ 265 ⁇ 266 ⁇ 263) Co., Ltd.
  • Each serum sample was subjected to quantitative PCR detection of hepatitis B virus nucleic acid, and was carried out in parallel using three different manufacturer reagents (Shenzhen Piki Bioengineering Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Daan Gene Co., Ltd.). The tests, test procedures and results are judged in strict accordance with the respective reagent instructions. The qualitative judgment of the nucleic acid of hepatitis B virus is based on the same test results of the two reagents.
  • the qualitative judgment of hepatitis B virus HBsAg, HBsAb, HBeAg, HBeAb, HBcAb is based on the same test results of the two reagents. Standard
  • Each serum sample was subjected to similar commercialization of hepatitis B virus pre-S1 antigen enzyme-linked immunoassay 1 1 test, using two different manufacturer reagents (Shanghai Fosun Changzheng Pharmaceutical Technology Co., Ltd. and Shanghai Alpha Biotechnology Co., Ltd.) for parallel detection.
  • the test procedures and results are judged in strict accordance with the instructions of each reagent.
  • hepatitis B virus pre-S1 antigen detection For each serum sample, the hepatitis B virus pre-S1 antigen detection, hepatitis B virus core antigen detection, and combined detection of hepatitis B virus pre-S1 disclosed in the present invention, as disclosed in Example 6, Example 7, and Example 8, respectively.
  • Antigen and core antigen (collectively referred to as nucleic acid related antigens). The test procedures and results are judged in strict accordance with the description of the corresponding examples.
  • the invention is a combo ELISA pre-SI detection reagent
  • Nucleic acid positive 110 107 3 103 7 70 40 66 44 94 16 Nucleic acid Yin 'f raw 6 0 6 0 6 0 6 0 6 0 6 0 6
  • Domestic 2 Hepatitis B virus pre-S1 antigen diagnostic kit produced by Shanghai Fosun Changzheng Pharmaceutical Technology Co., Ltd. (ELISA)
  • HBV combo ELISA hepatitis B virus pre-S1 antigen and core antigen detecting reagent
  • PreSl The hepatitis B virus pre-SI antigen detecting reagent disclosed in the present invention, as in Example 6
  • Core The hepatitis B virus core antigen detecting reagent disclosed in the present invention, as in Example 7
  • Sensitivity total number of positives detected / true positive (nucleic acid positive) total
  • the hepatitis B virus pre-S1 antigen detecting reagent of Example 7 has a greater sensitivity in sensitivity than the pre-commercial S1 antigen detecting reagent, because Example 7 is as
  • the hepatitis B virus pre-S1 antigen detection reagent is different from the traditional commercial reagents in that it uses the dual pre-S1 antibody sandwich detection mode instead of relying on traditional detection of the entire LHBs (ie, HBsAg-mediated detection on LHBs, Using a specific antibody against HBsAg as a capture antibody or reporter antibody, and then paired with a specific antibody against PreSl), an independent pre-S1 antigen detection reagent is formed, and the virus Dane particles are not caused by the mutation of HBsAg on LHBs.
  • the above S1 antigen is missed and has a high correlation with hepatitis B virus nucleic acid.
  • the data in the table is S/C. O,, S/C. 0. > 1 is a positive result, S/C. 0 ⁇ ⁇ 1 is a negative result; the virus content is detected by fluorescence quantitative PCR.
  • Example 11 Combined detection of hepatitis B virus pre-S1 antigen and core antigen (nucleic acid-related antigen) and hepatitis B virus "two pairs and a half, (HBsAg, HBsAb, HBeAg, HBeAb, HBcAb) Comparative analysis oo
  • Hepatitis B virus nucleic acid quantitative PCR detection reagents were purchased from Shenzhen Piki Bioengineering Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Daan Gene Co., Ltd.
  • Hepatitis B virus "Two-and-a-half" enzyme-linked immunosorbent assay was purchased from Beijing Wantai Biopharmaceutical Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Shanghai Industrial Kehua Biotechnology Co., Ltd.
  • Each serum sample was subjected to quantitative PCR detection of hepatitis B virus nucleic acid, and was carried out in parallel using three different manufacturer reagents (Shenzhen Piki Bioengineering Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Daan Gene Co., Ltd.). The tests, test procedures and results are judged in strict accordance with the respective reagent instructions. The qualitative judgment of the nucleic acid of hepatitis B virus is based on the same test results of the two reagents.
  • Hepatitis B virus "two-and-a-half" enzyme-linked immunosorbent assay for each serum sample using three different manufacturers reagents (Beijing Wantai Biological Pharmaceutical Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Shanghai Industrial Division Hua Biotechnology Co., Ltd.) performs parallel testing, and the test procedures and results are judged in strict accordance with the specifications of each reagent.
  • the qualitative judgment of hepatitis B virus HBsAg, HBsAb, HBeAg, HBeAb, HBcAb is based on the same results of the two reagents.
  • Screening PCR confirms hepatitis B virus nucleic acid-positive, "two-and-a-half" HBsAg-negative and semi-abnormal model specimens, and combines the pre-hepatitis B virus pre-S 1 antigen and core antigen as disclosed in Example 9 (collectively For the detection of nucleic acid related antigens).
  • the test procedure and the result judgment were carried out in strict accordance with the description of Example 9.
  • Hepatitis B virus nucleic acid quantitative PCR detection reagents were purchased from Shenzhen Piki Bioengineering Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Daan Gene Co., Ltd.
  • Hepatitis B virus "Two-and-a-half" enzyme-linked immunosorbent assay was purchased from Beijing Wantai Biopharmaceutical Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Shanghai Industrial Kehua Biotechnology Co., Ltd.
  • Each serum sample was subjected to quantitative PCR detection of hepatitis B virus nucleic acid, and was carried out using three different manufacturer reagents (Shenzhen Piki Bioengineering Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Daan Gene Co., Ltd.). Parallel testing, testing procedures and results are judged in strict accordance with the respective reagent instructions. The negative qualitative judgment of the hepatitis B virus nucleic acid was negative for the three reagents and negative for the quantitative PCR.
  • Example 5 Screening quantitative PCR confirms hepatitis B virus nucleic acid negative, "two-and-a-half" HBsAg positive, and performs the detection of the combined hepatitis B virus pre-S1 antigen and core antigen (collectively referred to as nucleic acid-related antigen) as disclosed in Example 5 of the present invention. .
  • the test procedure and the results were judged in strict accordance with the description of Example 5.
  • These specimens were subjected to partial nested PCR (see: Mar i e- Anne Pe t i t et al., JMV 2001).
  • a diagnostic kit for the combined detection of hepatitis B virus pre-S1 antigen and core antigen was prepared as in Example 9.
  • Example 9 Each of the sera was tested for the combined hepatitis B virus pre-S1 antigen and core antigen (collectively referred to as HBV combo ELISA) as disclosed in Example 9. The test procedures and results were judged in strict accordance with the description of Example 9.
  • the detection reagents associated with the hepatitis B virus pre-S1 antigen and the core antigen are particularly specific.
  • Example 14 Combined detection of pre-hepatitis B Application of S1 antigen and core antigen in chemical luminescence
  • composition and preparation of the combined detection kit for pre-S1 and core antigen of hepatitis B virus (chemiluminescence method) included in the present invention are as follows:
  • Dilute G8 with 7H11 (others may be used) and purchased anti-HBc monoclonal antibody as in Example 3 (Xiamen Bosheng Biotechnology Co., Ltd. ⁇ 0 ⁇ 050824) (Other anti-S1 antigens and anti-core antigen antibodies may also be used) Coated with a suitable concentration (final concentration of 5 ⁇ g/ml and 4 ⁇ g/ml, respectively) on a chemiluminescent plate (Xiamen Yixinmei Co., Ltd., NO.C0004).
  • lysislO or lysisl3 or lysisl6 or lysisl8 As shown in Example 5, lysislO or lysisl3 or lysisl6 or lysisl8.
  • 4D11-HRP was purchased with HRP-labeled anti-HBs monoclonal antibody, HRP-labeled anti-HBc monoclonal antibody CZ (Xiamen Bosheng Biotechnology Co., Ltd., NO.04042901) (Other anti-pre-treatments can also be used)
  • the S1 antigen and the antibody against the core antigen are formulated to a suitable concentration (1: 10,000 enzyme standard reagent, and the dilution is 20 mM Na 2 HP0 4 /NaH 2 with an H value of 7.4 containing 20% calf serum and 1% casein. P0 4 . Flush solution.
  • hepatitis B virus pre-S1 antigen and core antigen (HBcAg) diagnostic kit chemiluminescence method
  • Lysis Pipette 50 ⁇ l of sample lysate into the corresponding wells using a pipette.
  • washing After the incubation, the sealing film is peeled off, the liquid in the hole is sucked out, and washed with washing liquid for 5 times, soaking for 30 to 60 seconds each time.
  • Luminescence Add 100 ⁇ l of illuminant per well to the chemiluminometer to determine the luminescence intensity of each well.
  • the combined detection of hepatitis B virus pre-S1 and core antigen methods has a high correlation with virus content. It is possible to estimate the nucleic acid load of a sample by using a combined detection of pre-S1 and core antigens in combination with hepatitis B virus.
  • Example 15 Detection of HBsAg mutant strain by a method for jointly detecting pre-S1 antigen and core antigen of hepatitis B virus
  • Kit preparation Preparation of a Hepatitis B Virus Pre-S1 Antigen and Core Antigen Diagnostic Kit (Enzyme-Linked Immunoassay) As described in Example 9, a kit prepared by a similar method can also be used.
  • Hepatitis B virus nucleic acid quantitative PCR detection reagents were purchased from Shenzhen Piki Bioengineering Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Daan Gene Co., Ltd.
  • Hepatitis B virus "Two-and-a-half" enzyme-linked immunosorbent assay was purchased from Beijing Wantai Biopharmaceutical Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Shanghai Industrial Kehua Biotechnology Co., Ltd.
  • Each serum sample was subjected to quantitative PCR detection of hepatitis B virus nucleic acid, and was carried out in parallel using three different manufacturer reagents (Shenzhen Piki Bioengineering Co., Ltd., Yingke Xinchuang (Xiamen) Technology Co., Ltd. and Daan Gene Co., Ltd.). The tests, test procedures and results are judged in strict accordance with the respective reagent instructions. The qualitative judgment of the nucleic acid of hepatitis B virus is based on the same test results of the two reagents.
  • the qualitative judgments of hepatitis B virus HBsAg, HBsAb, HBeAg, HBeAb, HBcAb are based on the same test results of the two reagents.
  • the S region "A" epitope gene was amplified for sequencing.
  • the gene amplification method is as follows: Serum DNA by proteinase K method Extraction, respectively, using appropriate primers for nested PCR PCR reaction procedures are 95 ° C for 10 minutes, 95 ° C for 40 seconds, 55 ° C for 40 seconds, 72 ° C for 40 seconds, 35 cycles (two rounds of 25 cycles), 72 ° C10 min, S region outer primer aFl: 5'-GTCT GCGGCGTTTTATC-3 / and aRl: 5' -ACAGTGGGGGAAAGC-3 / , inner primer aF2 : S'-TGCC CGTTTGTCCTCTA-3 / and aR2 : 5'-AGA AACGGRCTGAGGC-3 '. Primer synthesis and sequencing were performed by Shanghai Yingjun Company.
  • the serum samples of the HBsAg "A" epitope mutation are subjected to the detection of the combined hepatitis B virus pre-S1 antigen and core antigen (called nucleic acid-associated antigen) as disclosed in the present invention.
  • the test procedure and the result judgment were carried out in strict accordance with the description of Example 9.
  • Table 13 Test results and analysis of HBsAg mutants by combined detection From the results of Table 13, the 13 samples of HBsAg reagent were negative, and the sequence analysis showed that the serum samples of patients with "A" epitope mutations were associated with The detection can detect 11 of them, and the detection rate is about 77°/. . This is because the core antigen HBcAg is usually encapsulated inside the HBV, which avoids the selection pressure of the body humor immunity and is therefore conservative. Although the pre-S1 antigen is easily mutated, its AA21-47 peptide (especially its anterior segment) is one of the binding sites of HBV and hepatocytes, and is quite conservative in each genotype.
  • the high-affinity monoclonal antibody against the 21-47 peptide of the pre-S1 antigen of HBV in the two forests was used as a capture antibody and an antibody, and a highly efficient HBcAg detection system was used to simultaneously detect the pre-S1 antigen and the core antigen, and effectively utilized the same.
  • the complementarity of the two antigens compensates for possible missed detections due to single antigenic variation or low titers.
  • the combined detection test is completely independent of HBsAg, and the target molecule detected is more conservative than HBsAg, so the combined detection can detect HBsAg-negative HBV infection specimens.

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Abstract

L'invention concerne un procédé pour détecter conjointement l'antigène preS1 et l'antigène de noyau de VHB, un coffret d'expérimentation, un substrat solide et une solution de lyse de virus de ceux-ci. Un procédé en sandwich à double anticorps est utilisé pour détecter l'antigène preS1 et l'antigène de noyau de VHB.
PCT/CN2007/002000 2006-06-27 2007-06-27 Procédé de détection conjointe de l'antigène pres1 et de l'antigène de noyau de vhb, coffret d'expérimentation, substrat solide et solution de lyse de virus Ceased WO2008003236A1 (fr)

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CN117089651A (zh) * 2023-08-21 2023-11-21 湖南郴新生物技术有限公司 一种乙肝病毒检测试剂盒及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1197927A (zh) * 1997-04-25 1998-11-04 武汉市第七医院 一种直接检测血清乙肝病毒核心抗原试剂
CN1512178A (zh) * 2002-12-27 2004-07-14 杜凤鸣 定量法乙肝病毒前s1抗原酶联免疫测定试剂盒及制备方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100345463B1 (ko) * 1998-11-19 2003-01-08 주)녹십자 B형간염바이러스의표면항원프리-s1에대한인간화항체및이의제조방법
CN100501399C (zh) * 2000-05-15 2009-06-17 上海新新医学生物工程公司 乙肝病毒前s1抗原酶联免疫测定试剂盒及制备方法
CN1131432C (zh) * 2000-11-09 2003-12-17 武汉市第七医院 一种直接检测血清乙肝病毒核心抗原的方法和试剂盒
CN100385237C (zh) * 2001-02-01 2008-04-30 上海新新医学生物工程公司 乙肝病毒前s1抗体酶免测定试剂盒及制备方法
EP1281761A1 (fr) * 2001-07-27 2003-02-05 Institut National De La Sante Et De La Recherche Medicale (Inserm) Polypeptides synthetiques derivées du proteine pre-S1 de Hepatitis B virus et utilisations associées

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1197927A (zh) * 1997-04-25 1998-11-04 武汉市第七医院 一种直接检测血清乙肝病毒核心抗原试剂
CN1512178A (zh) * 2002-12-27 2004-07-14 杜凤鸣 定量法乙肝病毒前s1抗原酶联免疫测定试剂盒及制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FAN Y. ET AL.: "Pre-S1 Ag of Hepatitis B Virus: Its Determination and Clinical Significance", CHINESE JOURNAL OF NOSOCOMIOLOGY, vol. 15, no. 3, 2005, pages 292 - 294 *
ZHANG G. ET AL.: "A study of ELISA clinical applications Significance for detecting serum HBcAg", JIANGXI JOURNAL OF MEDICAL LABORATORY SCIENCES, vol. 21, no. 5, October 2003 (2003-10-01), pages 373 - 374 *
ZHAO Y. ET AL.: "Study on the Immunogenicity of Fusion Protin CS1 of Hepatitis B Virus", CHINESE JOURNAL OF VIROLOGY, vol. 17, no. 4, December 2001 (2001-12-01), pages 333 - 337 *

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EP4092050A4 (fr) * 2020-01-19 2024-03-13 Xiamen Innodx Biotech Co., Ltd Procédé et anticorps pour la détection de hbcag
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