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WO2008053900A1 - Procédé d'analyse immunologique à sensibilité élevée et réactif d'analyse immunologique pour le virus de l'hépatite b - Google Patents

Procédé d'analyse immunologique à sensibilité élevée et réactif d'analyse immunologique pour le virus de l'hépatite b Download PDF

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Publication number
WO2008053900A1
WO2008053900A1 PCT/JP2007/071149 JP2007071149W WO2008053900A1 WO 2008053900 A1 WO2008053900 A1 WO 2008053900A1 JP 2007071149 W JP2007071149 W JP 2007071149W WO 2008053900 A1 WO2008053900 A1 WO 2008053900A1
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WIPO (PCT)
Prior art keywords
antibody
antigen
hepatitis
virus
saponin
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PCT/JP2007/071149
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English (en)
Japanese (ja)
Inventor
Naoko Matsubara
Yasuhiro Sugamata
Osamu Kusano
Noriko Shirata
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Advanced Life Science Institute Inc
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Advanced Life Science Institute Inc
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Priority to JP2008542142A priority Critical patent/JP5332011B2/ja
Publication of WO2008053900A1 publication Critical patent/WO2008053900A1/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
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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

Definitions

  • the present invention relates to a highly sensitive immunological analysis method for hepatitis B virus and a reagent for immunological analysis.
  • hepatitis B virus hereinafter referred to as “HBV”
  • HBV hepatitis B virus
  • HBV is a causative virus for post-transfusion hepatitis and is transmitted by blood transfusion during surgery. Therefore, it is extremely important to diagnose the presence or absence of blood virus infection by screening blood for transfusion.
  • Such infection diagnosis methods include immunological analysis methods such as antibody test methods for detecting antibodies against viruses present in test samples, and antigen test methods for detecting antigens such as viruses, or viruses. There are gene detection methods for detecting these genes.
  • Gene detection methods include the NAT and DNA probe methods, which are currently widely used in clinical practice.
  • NAT test methods such as PCR and TMA are highly sensitive detection methods for detecting gene fragments.
  • the processing time required for the method is 2 hours, and multiple operation steps are required. This is very complicated.
  • the complexity of the operation increases the chances of contamination with other positive specimens, increasing the possibility of generating false positive specimens.
  • skill of the operator is required to obtain a stable quantitative value.
  • the development of automated equipment has been used to prevent contamination and shorten the DNA extraction processing time.However, since it still requires expensive equipment, it is generally popular except for facilities that process samples in large quantities. Not done.
  • the DNA primer must match the target gene, it is necessary to use several types of primers, and the cost per test is higher than the immunological analysis method.
  • an antibody test method improves an antigen for detecting an antibody
  • an antigen test method improves an antibody that binds to an antigen. I have been.
  • attempts have been made to increase sensitivity by improving the chromogenic substrate and the luminescent substrate.
  • Patent Document 1 Japanese Patent No. 3468763
  • Patent Document 2 Japanese Patent No. 3396231
  • the conventional hepatitis B virus immunological analysis method uses an immunological analysis method that is less sensitive than the gene detection method. There was a case of virus contamination. Such false negative blood can be used for blood transfusions, which can cause post-transfusion infection.
  • the present inventors have conducted extensive research on increasing the sensitivity of the hepatitis B virus immunoassay in order to reduce false negatives of such an immunological assay. It was found that the sensitivity of the immunological analysis method is increased by performing an antigen-antibody reaction in which an antigen and an antibody are contacted in the presence of saponin, bile acid, or an anionic surfactant.
  • the present invention is based on these findings.
  • the present invention includes a sandwich method comprising a step of contacting a hepatitis B virus antigen and an anti-hepatitis B virus antibody in the presence of a saponin, a bile salt, or an anionic surfactant. Relates to a method for immunological analysis of hepatitis B virus.
  • the anti-hepatitis B virusless antibody is a soluble antibody.
  • the saponin is a triterpenoid saponin, in particular, a killer saponin or a tea seed-derived saponin.
  • the present invention provides a reagent for hepatitis B virus immunological analysis by a sandwich method, wherein a reagent for contacting a hepatitis B virus antigen with an anti-hepatitis B virus antibody is used as a saponin, bile salt, or
  • the present invention also relates to a reagent for hepatitis B winores immunological analysis characterized by containing an anionic surfactant.
  • the preferred embodiment of the reagent according to the present invention! / Is that the anti-hepatitis B virus antibody is a soluble antibody.
  • the saponin is a triterpenoid. It is a saponin, in particular, a saponin derived from quilla or a saponin derived from tea seed.
  • the present invention further relates to a hepatitis B virus immunological analysis kit containing a reagent for hepatitis B virus immunological analysis.
  • analysis in the present specification includes both “detection” for determining the presence or absence of an analysis target compound and “quantification” for determining the presence of the analysis target compound.
  • test in the present specification includes both “detection” for determining the presence or absence of an antigen or antibody and “quantification” for determining the amount of an antigen or antibody.
  • hepatitis B virus immunological analysis method can be used as a “diagnosis method of hepatitis B virus”.
  • the present invention it is possible to detect and quantify HBV antigens or antibodies with high sensitivity in comparison with the hepatitis B virus immunological analysis method by the sandwich method. Moreover, according to the present invention, it is possible to increase the sensitivity of the hepatitis B virus immunological analysis method without increasing nonspecific binding between the antigen and the antibody.
  • Saponins are a group of glycosides widely contained in plants, and have a structure in which an oligosaccharide is bound to a triterpene skeleton or a steroid skeleton, such as digitalis and soybean.
  • Saponins are roughly classified into triterpenoid saponins and steroid saponins according to the type of sapogenins. Sapogenin is an aglycone (non-sugar part) of saponin, and there are triterpenes and steroids.
  • a triterpenoid saponin is a glycoside having aglycone of a triterpene (sometimes referred to as a triterpenoid) such as a terpene hydrocarbon or alcohol having 30 carbon atoms.
  • Triterpenodosaponin may also be referred to as triterpene saponin.
  • sugar bonded to triterpene include glucose, rhamnose, arabinose, galactose, xylose, pentose, and hexose. Used in the method of the present invention
  • the triterpenoid saponins that can be used are not particularly limited as long as they are glycosides of triterpenes.
  • Saponin licorice-derived saponin, Hirosanega-derived saponin, saponin-derived saponin, Tochiba carrot-derived saponin, Japanese scorpion-derived saponin, arabi-derived saponin, honey-butterfly-derived saponin, egonoki-derived saponin, modama-derived saponin, jujube saponin, jujube Examples include tea seed-derived saponins, quilla-derived saponins, and the like. In particular, killer saponins and tea seed saponins are preferred.
  • Steroidal saponins are glycosides having steroids as aglycones, and are particularly distributed in plants such as the liliaceae, the genus Hydrangeaceae, and the genus Camellia.
  • sugar bonded to the steroid include glucose, rhamnose, arabinose, galactose, xylose, pentose, and hexose.
  • the steroid saponin that can be used in the method of the present invention is not particularly limited as long as it is a steroidal glycoside.
  • amoronin, digitonin, di-succin, gitonin, cammonin, noronin, sanoressa Mention may be made of saponins, sumilonins, tigonins, trilarins, trilins, euconins.
  • Steroid saponins also include glycosides having aglycone as a steroid alkaloid, which is a steroid containing a nitrogen atom.
  • the concentration of the saponin that can be used in the hepatitis B virus immunoassay method of the present invention in the reaction solution in which the antigen and the antibody are brought into contact with each other is not particularly limited, but preferably the lower limit is 0.001. % (Mass / volume percent) or more, and the upper limit is the concentration that can be dissolved in the reaction solution. Moreover, it is preferably from 0 ⁇ 01 to 10% (mass / volume percent), more preferably from 0.02 to 5% (mass / volume percent), and most preferably 0.03-3. % Mass / volume percent). Strength S enhances the reaction between antigen and antibody over a wide concentration range.
  • the step of contacting an antigen and an antibody in the presence of bile acid can also be performed using the method for immunoassay of hepatitis B virus.
  • Sensitivity can be increased.
  • Bile acids are produced in the liver of mammals, amphibians, reptiles, birds or fish, and in the form of sodium salts, the main component of bile is the gallbladder. It helps to absorb fat from the intestinal tract by making it into micelles by its surface-active action.
  • Bile acid has a structure in which a hydroxyl group is bonded to a steroid skeleton, and has a hydrophobic steroid skeleton and a hydrophilic group such as a hydroxyl group in the same molecule, and thus exhibits a surface-active action.
  • the salt include sodium salt. Particularly preferred are cholic acid, deoxycholic acid, and salts thereof.
  • the concentration of the bile acid antigen that can be used in the hepatitis B virus immunoassay method of the present invention in the reaction solution in which the antibody is brought into contact with the antibody is not particularly limited, but preferably the lower limit is 0. 001% (mass / volume percent) or more, and the upper limit is the concentration that can be dissolved in the solution. Moreover, it is preferably 0.01 to 10% (mass / volume percent), more preferably 0.02 to 5% (mass / volume percent), and most preferably 0.03-3. 2% mass / volume percent). It is possible to enhance the antigen-antibody reaction over a wide concentration range.
  • the step of contacting an antigen and an antibody in the presence of an anionic surfactant can also be performed. It is possible to increase the sensitivity of the virus immunological analysis method.
  • the anionic surfactant include alkylbenzene-based alkylaryl sulfonates, higher alcohol-based alkyl sulfates, alkyl ether sulfates, monoalkyl phosphates, and alpha-olefin alpha. Examples thereof include olefin sulfonate and normal paraffin-based alkane sulfonate.
  • sodium lauryl sulfate (NLS), sodium dodecyl sulfate (SDS), 1-octane sodium sulfonate, 1 sodium decane sulfonate, 1-sodium sodium decane sulfonate, sodium 1-dodecane sulfonate, etc. Can do.
  • concentration of the anionic surfactant that can be used in the hepatitis B virus immunological analysis method of the present invention in the reaction solution in which the antigen and the antibody are contacted is not particularly limited, but the lower limit is 0.
  • the step of bringing a hepatitis B virus antigen into contact with an anti-hepatitis B virus antibody comprises saponin, cholic acid, and an anionic surfactant.
  • saponin a mixture of two or more of these, it becomes possible to measure the antigen of hepatitis B violets more sensitively.
  • a mixture of saponin and an anionic surfactant is preferred! The effects obtained by adding saponin and the effects obtained by adding an anionic surfactant can be obtained, and in some cases, a synergistic effect can be obtained.
  • saponin and anionic surfactant used in the mixture all of the saponin and anionic surfactant which can be used alone can be used.
  • concentration of saponin and anionic surfactant is determined by the force used at the same concentration as that used alone.
  • the method for immunological analysis of hepatitis B virus by the sandwich method of the present invention comprises a method for treating hepatitis B virus antigen and anti-hepatitis B virus antibody in the presence of saponin, bile acid, or anionic surfactant.
  • a step of contacting The presence of saponin, bile acid, or anionic surfactant in the liquid phase where the antibody and the antigen bind to each other enhances the binding between the antigen and the antibody.
  • the antigen may be an antigen immobilized on a carrier or a soluble antigen.
  • the antibody may be either an antibody immobilized on a carrier or a soluble antibody, but is preferably a soluble antibody.
  • “soluble antibody” means an antibody not immobilized on a carrier.
  • the immunoassay method for hepatitis B virus immunoassay by the sandwich method of the present invention includes an antigen test for measuring an antigen using an antibody specific to the antigen from the viewpoint of the measurement target in the sample. It is possible to classify into an acupuncture method and an antibody test method for measuring a specific antibody against an antigen. Furthermore, the two-step method can be classified into a forward sandwich method and a reverse sandwich method.
  • the procedure of the antigen measurement method is not particularly limited as long as it is a method for detecting or measuring an antigen in a test sample.
  • the procedure can be performed as follows.
  • a capture antibody that binds to the antigen to be measured is immobilized on a carrier such as a microplate or beads. After that, blocking with bovine serum albumin is performed to prevent non-specific adsorption to the capture antibody or carrier.
  • a test sample containing the antigen to be measured is added to the plate or beads on which the capture antibody is immobilized, together with the primary reaction solution, and the capture antibody and the target antigen are brought into contact with each other to bind (antigen test primary reaction step). . Thereafter, antigens and contaminants not bound to the capture antibody are washed with a washing solution.
  • an antibody that recognizes the captured antigen and a labeled antibody bound with an enzyme such as horseradish peroxidase (HRP) are added, and the labeled antibody is bound to the captured antigen (antigen test secondary reaction step).
  • an immune complex of a capture antibody, an antigen, and a labeled antibody is formed on a carrier such as a microplate. It is possible to detect the signal by washing the unbound labeled antibody with a washing solution, adding a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody, and reacting the enzyme with the substrate.
  • the reactivity of the antigen-antibody reaction can be enhanced.
  • an antigen captured by the capture antibody and a labeled antibody are contacted with each other (antigen test secondary reaction step). The reactivity of the antibody reaction can be enhanced.
  • the antigen test primary reaction step and the antigen test secondary reaction step can be performed in a reaction solution usually used for antigen-antibody reaction.
  • the buffer used for the reaction solution is Although it is not particularly limited as long as it can stably maintain the components necessary for the original antibody reaction and does not inhibit the antigen-antibody reaction, specifically, a phosphate buffer, Tris buffer, glycine buffer or It can be appropriately selected from conventionally known buffers such as a hepes buffer.
  • the reaction solution also contains other components such as carrier proteins such as ushi serum albumin, antiseptics such as sodium azide to stabilize antigens and antibodies, and surface activity to suppress nonspecific reactions. It is also possible to contain an agent or the like.
  • the procedure of the antibody test method is not particularly limited as long as it is a method for detecting or measuring an antibody in a test sample.
  • it can be performed as follows.
  • a capture agent that binds to the antibody to be measured is immobilized on a carrier such as a microplate or beads. After that, blocking with bovine serum albumin is performed to prevent non-specific adsorption to the capture antigen or carrier.
  • a test sample containing the antibody to be measured is added to the plate or bead on which the capture antigen is immobilized, together with the primary reaction solution, and the capture antigen and the target antibody are brought into contact with each other to bind (antibody test primary reaction step). . Thereafter, antibodies and contaminants that have not bound to the capture antigen are washed with a washing solution.
  • an antibody that recognizes the captured antibody and a labeled antibody conjugated with an enzyme such as horseradish peroxidase (HRP) are added, and the labeled antibody is bound to the captured antibody (antibody test secondary reaction step).
  • an immune complex of a capture antigen, an antibody, and a labeled antibody is formed on a carrier such as a microplate. It is possible to detect the signal by washing the unbound labeled antibody with a washing solution, adding a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody, and reacting the enzyme with the substrate.
  • the reactivity of the antigen-antibody reaction can be enhanced.
  • the anti-hepatitis B virus antibody in the sample is soluble in the primary reaction step of the antibody test, and the saponin, bile acid, or anionic surfactant is present in the reaction solution. Therefore, it is possible to enhance the reactivity of antigen-antibody reaction.
  • the buffer solution, carrier protein, surfactant, etc. used in the antigen testing method can also be used.
  • the antigen test method and the antibody test method can be performed by a two-step forward sandwich method, a reverse sandwich method, and a one-step method.
  • the antigen test method and antibody test method exemplified above are test methods based on the forward sandwich method.
  • the forward sandwich method, reverse sandwich method, and one-step method will be described below using the antigen test method as an example, but the antibody test method can also be performed by the forward sandwich method, reverse sandwich method, and one-step method. is there.
  • the forward sandwich method has the same force as the procedure described as the antigen test method.
  • the capture antibody is immobilized on a carrier such as a microplate or a bead.
  • block with bovine serum albumin The sample to be measured is added to the plate or beads on which the capture antibody is immobilized together with the primary reaction solution, and the capture antibody and the target antigen are brought into contact with each other and bound (forward primary reaction step).
  • the plate and beads are washed with a washing solution.
  • a labeled antibody is added, and the labeled antibody is bound to the captured antigen (forward secondary reaction step).
  • an immunocomplex of a capture antibody, an antigen-labeled antibody is formed on a carrier such as a microplate.
  • a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody is added, and the signal is detected by reacting the enzyme with the substrate.
  • a capture antibody that binds to an antigen to be tested is immobilized on a carrier such as a microplate or a bead. After that, in order to prevent non-specific adsorption to the capture antibody and the carrier, blocking with bovine serum albumin is performed to prepare a solid-phase carrier.
  • the test sample containing the antigen to be tested is brought into contact with and bound to the labeled antibody in the reaction solution (reverse primary reaction step).
  • the reaction solution containing the antigen-labeled antibody immune complex in the test sample is brought into contact with a carrier on which the capture antibody is immobilized (reverse secondary reaction step).
  • an immune complex of a capture antibody, an antigen, and a labeled antibody is formed on a carrier such as a microplate. Wash unbound antibody, antigen, labeled antibody, etc. with washing solution. Next, by adding a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody, the signal can be detected by reacting the enzyme with the antibody.
  • a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody the signal can be detected by reacting the enzyme with the antibody.
  • a test sample containing an antigen to be measured is contacted with a labeled antibody in a reaction solution and allowed to bind (reverse)
  • a reaction solution containing an antigen-labeled antibody immune complex in a test sample is mixed with a carrier on which a capture antibody is immobilized.
  • the saponin, bile acid, or anionic surfactant is present in the reverse primary reaction step in which the antibody is soluble.
  • a capture antibody that binds to an antigen to be measured is immobilized on a carrier such as a microplate or a bead. Thereafter, in order to prevent non-specific adsorption to the capture antibody or the carrier, blocking with bovine serum albumin or the like is performed to prepare a solid phase carrier. Next, the reaction solution is added to the immobilized carrier, and the test sample containing the antigen and the labeled antibody are added simultaneously, or the test sample containing the antigen is added, and then the labeled antibody is added immediately. The immobilized capture antibody, antigen and labeled antibody are simultaneously contacted and reacted (one-step reaction process).
  • an immune complex of a capture antibody, an antigen, and a labeled antibody is formed on a carrier such as a microplate.
  • a carrier such as a microplate.
  • the unbound antibody, antigen, labeled antibody, etc. are washed with a washing solution.
  • a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody is added, and the signal can be detected by reacting the enzyme with the substrate. Even in the one-step method, the cleaning process is performed only once.
  • the one-step method in the presence of saponin, bile acid, or an anionic surfactant, a carrier on which a capture antibody is immobilized, a test sample containing an antigen, and a labeled antibody react with each other.
  • a carrier on which a capture antibody is immobilized By performing the (one-step reaction process), the reactivity of the antigen-antibody reaction can be enhanced.
  • the saponin, bile, or both of the reactivity of the immobilized capture antibody and the antigen-antibody reaction of the antigen in the test sample, as well as the antigen-antibody reaction of the antigen and labeled antibody in the test sample are detected.
  • the ability to add an acid or an anionic surfactant has a potentiating effect.
  • the hepatitis B virus antigen that can be used in the method for immunological analysis of hepatitis B virus by the sandwich method of the present invention is an epitope or antigenic determinant to which an antigen binding site of an anti-hepatitis B virus antibody binds.
  • the antigen is not particularly limited as long as it has an antigen.
  • the antigen used to measure the anti-hepatitis B virus antibody in the test sample by the antibody detection method can be a native antigen of hepatitis B virus, or a combination produced using Escherichia coli or the like. It is also possible to use a replacement HBV antigen.
  • hepatitis B virus antigens detected by the antigen testing method include HBs antigen, HBc antigen, HBe antigen and HBc-related antigen, and HBs antigen is particularly preferable.
  • the HBs antigen includes a LargeS protein consisting of 389 to 400 amino acid residues, a MiddleS protein consisting of 281 amino acid residues, and a SmallS protein consisting of 226 amino acid residues.
  • Antigenic determinants or epitopes of these HBs antigens include pre-S1 region, pre-S2 region, common antigenic determinant a and the like.
  • the antigenic determinant or epitope utilized in the present invention is not limited to these, and the force S that utilizes the antigenic determinant or epitope of all HBs antigens.
  • the anti-hepatitis B virus antibody used in the method for immunological analysis of hepatitis B virus by the sandwich method of the present invention is not particularly limited. Examples thereof include antibodies that recognize HBs antigen, HBc antigen, HBe antigen, and HBc-related antigen.
  • antibodies that bind to the HBs antigen include antibodies that bind to peptides consisting of amino acids 51 to 60 of the SmallS protein consisting of 226 amino acid residues, such as the 6G6 antibody, 15; An antibody that binds to a peptide consisting of amino acid residues of ⁇ 170, such as an HBsl21 antibody, an antibody that binds to a peptide consisting of amino acid residues of 31 to 50, such as an HBsl23 antibody, 1 1;! An antibody that binds to a peptide consisting of residues, for example, an HBsl36 antibody, 11;! To an antibody that binds to a peptide consisting of amino acid residues Nos.
  • HBs 128 antibody amino acids residues Nos. 1-226 It recognizes structural epitopes such as the common antigenic determinant a that bind to the full-length peptide of HBs and do not bind to peptides consisting of 20 amino acid residues that overlap by 10 amino acid residues from the amino acid sequence of the HBs antigen.
  • Body for example, a HBs605C3 antibodies and SF124CS antibody.
  • the type of antibody is not particularly limited.
  • blood of mammals of humans and mice A monoclonal antibody secreted from a hybridoma cell obtained by cell fusion with a myeloma cell may also be used.
  • Antibody is obtained by using proteases such as pepsin and papain to obtain antibody fragments such as F (ab ') and Fab.
  • heavy chains (H chains) of antibodies have heavy chains bonded to each other by S—S bonds, and the bonds are cleaved by a reducing agent.
  • these antibody fragments such as F (ab ′), Fab ⁇ Fab, F (abc ′), Fabc ′ and the like can be used.
  • the state of the antibody that binds to the antigen in the hepatitis B virus immunoassay method of the present invention may be either solid-phased on an insoluble carrier or the like, or may be in a soluble state. It is preferably soluble in the liquid.
  • soluble antibody means an antibody that is not immobilized on an insoluble carrier or the like during an antigen-antibody reaction.
  • the term “insoluble carrier” means a material for immobilizing an antibody or antigen such as a plate or bead, and a polymer compound such as dextran when preparing a labeled antibody. In some cases, an antibody, a labeling enzyme, and the like are bound, but such a polymer compound is not contained in the insoluble carrier.
  • Enzymes for labeling antibodies used in the hepatitis B virus immunoassay method of the present invention include alkaline phosphatase, ⁇ -galatatosidase, luciferase, and the like in addition to horseradish peroxidase (HRP). it can.
  • HRP horseradish peroxidase
  • luminescent substances such as ataridinium derivatives, fluorescent substances such as europium, and radioactive substances such as I 125 can be used as labeling substances.
  • the substrate and the luminescence inducing substance can be appropriately selected in accordance with the labeling substance.
  • the labeled antibody in the present invention includes those bound with substances that can be used for detection of antigen-antibody reaction signals such as haptens, low molecular weight peptides, and lectins as detection markers.
  • Haptens include biotin, dinitrophenyl (DNP) and FITC.
  • an avidin having affinity for biotin is labeled with an enzyme such as HRP, a fluorescent substance such as fluorescein, or a luminescent substance such as an ataridinium derivative. Signals can be detected by color reaction, fluorescence, luminescence, etc. after reaction.
  • the method for labeling the antibody is not particularly limited, and the antibody may be directly labeled with a label such as an enzyme, but the antibody and the label such as an enzyme are bound to a polymer compound such as dextran.
  • the labeled antibody may be bound to a polymer compound such as dextran. Even when an antibody is bound to such a high molecular compound, it is included in the “soluble antibody” in the present specification as long as the labeled antibody is soluble in the reaction solution.
  • the test sample analyzed by the hepatitis B virus immunological analysis method of the present invention may contain a hepatitis B virus antigen or anti-hepatitis B virus antibody measured by the immunological analysis method.
  • a test sample with a certain amount it is not particularly limited, and examples thereof include biological fluids generally used for clinical diagnosis, such as blood, serum, plasma, or urine.
  • biological fluids generally used for clinical diagnosis such as blood, serum, plasma, or urine.
  • the culture supernatant of cells containing hepatitis B virus antigen or anti-hepatitis B virus antibody is also included in the test sample.
  • the reagent for immunological analysis of hepatitis B virus by the sandwich method of the present invention may contain the saponin, bile acid, or anionic surfactant.
  • the immunological analysis reagent can contain a mixture of two or more of saponin, cholic acid, and an anionic surfactant.
  • the hepatitis B virus immunological analysis reagent is not particularly limited as long as it is a reagent used in an antigen-antibody reaction in which an antigen is brought into contact with an antibody.
  • it may be a reagent used for the antigen test method or a reagent used for the antibody test method. Further, it may be a reagent used in the forward sandwich method, the reverse sandwich method, and the one-step method.
  • Hepatitis B virus The buffer used for the immunological analysis reagent can be appropriately selected from conventionally known buffers such as phosphate buffer, Tris buffer, glycine buffer or hepes buffer.
  • the reagent for immunological analysis of hepatitis B virus includes other components, for example, carrier proteins such as urine serum albumin for stabilizing antigens and antibodies, preservatives such as sodium azide, and non-preservatives.
  • carrier proteins such as urine serum albumin for stabilizing antigens and antibodies
  • preservatives such as sodium azide
  • non-preservatives such as sodium azide
  • a surfactant or the like for suppressing a specific reaction can also be contained.
  • either a hepatitis B virus antigen or an anti-hepatitis B virus antibody to be contacted during the antigen-antibody reaction can be contained in the reagent.
  • the concentration of saponin or bile acid in the hepatitis B virus immunological analysis reagent is not particularly limited, but preferably the lower limit is 0.001% (mass / volume percent) or more, and the upper limit is dissolved in the solution. This is the concentration that can be done. Also, preferably 0.01 to 10% (mass / volume percent), more preferably 0.02 to 5% (mass / volume percent), and most preferably 0.03-3. % Mass / volume percent).
  • the concentration of the anionic surfactant of the reagent for immunological analysis of hepatitis B virus in the reaction of contacting the hepatitis B virus antigen and the anti-hepatitis B virus antibody is not particularly limited, but preferably The lower limit is 0.001% (mass / volume percent) or more, and the upper limit is the concentration that can be dissolved in the solution. Also, preferably from 0.01 to 10% (mass / volume percent), more preferably from 0.02 to 5% (mass / volume percent), and most preferably from 0.05 to 0.8. % (Mass / volume percent).
  • the concentration of each substance can be used at a concentration where each substance is added alone. It is.
  • the concentration of saponin, bile acid, or anionic surfactant in the hepatitis B virus immunological analysis reagent should be the same as that in the reaction by selecting an appropriate mixing ratio with the test sample. It is possible to adjust to. In addition, it is possible to use at the above concentration by attaching a user manual or the like indicating the mixing ratio with the test sample.
  • the immunological analysis kit of the present invention may contain a reagent for hepatitis VIII virus immunological analysis containing saponin, bile salt, or an anionic surfactant.
  • the reagent for hepatitis B virus immunological analysis may be provided in solution, but may also be provided in a lyophilized powder form.
  • the kit for immunological analysis of the present invention can contain a hepatitis virus antigen, an anti-hepatitis virus antibody, a carrier such as an antigen or antibody-immobilized plate or beads, instructions for use, and the like.
  • Hypridoma cell line 6G6 International Deposit No. FERM BP-10117 was established on September 9, 2004 by the National Institute of Advanced Industrial Science and Technology (National Institute of Advanced Industrial Science and Technology) (T 305-8566 Japan). The deposit was made internationally at Tsukuba, Ibaraki Pref.
  • Example 1 in the presence of saponin, the antigen test secondary reaction step of the antigen test method was performed, and HBV antigen was measured.
  • reaction buffer 0.3 M BES containing 1% BSA / 2% Mouse serum, pH 7.2
  • 75 L of healthy human serum or HBV positive specimen 25 L
  • the cells were washed 8 times with 0.05 mM Tween 20, 0.1% sodium dodecyl sulfate (SDS) in 10 mM phosphate buffer pH 7.3 (washing solution).
  • SDS sodium dodecyl sulfate
  • 10 mM phosphate buffer pH 7.3 washing solution.
  • the alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (SF12 4CS antibody) was diluted with 10 mM phosphate buffer ⁇ 7 ⁇ 3 containing 0.5% BSA and 2% mouse serum.
  • Saponin concentration 0 0.03% 0.05% 0.10% 0.203 ⁇ 4 0.40% 0.80% 1.60% 3.20%
  • HBV positive specimen 3 9.2 13.9 15.6 19.8 18.8 24.2 21.2 23.7 22.2
  • HBV positive specimen 6 60.8 91.9 97.2 135.9 150.3 165.3 145.8 156.5 159.5
  • Example 2 an antigen test secondary reaction step of the antigen test method was performed in the presence of bile acid, and HBV antigen was measured.
  • Each anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and HBs136 antibody equal volume mixture) in a 96-well microplate ⁇ 1110 0 Nunk Module, Maxisope surface) at a concentration of 68/111 Add 100 L to the well and incubate at 4 ° C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 ⁇ L of 10 mM phosphate buffer containing 0.5% sodium caseinate was added to each well, followed by blocking at 37 ° C. for 4 hours.
  • reaction buffer (0.3% BES containing 1% BSA / 2% Mouse serum, pH 7.2) and 75 ⁇ L of healthy human serum or HBV positive sample to each well, stirring The reaction was allowed to proceed for 1 hour at room temperature. After the reaction, the plate was washed 8 times with lOmM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecyl sulfate (SDS).
  • alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (SF 124CS antibody) was diluted with lOmM phosphate buffer ⁇ ⁇ 7 ⁇ 3 containing 0.5% BSA and 2% mouse serum, and sodium cholate (WAKO) was added to the reaction solution. Alternatively, sodium deoxycholate (WAKO) was added so that the concentration during the reaction was 0-0.40%. 100 L of this antibody dilution was added to each well and allowed to react for 1 hour at room temperature with stirring. After washing 8 times with the washing solution, 10 O ⁇ L of substrate solution (TROPIX, CDP-star with Emerald II) was added to each well and incubated at room temperature for 15 minutes. Luminescence intensity was measured with a luminometer (DIA—IATRON, Lum inous CT—9000D). The results are shown in Table 2. "RLU” and "S The “/ N ratio” was calculated in the same manner as in Example 1.
  • HBV positive specimen 2 36.4 48.4 59. 7 52.8 68 0 53.7 55.7 74.8 98.4 92.7
  • HBV fertile specimen 5 48.4 66.6 83. 5 70.4 94 2 68.4 70.7 116.4 126.9 120.8
  • HBV positive specimen 6 56.7 81.7 96. 2 81.2 08 4 80.5 76.1 122.8 139.2 123.1
  • the antigen test secondary reaction step of the antigen test method was performed, and HBV antigen was measured.
  • Each anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and HBs136 antibody equal volume mixture) in a 96-well microplate ⁇ 1110 0 Nunk Module, Maxisope surface) at a concentration of 68/111 Add 100 L to the well and incubate at 4 ° C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 ⁇ L of 10 mM phosphate buffer containing 0.5% sodium caseinate was added to each well, followed by blocking at 37 ° C. for 4 hours.
  • reaction buffer (0.3% BES containing 1% BSA / 2% Mouse serum, pH 7.2) and 75 ⁇ L of healthy human serum or HBV positive sample to each well, stirring The reaction was allowed to proceed for 1 hour at room temperature. After the reaction, it was washed 8 times with 10 mM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecyl sulfate (SDS).
  • Example 4 an antigen test secondary reaction step of the antigen test method was performed in the presence of an anionic surfactant, and HBV antigen was measured.
  • Each anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and HBs136 antibody equal volume mixture) in a 96-well microplate ⁇ 1110 0 Nunk Module, Maxisope surface) at a concentration of 68/111 Add 100 L to the well and incubate at 4 ° C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 ⁇ L of 10 mM phosphate buffer containing 0.5% sodium caseinate was added to each well, followed by blocking at 37 ° C for 4 hours.
  • reaction buffer (0 ⁇ 3M BES containing 1% BSA / 2% Mouse serum, pH 7.2) and 75 L of healthy human serum or HBV positive specimen to each well, and stir The reaction was allowed to proceed for 1 hour at room temperature. After the reaction, the plate was washed 8 times with 10 mM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecyl sulfate (SDS).
  • alkaline phosphatase-labeled anti-HBsAg monoclonal antibody S F124CS antibody was added to 0.5% BSA, 2% mouse serum, and 0.2% SDS, 1-octane sodium sulfonate, 1-decane sodium sulfonate, 1-un Sodium decane sulfonate or lOmM phosphate buffer pH 7.3 containing sodium 1-dodecane sulfonate After dilution, 100 L was added to each well, and the mixture was allowed to react at room temperature for 1 hour with stirring.
  • HBV positive specimen 2 112.0 278.7 251.4 211.9 358.6 322.6
  • HBV positive specimen 3 36.0 79.0 69.6 56.5 102.9 92.0
  • Example 5 an antigen test secondary reaction step of the antigen test method was performed in the presence of saponin or cholic acid, and HBV antigen was measured.
  • Anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and H Bsl36 antibody equivalent mixture)
  • reaction buffer 0.3M BES containing 1% BSA / 2% Mouse serum, pH 7.2
  • 75 L of healthy human serum or HBV positive specimen was added to each well, and stir at room temperature. For 1 hour.
  • Example 6 the antigen test secondary reaction step of the antigen test method was performed in the presence of saponin and an anionic surfactant, and HBV antigen was measured.
  • Anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and H Bsl36 antibody equivalent mixture)
  • reaction buffer 0.3M BES containing 1% BSA / 2% Mouse serum, pH 7.2
  • 75 L of healthy human serum or HBV positive specimen was added to each well, and stir at room temperature. For 1 hour.
  • the HBV antigen-positive specimen can be detected with higher sensitivity than the control solution when an antibody diluted with a solution containing saponin, NLS and deoxycholate is used, and NLS is detected in saponin or deoxycholate.
  • detection can be performed with higher sensitivity than when each is used alone.
  • saponin and an anionic surfactant has the effect of enhancing the antigen-antibody reaction. Admitted.
  • Example 7 in the presence of saponin, the antigen test primary reaction step of the antigen test method was performed, and the HBV antigen was measured.
  • reaction buffer containing 1% BSA / 2% Mouse serum (3% BES, ⁇ 7 ⁇ 2) ) 25 L and normal human serum or HBV positive sample 75 were added to each well and allowed to react at room temperature for 1 hour with stirring.
  • the HBV antigen-positive specimen is compared with the case where the reaction with the immobilized antibody is performed in the presence of the reaction buffer containing saponin, compared with the case where the reaction is performed with the reaction buffer without saponin. Improved reactivity. On the other hand, the reactivity was not improved by the addition of C14APS.
  • HBV positive specimen 2 46.0 37.9 44.2
  • Example 8 in the presence of saponin, the antigen test primary reaction step of the antigen test method was performed, and the HBV antigen was measured.
  • reaction buffer containing 0-10% (final concentration 0-2.5% in the reaction solution) of saponin (from Kiraja; SIGMA) (including 1% BSA / 2% Mouse serum, 0 ⁇ 3 ⁇ BES PH 7.2) 25 ⁇ L of healthy human serum or 75 ⁇ L of HBV positive specimen was added to each well and allowed to react at room temperature for 1 hour with stirring. After the reaction, the cells were washed 8 times with a 10 mM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecinole sulfate (SDS).
  • saponin from Kiraja; SIGMA
  • the HBV antigen-positive specimen is compared with the case where the reaction with the immobilized antibody is performed in the presence of the reaction buffer containing saponin, compared with the case where the reaction is performed with the reaction buffer without saponin. Improved reactivity.
  • HBV positive specimen 2 16.0 25.2 35.4 26.9 31.9 42.3 39.5 30.7 29.6 38.5 32.2 44.0
  • the present invention provides a method for immunological analysis of hepatitis B virus by the Sandwich method for highly sensitive detection or quantification of hepatitis B virus antigens in blood. Diagnose the presence of infection and screen blood for transfusion quickly and accurately.
  • the present invention can provide a highly sensitive reagent for hepatitis B virus immunological analysis and a hepatitis B virus immunological analysis kit.

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Abstract

Le but est de fournir un procédé d'analyse immunologique à sensibilité élevée pour un virus de l'hépatite B ; un réactif d'analyse immunologique à sensibilité élevée pour un virus de l'hépatite B ; et un kit d'analyse immunologique pour un virus de l'hépatite B, chacun utilisant une réaction antigène-anticorps. Le but est atteint en réalisant une réaction antigène/anticorps dans laquelle un antigène est mis en contact avec un anticorps en présence d'une saponine, un sel biliaire ou un agent tensioactif anionique. On décrit particulièrement un procédé d'analyse immunologique pour un virus de l'hépatite B par un procédé sandwich impliquant l'étape consistant à mettre en contact un antigène avec un anticorps soluble, où on laisse de préférence une saponine, un sel biliaire ou un agent tensioactif anionique exister dans le système réactionnel. On décrit en outre, un réactif d'analyse immunologique à sensibilité élevée pour un virus de l'hépatite B ; et un kit d'analyse immunologique pour un virus de l'hépatite B, chacun utilisant une réaction antigène-anticorps.
PCT/JP2007/071149 2006-10-30 2007-10-30 Procédé d'analyse immunologique à sensibilité élevée et réactif d'analyse immunologique pour le virus de l'hépatite b Ceased WO2008053900A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011069668A (ja) * 2009-09-24 2011-04-07 Yazaki Corp 液面レベル検出装置の組付け構造、及び液面レベル検出装置
EP2506013A3 (fr) * 2011-03-31 2013-02-27 FUJIFILM Corporation Procédé immunochromatographique hautement sensible
WO2019194280A1 (fr) * 2018-04-06 2019-10-10 富士レビオ株式会社 Méthode de dosage immunologique pour antigène du virus de l'hépatite b
CN111417854A (zh) * 2017-11-30 2020-07-14 富士瑞必欧株式会社 乙型肝炎病毒s抗原的测定方法和测定试剂盒

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000105233A (ja) * 1998-09-29 2000-04-11 Sekisui Chem Co Ltd 小粒子化ＨＢｓ抗原の調製法並びにこれを用いた免疫測定試薬の製造方法、免疫測定試薬及び免疫測定方法
JP2002277472A (ja) * 1997-08-04 2002-09-25 Sentan Seimei Kagaku Kenkyusho:Kk ウイルスの検出又は測定方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022585A1 (fr) * 2002-09-06 2004-03-18 Advanced Life Science Institute, Inc. Proteine pre-noyau du vhb capable de former des particules
JP4430677B2 (ja) * 2004-09-22 2010-03-10 株式会社先端生命科学研究所 Ｂ型肝炎ウイルスｓ抗原の検出法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002277472A (ja) * 1997-08-04 2002-09-25 Sentan Seimei Kagaku Kenkyusho:Kk ウイルスの検出又は測定方法
JP2000105233A (ja) * 1998-09-29 2000-04-11 Sekisui Chem Co Ltd 小粒子化ＨＢｓ抗原の調製法並びにこれを用いた免疫測定試薬の製造方法、免疫測定試薬及び免疫測定方法

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011069668A (ja) * 2009-09-24 2011-04-07 Yazaki Corp 液面レベル検出装置の組付け構造、及び液面レベル検出装置
EP2506013A3 (fr) * 2011-03-31 2013-02-27 FUJIFILM Corporation Procédé immunochromatographique hautement sensible
CN111417854A (zh) * 2017-11-30 2020-07-14 富士瑞必欧株式会社 乙型肝炎病毒s抗原的测定方法和测定试剂盒
CN111417854B (zh) * 2017-11-30 2023-12-08 富士瑞必欧株式会社 乙型肝炎病毒s抗原的测定方法和测定试剂盒
WO2019194280A1 (fr) * 2018-04-06 2019-10-10 富士レビオ株式会社 Méthode de dosage immunologique pour antigène du virus de l'hépatite b
JPWO2019194280A1 (ja) * 2018-04-06 2021-05-20 富士レビオ株式会社 Ｂ型肝炎ウイルス抗原の免疫測定方法
JP7320492B2 (ja) 2018-04-06 2023-08-03 富士レビオ株式会社 Ｂ型肝炎ウイルス抗原の免疫測定方法

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