WO2019194280A1 - Méthode de dosage immunologique pour antigène du virus de l'hépatite b - Google Patents

Méthode de dosage immunologique pour antigène du virus de l'hépatite b Download PDF

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WO2019194280A1
WO2019194280A1 PCT/JP2019/014975 JP2019014975W WO2019194280A1 WO 2019194280 A1 WO2019194280 A1 WO 2019194280A1 JP 2019014975 W JP2019014975 W JP 2019014975W WO 2019194280 A1 WO2019194280 A1 WO 2019194280A1
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antigen
antibody
hbv
water
immunoassay
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PCT/JP2019/014975
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English (en)
Japanese (ja)
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友教 西井
久美子 飯田
慎太郎 八木
千春 大植
克己 青柳
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富士レビオ株式会社
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Priority to JP2020512321A priority Critical patent/JP7320492B2/ja
Publication of WO2019194280A1 publication Critical patent/WO2019194280A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • 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

Definitions

  • the present invention relates to an immunoassay method for hepatitis B virus (HBV) antigen.
  • HBV hepatitis B virus
  • HBV When infected with HBV, HBV is detected in the patient's blood, but when the HBV growth is suppressed by the patient's immune system, HBV becomes negative and anti-HBV antibody becomes positive. Often leads to a state called. However, it has been found that there are patients whose HBV continues to proliferate after seroconversion and hepatitis B progresses. After seroconversion, the HBV concentration in the blood becomes low, so that it is difficult to detect HBV by ordinary immunoassay, and therefore a highly sensitive measurement method is required.
  • Patent Document 1 As a method for highly sensitive detection of HBV antigen, a method of pretreating a specimen with a surfactant is known (Patent Document 1). However, a more sensitive measurement method is desired.
  • a labeled antibody used for immunoassay a method using a combination of a water-soluble carrier, a plurality of labeled enzymes, and a plurality of antibody fragments (Patent Document 3 and Patent Document 4) is also known. ). Furthermore, a method of shortening the antigen-antibody reaction by performing immunoassay in the presence of dextran is also known (Patent Document 5). However, even when these known methods are applied to the immunoassay of HBV antigen, further improvement in sensitivity is desired.
  • An object of the present invention is to provide an immunoassay method for HBV antigen that can be carried out by a general-purpose immunoassay device and can detect HBV antigen with high sensitivity.
  • the inventors of the present application reacted HBV antigen in a sample with a labeled enzyme-anti-HBV antibody or an antigen-binding fragment thereof-water-soluble carrier conjugate in the presence of a water-soluble polymer.
  • HBV antigen can be detected with high sensitivity, and the present invention was completed.
  • the present invention provides the following. (1) An immunoassay method for hepatitis B virus (HBV) antigen in a sample isolated from a living body, In the presence of a water-soluble polymer, the HBV antigen in the sample is reacted with a labeling substance-anti-HBV antibody or antigen-binding fragment thereof-water-soluble carrier conjugate, and the conjugate bound to the HBV antigen is reacted with An immunoassay method comprising detecting.
  • HBV hepatitis B virus
  • the labeling substance is a labeling enzyme, and the labeling enzyme is at least one selected from the group consisting of horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, and luciferase.
  • the water-soluble carrier is dextran, aminodextran, Ficoll (trade name), dextrin, agarose, pullulan, various celluloses, chitin, chitosan, ⁇ -galactosidase, thyroglobulin, hemocyanin, polylysine, polypeptide and DNA, and these The method according to (1) or (2), wherein the method is at least one selected from the group consisting of the modified compounds.
  • the water-soluble polymer is at least one selected from the group consisting of dextran, aminodextran, and modified products thereof.
  • the antibody that specifically binds to the HBV antigen is an antibody that specifically binds to a hepatitis B virus surface antigen.
  • a kit for immunoassay of a hepatitis B virus (HBV) antigen in a sample An immunoassay kit comprising a labeled body fluid comprising a water-soluble polymer and a labeling substance-anti-HBV antibody or antigen-binding fragment thereof-water-soluble carrier conjugate.
  • a novel immunoassay method for HBV antigen and a kit for the same which can be carried out with a general-purpose immunoassay device and can detect HBV antigen with high sensitivity, are provided.
  • the HBV antigen in the blood can be measured with high sensitivity, so that the prognosis after seroconversion can be accurately predicted.
  • the immunoassay method of the present invention is an immunoassay method for HBV antigen in a sample separated from a living body.
  • the sample is not particularly limited as long as it can contain HBV, and examples thereof include serum, plasma, whole blood, urine, feces, oral mucosa, pharyngeal mucosa, intestinal mucosa and various biopsy samples.
  • the sample is serum or plasma. These samples can be diluted and used for immunoassay as desired.
  • the HBV antigen used as the object of an immunoassay will not be specifically limited if it is an antigen which can detect HBV,
  • a surface antigen (HBsAg) and a core related antigen (HBcrAg) can be mentioned.
  • the principle of the immunoassay method for HBV antigen in the method of the present invention can employ either a sandwich method or a competitive method.
  • a sandwich method in which detection is performed using a detection antibody is preferable.
  • the sandwich method the forward sandwich method (capture antibody and antigen in the sample, the reaction of the antigen bound to the capture antibody and the detection antibody are sequentially performed), the reverse sandwich method (detection antibody and sample in advance) And reacting the produced antigen-antibody complex with the capture antibody) and a one-step method (the reaction of the capture antibody, the antigen in the sample, and the detection antibody is simultaneously performed in one step).
  • any of these can be employed.
  • the forward sandwich method can be performed as follows. First, a capture antibody that binds to the HBV antigen is immobilized on an insoluble carrier such as a microplate or magnetic beads. Next, in order to prevent non-specific adsorption to the capture antibody or the insoluble carrier, the insoluble carrier is blocked with an appropriate blocking agent (for example, bovine serum albumin or gelatin). A buffer and a sample are added to a plate or bead on which the capture antibody is immobilized, and the capture antibody and the HBV antigen in the sample are brought into contact with each other to be bound (primary reaction step).
  • an appropriate blocking agent for example, bovine serum albumin or gelatin
  • an appropriate washing solution for example, a phosphate buffer containing a surfactant.
  • an antibody that recognizes the captured HBV antigen and a detection antibody in which a labeling substance is bound directly or indirectly are added, and the detection antibody is bound to the captured antigen (secondary reaction step).
  • an immune complex containing the capture antibody-antigen-detection antibody is formed on a carrier such as a microplate.
  • the remaining detection antibody labeling substance is detected by a predetermined method.
  • the labeling substance is an enzyme
  • a signal is detected by adding a chromogenic substrate or a luminescent substrate corresponding to the enzyme and reacting the enzyme with the substrate.
  • the capture antibody refers to an antibody that captures an HBV antigen in a specimen. In the sandwich method using the insoluble carrier, it is a solid phase antibody that is immobilized on an insoluble carrier.
  • the capture antibody any of an anti-HBV antibody that reacts with an HBV antigen or an antigen-binding fragment thereof can be used.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • a conjugate (labeled antibody) of an antibody that specifically reacts with the target antigen and a labeling substance is widely used.
  • a conjugate (label) of a labeling substance, an anti-HBV antibody or an antigen-binding fragment thereof, and a water-soluble carrier is used as a function of a labeled antibody in such a normal immunoassay.
  • water-soluble carrier in the polymer label examples include dextran, aminodextran, Ficoll (trade name), dextrin, agarose, pullulan, various celluloses (for example, hemicellulose and ligurin), chitin, chitosan, ⁇ -galactosidase, thyroglobulin, Examples include hemocyanin, polylysine, polypeptide and DNA, and modified products thereof (for example, DEAE Dextran, dextran sodium sulfate, etc.). Of these, dextran and aminodextran and their modified products are preferred, and dextran is particularly preferred.
  • the weight average molecular weight of the water-soluble carrier is not particularly limited, but is preferably 6,000 to 4,000,000, particularly preferably 20,000 to 150,000, from the viewpoint of sensitivity of immunoassay and water solubility.
  • any labeling substance used in a known immunoassay can be used, for example, a luminescent substance such as an enzyme or an acridinium derivative, A fluorescent substance such as europium, a radioactive substance such as I 125 , a low molecular weight labeling substance such as biotin, dinitrophenyl (DNP), and FITC, a low molecular weight peptide, a lectin, and the like can be used.
  • a luminescent substance such as an enzyme or an acridinium derivative
  • a fluorescent substance such as europium
  • a radioactive substance such as I 125
  • a low molecular weight labeling substance such as biotin, dinitrophenyl (DNP), and FITC
  • a low molecular weight peptide such as biotin, dinitrophenyl (DNP), and FITC
  • a low molecular weight peptide such as biotin, dinitrophenyl (DNP), and FITC
  • Preferred examples of the enzyme include, but are not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase and luciferase.
  • labeling enzyme an enzyme as a representative example of a labeling substance (hereinafter also referred to as “labeling enzyme”) will be described, but the labeling substance is not limited to an enzyme.
  • the anti-HBV antibody or antigen-binding fragment thereof in the polymer label is not particularly limited as long as it is an antibody or antigen-binding fragment thereof that reacts with an HBV antigen by an antigen-antibody reaction.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the antigen-binding fragment is not particularly limited as long as it is a fragment that reacts with HBV to be measured and antigen-antibody, and examples thereof include Fab fragment, Fab ′ fragment, and F (ab ′) 2 fragment.
  • antigen-binding fragments can be easily prepared by a known method in which an antibody is treated with a reducing agent such as 2-mercaptoethylamine or a proteolytic enzyme such as papain or pepsin.
  • a reducing agent such as 2-mercaptoethylamine or a proteolytic enzyme such as papain or pepsin.
  • antibody fragments are preferably used from the viewpoint of detection sensitivity, and Fab ′ fragments are particularly preferable.
  • the ratio of the label enzyme, anti-HBV antibody or antigen-binding fragment thereof and water-soluble carrier in the polymer label is such that the label enzyme and the antibody bind to one molecule of the water-soluble carrier in order to improve the detection sensitivity.
  • the number of molecules of the antigen-binding fragment thereof is preferably set to be as large as possible.
  • the labeling enzyme is preferably 200 parts by weight to 2000 parts by weight, more preferably 400 parts by weight.
  • the amount of the antibody or antigen-binding fragment is preferably about 700 to 1400 parts by weight, and more preferably about 1000 to 1300 parts by weight.
  • a water-soluble carrier is oxidized with an oxidizing agent such as sodium periodate to form an aldehyde group, which is reacted with chlorohydrazine to form hydrazine
  • the labeling enzyme is oxidized with an oxidizing agent such as sodium periodate to generate an aldehyde group in the sugar chain.
  • This aldehyde group is reacted with a hydrazine residue of a water-soluble carrier to form a hydrazone and bonded, and then the resulting water-soluble carrier-labeled enzyme conjugate is converted to EMCS (N- ⁇ -reimidocaproyl-oxy Treatment with a protein coupling agent such as sulfosuccinimide ester), reacting this with an antibody or a fragment thereof, and covalently binding the labeled enzyme to the antibody or a fragment thereof, thereby covalently binding the three. it can.
  • an antibody or a fragment thereof can be obtained by binding two or more water-soluble carriers to each other via a labeling enzyme.
  • the method for covalently bonding the three parties is not limited to the above method.
  • a thiol group is introduced into an amino group (an amino group of a labeling enzyme, an antibody or a fragment thereof, or an amino group introduced into a water-soluble carrier), while a maleimide group or a pyridyl disulfide group is introduced into the amino group of another substance.
  • other known methods such as a method of reacting them.
  • the thiol group can be introduced using, for example, a commercially available reagent such as S-acetylmethylcaptosuccinic anhydride or N-succinimidyl 3- (2-pyridyldithio) propionate, and the maleimide group is introduced into the amino group.
  • a commercially available reagent such as S-acetylmethylcaptosuccinic anhydride or N-succinimidyl 3- (2-pyridyldithio) propionate
  • the maleimide group is introduced into the amino group.
  • Can be carried out using commercially available reagents such as N- (6-maleimidocaproyloxy) succinimide and N- (4-maleimidobutyryloxy) succinimide.
  • the introduction of the pyridyl disulfide group is, for example, N -Succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N- ⁇ 6- [3- (2-Pyridyldithio) propionamido] hexanoyloxy ⁇ sulfosuccinimide, sodium salt (Sulfo-AC5- It can be performed using a commercially available reagent such as SPDP).
  • the ratio of the labeling enzyme, anti-HBV antibody or antigen-binding fragment thereof, and water-soluble carrier to be used in these reactions is appropriately selected so as to achieve the above-mentioned preferable three ratios.
  • the ratio of the three to be used for the reaction is that when the water-soluble carrier is 100 parts by weight, the labeling enzyme is preferably 200 parts by weight to 2000 parts by weight, more preferably about 400 parts by weight to 1200 parts by weight.
  • the sex fragment is preferably 700 to 1400 parts by weight, more preferably about 1000 to 1300 parts by weight.
  • the final concentration of the polymer label in the reaction solution at the time of immunoassay is not particularly limited, but from the viewpoint of sensitivity of immunoassay, the weight of the labeled enzyme is preferably 0.1 to 10 ⁇ g / mL, particularly 0.5 to 5.0 ⁇ g / mL, more preferably 1.0 to 3.0 ⁇ g / mL is preferable.
  • the reaction between the HBV antigen and the polymer label is performed in the presence of a water-soluble polymer. That is, an immune reaction is performed under conditions where a water-soluble polymer and a polymer label coexist.
  • water-soluble polymer examples include dextran, aminodextran, Ficoll (trade name), dextrin, agarose, pullulan, various celluloses (for example, hemicellulose and ligrin), chitin, chitosan, ⁇ -galactosidase, thyroglobulin, hemocyanin, polylysine, Examples thereof include polypeptides and DNA, and modified products thereof (for example, DEAE Dextran, dextran sodium sulfate, etc.). Of these, dextran and aminodextran and their modified products are preferred, and dextran is particularly preferred.
  • the weight average molecular weight of the water-soluble polymer is not particularly limited, but is preferably 20,000 to 4,000,000, particularly 700,000 to 3,000,000, and more preferably 1,000,000 to 1,000,000 from the viewpoint of immunoassay sensitivity and water solubility. 2.5 million is preferred.
  • the final concentration of the water-soluble polymer in the reaction solution of the antigen-antibody reaction in the immunoassay is not particularly limited, but is preferably 0.01 to 10.0% by weight, particularly 0.1% from the viewpoint of the sensitivity of the immunoassay. It is preferably -5.0% by weight, more preferably 0.5-3.0% by weight. In the following description, “%” indicating concentration means “% by weight” unless otherwise specified.
  • the reaction between the HBV antigen and the polymer label is preferably performed in the presence of a zwitterionic surfactant in addition to the water-soluble polymer.
  • a zwitterionic surfactant in addition to the water-soluble polymer.
  • amphoteric surfactants include (i) an amphoteric surfactant having an alkyl group having 10 or more carbon atoms in the molecule and quaternary ammonium, and (ii) a steroid skeleton and a quaternary quaternary in the cocoon molecule. Mention may be made of amphoteric surfactants with secondary ammonium.
  • Preferred examples of the zwitterionic surfactant (i) include C10APS (N-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), C12APS (N-dodecyl-N, N- Dimethyl-3-ammonio-1-propanesulfonate), C14APS (N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), C16APS (N-hexadecyl-N, N-dimethyl- 3-ammonio-1-propanesulfonate) and the like.
  • Preferred examples of the zwitterionic surfactant (ii) include CHAPS (3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonate) and CHAPSO (3-[(3-chola Midpropyl) dimethylammonio] -1-hydroxypropanesulfonate) and the like.
  • CHAPS (3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonate)
  • CHAPSO 3-[(3-chola Midpropyl) dimethylammonio] -1-hydroxypropanesulfonate
  • amphoteric surfactant having an alkyl group having 10 or more carbon atoms and quaternary ammonium in the molecule (1) is particularly preferable.
  • the final concentration of the zwitterionic surfactant in the reaction solution at the time of immunoassay is not particularly limited, but is preferably 0.1 to 10%, particularly 1.0 to 7.0%, more preferably 2.0 to 5.0% is preferable.
  • the immunoassay method of the present invention can be carried out in the same manner as a normal sandwich immunoassay method using a labeled antibody, except that the above-mentioned water-soluble polymer and the above-mentioned polymer labeled substance are allowed to coexist. it can.
  • an anti-HBV antibody capture antibody
  • a solid phase carrier such as magnetic beads
  • a sample containing the HBV antigen to bind the HBV antigen in the sample to the solid phase
  • a liquid containing the water-soluble polymer and the polymer labeled body is added thereto and reacted, and after washing, the labeled enzyme bound to the solid phase is quantified to perform immunoassay.
  • Each antigen-antibody reaction may be the same as in the ordinary sandwich method, for example, room temperature to 37 C., preferably 37.degree. C., for about 3 to 120 minutes, preferably about 5 to 10 minutes. However, it is not limited to these conditions.
  • Quantification of the labeled enzyme bound to the solid phase can be performed by a conventional method depending on the type of the labeled enzyme used. For example, when alkaline phosphatase is used as the labeling enzyme, AMPPD (3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane disodium salt) is used as a substrate.
  • the amount of labeled enzyme bound to the solid phase can be quantified by measuring the amount of light emitted having a maximum absorption at a wavelength of 463 nm, which is emitted when AMPPD is decomposed.
  • the sample separated from the living body can be subjected to the above immunoassay as it is or after being diluted as desired, but prior to this, the sample may be subjected to pretreatment.
  • the pretreatment can be performed by treating the sample with a pretreatment liquid.
  • the pretreatment liquid (1) at least one surfactant selected from the group consisting of anionic surfactants, amphoteric surfactants and nonionic surfactants, and / or (2) acidic properties And at least one selected from the group consisting of an agent, an alkalinizing agent and a protein denaturant.
  • the pretreatment liquid may contain both of the above (1) and (2), or may contain only one of them.
  • any of an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant is used as the surfactant.
  • anionic surfactants are particularly preferred.
  • sodium dodecyl sulfate (SDS), N-lauroyl sarcosine (NLS), lithium dodecyl sulfate, sodium dodecylbenzenesulfonate, deoxycholic acid, etc. can be preferably used, and SDS is particularly preferably used. it can.
  • a cationic surfactant is used to reduce the influence of the anionic surfactant introduced into the reaction system after the pretreatment.
  • a cationic surfactant can be used in place of the anionic surfactant.
  • a cationic surfactant having a single-chain alkyl group having 10 or more carbon atoms and a tertiary amine or quaternary ammonium salt in the same molecule is particularly preferable. .
  • surfactants examples include decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride (C16TAC), decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride.
  • examples include bromide, hexadecyltrimethylammonium bromide (CTAB), laurylpyridinium chloride, tetradecylpyridinium chloride, cetylpyridinium chloride and the like.
  • the addition amount of the cationic surfactant is preferably from 0.1% to 15%, more preferably from 0.5% to 10%, in terms of the concentration when mixed with the specimen.
  • cationic surfactant In addition to the cationic surfactant, other surfactants such as an amphoteric surfactant and a nonionic surfactant may be further contained. By adding other surfactants, it becomes possible to detect the HBV antigen with higher sensitivity.
  • the pretreatment liquid contains an acidifying agent
  • hydrochloric acid, sulfuric acid, acetic acid, citric acid or the like can be suitably used as the acidifying agent.
  • the acidifying agent is used, the normality of the acid in the pretreatment liquid is preferably more than 0.05N and 0.5N or less, particularly 0.1N or more and 0.4N or less as the concentration during the pretreatment. By setting the normality of the acid to more than 0.05N and 0.5N or less, the effect of the pretreatment can be sufficiently obtained, and the influence on the subsequent reaction step can be minimized.
  • the alkalizing agent is not particularly limited, and examples thereof include sodium oxide, potassium hydroxide, and magnesium hydroxide.
  • the normality of the alkali in the pretreatment liquid is preferably more than 0.05N and 0.5N or less, particularly 0.1N or more and 0.4N or less as the concentration during the pretreatment.
  • the pretreatment reagent may contain other protein denaturing agents such as urea and thiourea as necessary.
  • concentration of the denaturing agent is preferably 0.1 M or more, more preferably 0.5 M or more and less than 4 M in terms of the concentration during treatment.
  • any of monosaccharides, disaccharides, citric acid, and citrates, or a combination thereof may be added to the pretreatment reagent.
  • the pretreatment reagent may contain a chelating agent such as EDTA.
  • the pretreatment reagent may contain a reducing agent as necessary.
  • a reducing agent any of those usually used for cleaving the S—S bond of a protein can be used, and is not particularly limited.
  • existing reducing agents such as 2- (diethylamino) ethanethiol hydrochloride (DEAET), tris (2-carboxyethyl) phosphine hydrochloride (TCEP), dithiothreitol (DTT), 2-mercaptoethanol, etc. are all used. Is possible.
  • the volume ratio of the sample to be mixed in the pretreatment step and the pretreatment reagent is preferably 1:10 to 10: 1, particularly 1: 5 to 5: 1, and more preferably 1: 3 to 3: 1.
  • the biological sample and the pretreatment reagent may be mixed and then heated.
  • a surfactant is used as the pretreatment reagent, it is preferable to heat to increase the effect.
  • the temperature in the pretreatment step can be 95 ° C. or less, particularly 20 to 90 ° C., more preferably 20 to 80 ° C., 25 to 70 ° C., 25 to 60 ° C., 30 to 50 ° C., or 35 to 45 ° C.
  • the pretreatment process can be performed in a shorter time if heated under higher temperature conditions, but can also be performed under room temperature conditions. In this case, the reaction time becomes longer, but high temperature heating is possible. There is an advantage that no device is required. There is no particular upper limit for the reaction time in the pretreatment step, but it is usually 60 minutes or less, particularly 30 minutes or less.
  • a labeled antibody an antibody labeled with one molecule of a labeling enzyme
  • a water-soluble polymer Compared with conventional immunoassay methods, the detection sensitivity of HBV antigen is high.
  • a commercially available general-purpose automatic immunoassay device can be used as it is.
  • the immunoassay kit of the present invention includes a labeled body fluid containing at least the above-described polymer label and the above-mentioned water-soluble polymer as a constituent reagent.
  • the immunoassay kit of the present invention is not particularly limited except that it is provided with the labeled body fluid and is capable of immunoassay of HBV antigen in a sample, and can be used for normal immunoassay such as ELISA, CLEIA, and immunochromatography.
  • the structure which should be provided can be provided.
  • a magnetic particle solution containing magnetic beads on which a capture antibody is immobilized, a washing solution, and an enzyme substrate solution can be provided.
  • the immunoassay kit of the present invention may further include a pretreatment liquid for pretreatment of the sample, if necessary.
  • the concentration of the polymer label in the labeled body fluid is not particularly limited, but from the viewpoint of sensitivity of immunoassay, 0.1 to 10 ⁇ g / mL is preferable, 0.5 to 5.0 ⁇ g / mL is particularly preferable, and 1.0 to 3.0 ⁇ g / mL is particularly preferable.
  • the concentration of the water-soluble polymer in the labeled body fluid is preferably 0.01 to 10.0%, particularly preferably 0.1 to 5.0%, more preferably 0.5 to 3.0%. It is preferable that the labeling body further contains an amphoteric surfactant.
  • the concentration of the zwitterionic surfactant in the labeled body fluid is preferably 0.1 to 10%, particularly 1.0 to 7.0%, and more preferably 2.0 to 5.0%.
  • NH 2 NH 2 HCl 0.63 g NH 2 NH 2 HCl was added and stirred in the dark at 25 ° C. for 60 minutes. 10 mg of DMAB (dimethylamine borane) was added, and the mixture was further stirred for 60 minutes in the dark at 25 ° C. Dialysis with 4 L of ion-exchanged water was performed for 2 hours in the dark using a CE 50k (cellulose ester having a molecular weight of 50,000) dialysis membrane, and then allowed to stand at 4 ° C. overnight. Using a PD-10 column (trade name), the buffer was exchanged with 0.1 M phosphate buffer (pH 6.0) to obtain 2.5 mL of a solution. The concentration was adjusted to 2.5 mg / mL to obtain a hydrazinated dextran solution.
  • DMAB dimethylamine borane
  • the 2.5 mg / mL hydrazinated dextran prepared in (1) was added so that the molar ratio of hydrazinated dextran to ALP-50 was 1: 4, and the mixture was stirred in the dark at 25 ° C. for 16 hours.
  • 21 mg of DMAB was added and stirred for 60 minutes in the dark at 25 ° C.
  • 2.5 mL of 1.5 M Tris buffer (pH 9.0) was added, and the mixture was stirred in the dark at 25 ° C. for 2 hours.
  • Fab ′ Three kinds of mouse anti-HBsAg monoclonal antibodies (antibodies A, B and C) prepared according to a conventional method and one kind of mouse anti-HBcrAg monoclonal antibody (antibody c) were added to 1 mM EDTA and 0.1 M, respectively.
  • An antibody solution was prepared with a phosphate buffer (pH 6.0).
  • a 2-mercaptoethylamine solution was added to each antibody solution and reacted at 37 ° C.
  • the buffer was exchanged with 1 mM EDTA, 0.1 M phosphate buffer (pH 6.3) using a PD-10 column, and the solution was recovered.
  • the concentration was adjusted with 1 mM EDTA, 0.1 M phosphate buffer (pH 6.3).
  • the epitope sequences of antibodies A, B and C on HBsAg (SEQ ID NO: 1) are as shown in Table 1-1.
  • the epitope sequence of antibody c on hepatitis B virus core antigen (HBcAg) (SEQ ID NO: 2) is as shown in Table 1-2.
  • BSA was added to a final concentration of 3.3%, and then filtered through a 0.45 ⁇ m pore filter to obtain 74 mL of a 200 ⁇ g / mL dextran-enzyme-antibody (Fab) conjugate (polymer label) solution. .
  • Fab dextran-enzyme-antibody
  • HBsAg hepatitis B virus s antigen
  • mice anti-HBsAg antibodies Three types of mouse anti-HBsAg antibodies (antibodies A, B, and C) were mixed at a molar ratio of 1: 1: 2, and then labeled with alkaline phosphatase according to a conventional method. Is diluted with a labeled diluent (50 mM MES, 6% BSA, 600 mM NaCl, 4.0% C14APS (pH 6.8)) to prepare a single molecule labeled body fluid. did. In addition, the polymer label prepared in Example 2 was diluted with a label diluent to 1.5 ⁇ g / mL to prepare a polymer label solution.
  • a labeled diluent 50 mM MES, 6% BSA, 600 mM NaCl, 4.0% C14APS (pH 6.8)
  • HBsAg was performed using Lumipulse Presto II (Fujirebio Inc.). 100 ⁇ L of the sample after the pretreatment was mixed with 50 ⁇ L of the sample diluent (10 mM Tris, 3.2% CHAPS, 2.3% Tween 20 (trade name, pH 7.2). Next, the mixture was mixed with 50 ⁇ L of the magnetic particle solution and 37 ° C. The magnetic beads were collected and washed 5 times with Lumipulse washing solution (Fujirebio Co., Ltd.) 50 ⁇ L of each labeled body fluid was added and reacted for 8 minutes at 37 ° C. The magnetic beads were collected.
  • the sample diluent 10 mM Tris, 3.2% CHAPS, 2.3% Tween 20 (trade name, pH 7.2).
  • the mixture was mixed with 50 ⁇ L of the magnetic particle solution and 37 ° C.
  • the magnetic beads were collected and washed 5 times with Lumipulse washing solution (Fujirebio Co., Ltd.) 50
  • Lumipulse substrate containing AMPPD (3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane disodium salt) after magnetizing and washing 5 times 50 ⁇ L of the solution (Fujirebio Inc.) was added and allowed to react for 4 minutes at 37 ° C. Due to the catalytic action of alkaline phosphatase in which AMPPD was bound to the magnetic beads. The amount of luminescence of light having a maximum absorption at a wavelength of 463 nm emitted by the decomposition was measured, and the measurement result was output as the luminescence intensity (count) of the substrate. In addition, the result of a display shows the average value of double measurement.
  • HBcrAg hepatitis B virus core-related antigen
  • mouse anti-HBcrAg antibody (antibody c) was labeled with alkaline phosphatase according to a conventional method, and the obtained labeled antibody (hereinafter also referred to as “single molecule label”) was labeled with a diluted diluent (20 mM MES, 3% BSA). , 300 mM NaCl, 0.4% N-lauroyl sulfate, 0.2% C14APS (pH 7.5)) was diluted to 1.0 ⁇ g / mL to prepare a single molecule labeled body fluid.
  • the polymer labeled body of antibody c prepared in Example 2 was diluted with a labeled body diluent to 1.0 ⁇ g / mL to prepare a polymer labeled body fluid.
  • a labeled body diluent 1.0 ⁇ g / mL
  • dextran 2000k manufactured by Sigma, weight average molecular weight 2 million
  • a method using a single molecule label is a comparative example, and a method in which the amount of dextran added is 0% (no addition) is also a comparative example.
  • HBcrAg The measurement of HBcrAg was performed using Lumipulse L2400 (manufactured by Fujirebio). Three HBcrAg-negative serum samples (4, 5, 6) and two HBcrAg-positive serum samples (7, 8) were diluted 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 times with negative serum, respectively. 30 ⁇ L of each sample was mixed with 90 ⁇ L of the pretreatment solution (0.71 M urea, 0.22 N HCl, 3.9 mM CHAPS, 4.3 mM DEAET), and heated at 37 ° C. for 6.5 minutes.
  • the pretreatment solution (0.71 M urea, 0.22 N HCl, 3.9 mM CHAPS, 4.3 mM DEAET
  • a neutralizing solution (0.7 M Bicine, 10% sodium N-lauroyl sulfate (pH 10)) was added and mixed, followed by heating at 37 ° C. for 20 seconds.
  • a neutralizing solution 0.7 M Bicine, 10% sodium N-lauroyl sulfate (pH 10)
  • the same treatment was performed on standard solutions with HBcrAg concentrations of 0, 5, and 250 kU / mL in Lumipulse HBcrAg standard solution (Fujirebio Co., Ltd.).
  • 150 ⁇ L of the sample after pretreatment was mixed with 50 ⁇ L of the magnetic particle solution and reacted at 37 ° C. for 8 minutes.
  • the magnetic beads were collected and washed 5 times with Lumipulse washing solution (Fujirebio Co., Ltd.), and 50 ⁇ L of each labeled body fluid was added and reacted at 37 ° C. for 8 minutes.
  • AMPPD (3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane-2 50 ⁇ L of Lumipulse substrate solution (manufactured by Fujirebio) containing sodium salt was added and reacted at 37 ° C. for 4 minutes.
  • the amount of light emitted having a maximum absorption at a wavelength of 463 nm was measured by decomposing AMPPD by the catalytic action of alkaline phosphatase bound to magnetic beads. The measurement result was output as the luminescence intensity (count) of the substrate. Table 3 shows the measurement results under each condition.

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Abstract

L'invention concerne une méthode de dosage immunologique pour un antigène du virus de l'hépatite B (VHB), qui peut être réalisée à l'aide d'un dispositif de dosage immunologique universel et permet la détection de l'antigène du VHB à une sensibilité élevée, et un kit associé. La méthode de dosage immunologique, dans laquelle un antigène du VHB dans un échantillon isolé à partir d'un organisme vivant doit être analysé immunologiquement, comprend, en présence d'un polymère soluble dans l'eau, faire réagir l'antigène du VHB dans l'échantillon avec un conjugué d'une enzyme de marquage/anticorps anti-VHB ou d'un fragment de liaison à l'antigène de celui-ci/un support soluble dans l'eau, puis détecter le conjugué lié à l'antigène du VHB.
PCT/JP2019/014975 2018-04-06 2019-04-04 Méthode de dosage immunologique pour antigène du virus de l'hépatite b WO2019194280A1 (fr)

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CN111830250A (zh) * 2020-08-04 2020-10-27 珠海市丽拓生物科技有限公司 一种信号放大的酶标二抗的制备方法
CN112526136A (zh) * 2020-11-03 2021-03-19 广州市达瑞生物技术股份有限公司 一种联合检测乙型肝炎病毒核心相关抗原的样本预处理液及试剂盒
WO2022211084A1 (fr) * 2021-04-01 2022-10-06 富士レビオ株式会社 Procédé de traitement d'un échantillon biologique
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

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WO2008053901A1 (fr) * 2006-10-30 2008-05-08 Advanced Life Science Institute, Inc. Procédé d'analyse de l'antigène s du virus de l'hépatite b
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JPH10227796A (ja) * 1997-02-13 1998-08-25 Sekisui Chem Co Ltd ＨＢｓ抗原の定量方法及びＨＢｓ抗原測定用免疫学的キット
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
WO2008053900A1 (fr) * 2006-10-30 2008-05-08 Advanced Life Science Institute, Inc. Procédé d'analyse immunologique à sensibilité élevée et réactif d'analyse immunologique pour le virus de l'hépatite b
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JP2017032583A (ja) * 2013-01-28 2017-02-09 シスメックス株式会社 ＨＢｓ抗原を検出するための前処理用試薬キットおよびＨＢｓ抗原検出用試薬キット

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111830250A (zh) * 2020-08-04 2020-10-27 珠海市丽拓生物科技有限公司 一种信号放大的酶标二抗的制备方法
CN112526136A (zh) * 2020-11-03 2021-03-19 广州市达瑞生物技术股份有限公司 一种联合检测乙型肝炎病毒核心相关抗原的样本预处理液及试剂盒
WO2022211084A1 (fr) * 2021-04-01 2022-10-06 富士レビオ株式会社 Procédé de traitement d'un échantillon biologique
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

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