WO2006033368A1 - B型肝炎ウイルスs抗原の検出法 - Google Patents
B型肝炎ウイルスs抗原の検出法 Download PDFInfo
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- WO2006033368A1 WO2006033368A1 PCT/JP2005/017420 JP2005017420W WO2006033368A1 WO 2006033368 A1 WO2006033368 A1 WO 2006033368A1 JP 2005017420 W JP2005017420 W JP 2005017420W WO 2006033368 A1 WO2006033368 A1 WO 2006033368A1
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/01—DNA viruses
- C07K14/02—Hepadnaviridae, e.g. hepatitis B virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
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- C12P21/00—Preparation of peptides or proteins
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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
- G01N33/531—Production of immunochemical test materials
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a probe that recognizes a novel epitope of s antigen (HBs antigen) of hepatitis B virus (HBV), and a method for detecting HBV or HBs antigen using the probe.
- HBs antigen hepatitis B virus
- Diagnosis of virus infection is performed mainly by a method for detecting viruses and virus-related components (proteins and nucleic acids) and a method for detecting specific antibodies produced by living bodies due to virus infection.
- HBV DNA HBV DNA Quantity measurements are used.
- HBs antigens are the main constituent coat proteins on the surface of infectious HBV particles and contain HBV-DNA core particles It is included in the lipid bilayer derived from hepatocytes enveloping In the blood of HBV-infected individuals, there are small infectious small spherical particles and tubular particles such as HBs antigen. Small spherical particles are most abundant in the blood, and HBV particles are observed at a ratio of about 1000 to 1 to several. Currently marketed HBs antigen testing agents mainly detect HBs antigens in the form of small spherical particles.
- the HBs antigen is a membrane protein that penetrates a lipid bilayer membrane consisting of a total length of 226 amino acid residues (amino acid numbers: 1 to 226) four times.
- a lipid bilayer membrane consisting of a total length of 226 amino acid residues (amino acid numbers: 1 to 226) four times.
- the HBs antigen has a lipid bilayer that is the outer (ER / lamen side) region of the lipid bilayer consisting of Nos. 1 to 11 on the N-terminal side and the 12th to 28th forces A hydrophobic transmembrane region that penetrates, inside the lipid bilayer consisting of 29th to 80th Proposed that it consists of the 81-97th hydrophobic transmembrane region, the 98th-156th hydrophilic ER / Remen region, and the 157th and subsequent hydrophobic transmembrane regions. (Fig. 1).
- the main common antigenic determinant a used for the detection of HBs antigen in the conventional method is the amino acid number, the 98th force located on the ER / lemme side, that is, on the surface of the virus particle is also included in the 156th amino acid. Located on 110-156.
- This common antigenic determinant a has a complex higher-order structure and has been reported to have at least 4 epitopes (Non-Patent Document 2).
- HBV is a DNA virus that replicates DNA from DNA when the virus grows, and further synthesizes DNA from RNA using reverse transcriptase, so that mutations comparable to RNA viruses occur. It is known to wake up. For this reason, it is thought that mutants with a wide variety of mutations are mixed in individuals infected with HBV. In such a state, when selective pressure such as neutralizing antibody is applied from the outside, the H BV strain sensitive to the pressure decreases, and on the contrary, it is resistant to this! Phenomenon that happens.
- escape mutants which have been a problem, have escaped the ability of antibodies to recognize the antigenic determinant a before mutation due to the substitution, deletion, or insertion of amino acids in the aforementioned common common antigenic determinant a. It is a mutant strain that can maintain infection.
- escape mutants cannot be detected by the conventional HBs antigen test method.
- escape mutants of common antigenic determinant a have reduced reactivity with monoclonal antibodies against wild-type common antigenic determinant a, so wild-type common antigenic determinant a used in conventional HBs antigen testing methods Monoclonal antibodies against can not recognize escape mutant HBs antigens and miss HBV infection.
- the 145Arg mutant in which the 145th amino acid was mutated from the wild strain Gly to Arg, showed a marked decrease in reactivity with monoclonal antibodies against the common antigenic determinant a.
- Non-Patent Document 2 it was reported that hepatitis B infection was caused by transfusion of blood that was negative for HBs antigen following the screening test for HBV using this conventional HBs antigen measurement reagent.
- HBs antigen in acute infection with HBV, a phenomenon has been reported in which HBs antigen is positive in the early stage of infection, and then HBs antigen is negative, but HBs antibody is positive.
- the reason why the HBs antibody becomes positive is that an antibody against the common antigenic determinant a of the HBs antigen is produced in the body of the infected person.
- the patient's own antibody against the common antigenic determinant a of this patient binds to the same region as the common antigenic determinant a recognized by the monoclonal antibody used in the HBs antigen test drug. It is thought that the sensitivity of the HBs antigen test drug is reduced and detection by the test drug is hindered.
- Non-patent literature 1 Howard et ai. Viral Hepatitis ana Liver Disease, ed by Zuckerman AJ, Alan R), pl094—1101, Liss Inc, New York, 1988.
- Non-Patent Document 2 Hiroaki Okamoto Japanese Clinical Molecular Hepatitis Virology Basic, Clinical, Prevention Shimo A, B, D, Hepatitis E virus edition, p212-222, published on October 26, 1995.
- Non-Patent Document 3 Thiers et al. Lancet, ii, 1273-1276, 1988.
- the conventional HBs antigen test using a monoclonal antibody against the wild-type common antigenic determinant a cannot be detected as an escape mutant on the common antigenic determinant a.
- Blood used in blood transfusions can cause hepatitis B infection.
- An object of the present invention is to develop a probe capable of detecting such an escape mutant of HBV and a method for measuring HBs antigen using the probe.
- the present invention uses a probe capable of measuring an HBs antigen without being disturbed by the patient's own antibody against the common antigenic determinant a, even if it is an HBs antibody-positive infected patient sample, and the probe.
- a probe capable of measuring an HBs antigen without being disturbed by the patient's own antibody against the common antigenic determinant a, even if it is an HBs antibody-positive infected patient sample, and the probe.
- the probe was to develop a method for measuring HBs antigen.
- the present inventors have succeeded in solving the above problems by using an antibody that recognizes an epitope present on a peptide having the amino acid sequence shown in SEQ ID NO: 1 as a probe.
- the present invention relates to a probe that recognizes an epitope present on a peptide consisting of an amino acid sequence corresponding to the 26th to 80th amino acids of hepatitis B virus s antigen, and particularly has the amino acid sequence of SEQ ID NO: 1. It relates to a probe that recognizes an epitope present on a peptide.
- the present invention also relates to a method for detecting hepatitis B virus or hepatitis B virus s antigen using the probe.
- the position of the partial amino acid sequence in the antigen is represented with the N-terminal amino acid residue of the HBs antigen having 226 amino acid residue power as the first.
- the S region of the HBV gene contains Pre-Sl, Pre-S2 and S genes, which are controlled by the Pre Sl + Pre-S2 + S gene !, 389-400 LargeS protein consisting of amino acid residues, Middle protein consisting of 281 amino acid residues controlled by Pre—S2 + S gene, and SmallS protein consisting of 226 amino acid residues controlled by S gene Coded (Mitamura Junji, Japanese Clinical Molecular Hepatitis Virus Pathology Basic / Clinical 'Prevention, Volume 2, pages 13-27, published October 26, 1995).
- HBs antigen when it is described as HBs antigen, it means SmallS protein, and it will be expressed as such in this specification unless otherwise specified.
- the detection method of the present invention can detect any protein of LargeS protein, Middle protein, and SmallS protein, the detected hepatitis B virus s antigen (HBs antigen) Contains 3 proteins.
- the probe of the present invention is a probe capable of recognizing an epitope on a peptide having the amino acid sequence shown in SEQ ID NO: 1, and typically a polyclonal capable of specifically recognizing the epitope.
- An antibody or a monoclonal antibody More specifically, on September 9, 2004, it was accepted by the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st, 1st, 1st, 1st, Tsukuba, Ibaraki). No. FERM ABP—deposited as 10115, 1 10116 and 1 10117 It is a monoclonal antibody produced by either 1C10, 4A3 or 6G6 which is a hyperidoma cell line.
- the amino acid sequence represented by SEQ ID NO: 1 corresponds to the 26th to 80th amino acid sequence of the HBs antigen, that is, the hydrophilic region present inside the lipid bilayer of the HBs antigen. Since the epitope is located inside the HBV virus particle, small spherical particle or tubular particle, unlike the region located on the ER / Remen side containing the common antigenic determinant a of the HBs antigen, it induces the aforementioned escape mutant. Select the neutralizing antibody for the external force to be obtained.
- mutations on the epitope do not receive selective pressure, so only specific mutants become dominant and do not react with HBs antigen measurement reagents. If only increases, the phenomenon is considered to hardly occur in the epitope in the present invention.
- the probe of the present invention when used to detect an HBs antigen, the antibody hardly interferes with the antibody against the patient's own HBs antigen. This is because, in Epitopoca HBV particles, small spherical particles and tubular particles recognized by the probe of the present invention, V, the deviation is located inside them, and therefore, compared with the common antigenic determinant a of the HBs antigen. This is thought to be due to the suppression of antibody production against the patient's own epitopes, which are less likely to act as immunogens in the infected body.
- HBV particles and small spherical particles in the sample are present! / ⁇ to detect HBs antigen present in the tubular particles using the probe of the present invention, inside the lipid bilayer, spherical or tubular particles. It is necessary to make the epitope on the amino acid sequence from the 26th force to the 80th force of the localized HBs antigen accessible to the probe.
- Another embodiment of the present invention is a denaturing agent capable of denaturing lipid bilayers or protein aggregates when detecting the HBs antigen in this specimen using the probe of the present invention, typically Is a method for detecting HBV or HBs antigens in a sample, including adding a protein denaturant such as a surfactant or chaotropic ion, particularly a surfactant, to the sample.
- a protein denaturant such as a surfactant or chaotropic ion, particularly a surfactant
- the present invention provides an HBV particle, a small spherical particle and a tube by using a modifier.
- Denature the particles to expose the region consisting of the 26th to 80th amino acid sequences of the HBs antigen existing inside them, that is, the hydrophilic region present inside the lipid bilayer of the HBs antigen.
- the denaturing agents that can be used here disrupt the lipid bilayer of the HBV virus particles and inactivate the probe itself, while separating the binding (aggregation) between HBs antigens in small spherical and tubular particles.
- Sena! / A denaturant, and typically a surfactant such as sodium dodecyl sulfate can be used.
- the present invention includes a method for detecting an HBV virus and a configuration for using the probe of the present invention, a denaturant, and a probe that can specifically recognize an HBV antigen other than the HBs antigen. That is, a reagent for detecting HBV comprising the probe of the present invention, a denaturing agent, and a probe for HBV antigens other than HBs antigen is also provided.
- HBV virus antigens other than HBs antigen such as HBcr antigen
- HBV-infected specimens that can be erroneously determined to be HBV-negative by detection of only the HBs antigen can be reliably regarded as HBV-positive.
- HBs antigens are known to cause mutations as frequently as RNA viruses. Therefore, there is a mutant HBs antigen in which the 26th force of the HBs antigen is different from the amino acid sequence of SEQ ID NO: 1 in the 80th amino acid sequence.
- HBs antigens having strong mutations are identified, their amino acid sequences can be expressed and purified in E. coli according to the disclosure of the present specification. It is also possible to obtain a probe for the purified mutant HBs antigen and thereby detect the HBs antigen. Therefore, in the present invention, the sequence corresponding to the 26th to 80th amino acid residues of the HBs antigen is not limited to that shown in SEQ ID NO: 1. Therefore, the present invention includes the amino acid sequence shown in SEQ ID NO: 1. It is not limited to probes that recognize epitopes on powerful peptides, nor is it limited to detection methods using the probes.
- the probe of the present invention and the detection method of HBV virus or HBs antigen using the probe are based on the common antigenic determinant a of HBs antigen that cannot be detected by the conventional HBs antigen detection method. Escape mutants can be detected with high sensitivity, and HBV infection can be reliably determined. Moreover, even when the patient's own antibody against the common antigenic determinant a of the HBs antigen inhibits the detection of the HBs antigen, the detection method of the present invention can reliably determine HBV infection.
- HBV infection can be more reliably detected by measuring HBs antigen and other antigen of HBV simultaneously in combination with a probe that recognizes other antigen of HBV.
- the probe of the present invention is an amino acid sequence corresponding to the 26th to 80th amino acids of the HBs antigen, for example, as long as it specifically recognizes an epitope on the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
- the power that can be used for any antibody Typically, antibodies obtained using the peptide or HBs antigen as an antigen, particularly monoclonal antibodies are useful.
- the peptide having the amino acid sequence ability represented by SEQ ID NO: 1 can be prepared by a recombinant gene technique using a gene encoding the peptide or a chemical synthesis method. It can be obtained using various methods or devices known per se.
- the gene fragment containing the base sequence encoding the amino acid sequence of SEQ ID NO: 1 can be prepared by separating a viral gene from HBV patient serum and amplifying the target gene by PCR. Furthermore, it can be cloned into an expression vector using a restriction enzyme site derived from a linker added at the time of PCR preparation or a restriction enzyme site derived from a plasmid into which the gene fragment is inserted.
- This expression vector is introduced into a host such as Escherichia coli by transformation, and the Escherichia coli is cultured to obtain an antigen of HBs (26-80) located inside the lipid bilayer membrane.
- the method for collecting and purifying the target protein from the cells obtained by culturing can be achieved by conventional techniques such as ultrasonic disruption of cells, centrifugation, and various chromatographic operations. In other words, when the target protein is efficiently expressed by the method described above, many proteins are found in the cells. Insoluble granules are formed.
- the cells are suspended in a buffer solution of physiological conditions such as physiological saline, then the cells are disrupted by sonication, and the disrupted cells are centrifuged to recover the insoluble fraction. To do.
- the recovered insoluble fraction can be extracted with 6M urea to obtain a highly pure 1 ⁇ 13 ⁇ 43 (26-80) antigen by gel filtration and then used as an immunogen.
- the probe of the present invention for example, a polyclonal antibody, can be used for the above-mentioned HBs (26-80) antigen or polypeptide (hereinafter referred to as the present antigen) alone or BSA to animals such as rats, rabbits, goats, and sheep.
- the present antigen an antigen conjugated with KLH, etc.
- it can be prepared by mixing with an adjuvant such as Freund's complete adjuvant and periodically immunizing and collecting serum.
- an adjuvant such as Freund's complete adjuvant
- Hypridoma The production of monoclonal antibodies by Hypridoma is well known.
- the antigen is immunized periodically by mixing it with an adjuvant such as Freund's complete adjuvant alone or as an antigen combined with BSA, KLH or the like.
- an adjuvant such as Freund's complete adjuvant alone or as an antigen combined with BSA, KLH or the like.
- this antigen is administered into the tail vein as the final immunization, and the spleen is aseptically removed and then fused with an appropriate mouse myeloma cell line to obtain a hyperidoma.
- This method can be performed according to the method of Kohler and Milstein (Nature 256: 495-497, 1975).
- the hyperidoma obtained by the above method is cultured in an appropriate culture solution, and then a hybridoma cell line producing an antibody exhibiting a specific reaction against the present antigen is selected and cloned. .
- a hybridoma cell line producing an antibody exhibiting a specific reaction against the present antigen is selected and cloned.
- soft agar method (Eur J Immunol. 6: 511-519, 1976) can be used for the cloning of antibody-producing hyperpridoma.
- This hybridoma can be cultured in the culture medium or in the peritoneal cavity of mice to produce monoclonal antibodies in the culture medium or ascites.
- Polyclonal antibodies in serum and monoclonal antibodies produced in culture medium or ascites can be purified by a method such as protein A column chromatography.
- Polyclonal antibodies can be purified only for antibodies that react with a specific antigen by a method such as affinity chromatography using a carrier-fixed antigen. In this way, an antibody that does not react with a specific antigen can be obtained.
- molecules used as probes can be prepared.
- the recombinant antibody is described in detail in Hoogenboon's review (Trends in Biotechnology, 15: 62-70, 1997).
- the denaturant used in the present invention can break down the structure of the lipid bilayer membrane of HBV particles, or separate the binding (aggregation) between HBs antigens in small spherical particles and tubular particles that also have HBs antigenic power.
- Any modifier can be used.
- urea, acidifying agents, alkali agents, etc. can be used, but surfactants are particularly effective.
- nonionic surfactants such as Tween 20 and Nonidet P-40 are not so strong in surface activity, but can sufficiently destroy the lipid bilayer structure and expose the epitope in the present invention.
- anionic surfactants such as SDS and Sarcosyl are considered to have a strong surface-active effect. Such surfactants can also expose the epitope in the present invention. When treated with a strong detergent, the structural epitope of the protein may be destroyed and the antibody recognizing the structural epitope may not be able to bind to the antigen.
- the denaturing agent not only efficiently releases the HBs antigen present in the specimen, but also has a role of allowing the monoclonal antibody to easily bind to the HBs antigen.
- a probe that specifically binds to an antigen denatured with a surfactant as described above.
- an antibody it is necessary that the probe is exposed by such a denaturation treatment and can be bound to the denatured epitope in the present invention.
- an antibody suitable for the immunoassay of the present invention can be obtained by immobilizing the modified peptide antigen and screening for a monoclonal antibody that reacts in a solution containing a surfactant. Since such an antibody contains a surfactant in the screening solution, the monoclonal antibody itself can obtain an antibody resistant to the modification action of the surfactant.
- HBs antigens in samples treated with denaturants may be enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay, radioimmunoassay, aggregate-based assay, or other It is possible to detect by the well-known immunoassay method.
- ELISA enzyme-linked immunosorbent assay
- enzyme-linked immunosorbent assay enzyme-linked immunosorbent assay
- radioimmunoassay radioimmunoassay
- aggregate-based assay or other It is possible to detect by the well-known immunoassay method.
- a labeled antibody for detection, for example, a fluorescent substance, a chemiluminescent substance, a radioactive substance, an enzyme, or the like is used as the label.
- a method based on the sandwich reaction system of ELISA is used in order to detect HBs antigen in a specimen, it is carried out by the following steps. First, an antibody that recognizes an epitope located inside the lipid bilayer is bound to a solid support (for example, the inner wall of a microtiter well). Next, to eliminate non-specific reactions, block with bovine serum albumin. A specimen treated with a surfactant or the like is added to the support, and the HBs antigen is captured by the immobilized antibody. The HBs antigen can be detected by reacting the labeled antibody against the captured HBs antigen. Any antibody can be used as long as it binds to the epitope located inside the lipid bilayer membrane. As the labeled antibody, any antibody that binds to the HBs antigen can be used. This combination is free, and a combination with high sensitivity and high specificity can be selected.
- solid supports examples include polystyrene, polycarbonate, polypropylene, polyvinyl microtiter plates, test tubes, pills, beads (lattus particles, red blood cells, metal compounds, etc.), membranes (ribosomes, etc.) ) And filters.
- Samples capable of measuring HBs antigen in the present invention include whole blood, plasma, serum, urine, saliva , Biological fluids such as cerebrospinal fluid, and tissues such as liver tissue.
- a method for treating HBs antigen in a sample into a state suitable for binding reaction with a probe such as a monoclonal antibody without complicated operation is important.
- a probe such as a monoclonal antibody without complicated operation
- pATtrpE DNA an expression vector
- restriction enzyme reaction solution 20 ⁇ 1 [50 mM Tris—HCl (pH 7.5), lOmM MgC12, ImM dithiothreitol, lOOmM NaCl, 15 units Of EooRI and 15 units of BamHI enzyme) at 37 ° C for 1 hour, water 39 1 in the reaction mixture, heat-treated at 70 ° C for 5 minutes, and then bacterial alkaline phosphatase (BAP) 1 ⁇ 1 (250 units ⁇ ⁇ 1) was added and incubated at 37 ° C for 1 hour.
- BAP bacterial alkaline phosphatase
- the sensitive Escherichia coli strain used for transformation was the salt-calcium calcium method [Mandel, M. and Higa,
- Transformation E. coli was spread on an LB plate (1% tryptone, 0.5% NaCl, 1.5% agar) containing 25 g / ml ampicillin and incubated at 37 ° C. A colony of bacteria produced on the plate was picked from one platinum loop, transferred to LB medium containing 25 ⁇ g Zml ampicillin, and cultured at 37 ° C.
- 2YT medium expression plasmids pATtrpE-HBs 3ml containing ampicillin 50 i u gZml Escherichia coli HB101 strain with (26-80) was inoculated into (1.6% tryptone, 1% yeast extract, 0. 5% Na C1) Incubate at 37 ° C for 9 hours.
- This insoluble fraction was dissolved in 3 ml of PB S containing 8 M urea, 10 mM dithiothreitol, and ImM EDTA, and gel filtration was performed on a Cefacryl S300HR column in the presence of 6 M urea, and tr pE-HBs (26-80) The fusion antigen was purified almost singly.
- the polypeptide [trpE-HBs (26-80)] prepared by the above method is dissolved in 6M urea, and the final concentration is 0.2-0.1 in 10 mM phosphate buffer (pH 7.3) (PBS) containing 0.15 M NaCl. 1. Dilute to OmgZml, mix with equal volume of Freund's adjuvant, and administer 10-20 g intraperitoneally to 4-6 weeks old BALBZc mice. Similar booster immunizations were performed every 2-4 weeks, and HBs 10 / z g dissolved in PBS was administered into the tail vein as the final immunization.
- PBS phosphate buffer
- the spleen was aseptically removed from this mouse, the spleen was loosened into individual cells using scissors and metal mesh, and washed three times with RPMI-1640 medium.
- the mouse myeloma cell line Sp2 / 0Agl4 in the logarithmic growth phase was washed with RPMI-1640 medium three times, and then the cells and spleen cells were mixed at a cell number ratio of 1: 5.
- PEG polyethylene glycol
- the fused cells were removed from PEG by centrifugation (200 X g, 5 min), and then added to RPMI-1 640 medium containing 10% urine fetal serum and hypoxanthine, aminopterin and thymidine (HAT). Suspended and seeded into 96-well cell culture plates. After culturing for about 10 days to grow only the hyperpridoma, clones producing the desired antibody were searched by ELISA to obtain a hyperidoma producing a monoclonal antibody having the desired reaction specificity.
- the obtained hybridoma was single-cloned by the limiting dilution method to establish an antibody-producing hybridoma.
- the resulting hyperpridoma was named 6G6, 4 A3, 1C10. These hybridoma cells were deposited on September 9, 2004 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary.
- the hyperpridoma obtained by the method described in Example 2 was preliminarily administered by intraperitoneal administration of pristane! And transplanted into the peritoneal cavity of BALBZc mice to obtain a monoclonal antibody produced in ascites. . From the monoclonal antibody, the IgG fraction was separated and purified by affinity chromatography using a protein A sepharose column.
- the obtained monoclonal antibody was subjected to epitope analysis using a TrpE-HBs (26-80) antigen and a synthetic peptide having a strength of 20 amino acids synthesized by a sequence derived from the HBs region. It was found that this is a monoclonal antibody that recognizes an epitope (amino acid number: 26 to 80) located inside the lipid bilayer of the HBs antigen o
- the (sub) class of each monoclonal antibody was identified by an isotype typing kit (Zymed) using anti-mouse Ig isotype antibodies.
- Table 2 6G6 and 4A3 were IgGl and ⁇ , and IC10 was IgG2a and ⁇ .
- Table 3 shows the results of measuring HBs-positive sera using various surfactants.
- HBs antigen-positive serum was measured using a buffer containing a surfactant, HBs antigen could not be detected, but various types of surfactant (anionic, When measured using a buffer added with (cationic, amphoteric, nonionic), a sufficient signal was obtained, and the HBs antigen could be clearly detected. This revealed that various surfactants exposed and detected novel epitopes existing inside the lipid bilayer of HBs antigen.
- 5C3 which is a peroxidase-labeled monoclonal antibody, expresses and purifies an antigen having the amino acid sequence of amino acids 1 to 226, which is the entire length of the HBs antigen, in the same manner as in Example 1, and the recombinant antigen Is a monoclonal antibody obtained by immunizing mice. It was confirmed that this antibody binds to the above recombinant HBs antigen. However, as a result of investigating the binding between the antibody and a synthetic peptide having 20 amino acid strength overlapping by 10 amino acids based on the amino acid sequence of 1 to 226 of the HBs antigen in the same manner as in Example 3, The antibody did not react with any synthetic peptide. Therefore, 5C3 is considered to recognize structural epitopes that are not continuous Epitopes of the amino acid sequence of the HBs antigen.
- a specimen that is negative for HBs antigen but considered to be infected with HBV was measured by a method improved from the method of Example 4.
- Anti-HBs antigen monoclonal antibody 6G6 was diluted with 10 mM sodium phosphate buffer (pH 7.3) containing 0.15 M NaCl to a final concentration of 6 ⁇ g Zml, and a 96-well microphone plate (Nunk) 100) was dispensed per well. After standing at 4 ° C, wash twice with 0.35 ml of lOmM sodium phosphate buffer (pH 7.3) containing 0.15 M NaCl, containing 0.5% sodium caseinate and 3% sucrose. Add 0.35 ml of lOmM sodium phosphate buffer (PH7.3) (blocking solution) and let stand at room temperature for 2 hours. It was.
- the buffer solution (0.15 M NaCl, 10 mM EDTA—2Na, 0.2% procrine, 1% BSA, 0.1% sodium caseinate, 3% horse Serum, 2% mouse serum, 10% Brij35 containing lOOmM sodium phosphate buffer (pH 7.0) 5 0 1 and measurement sample 50 1 were added to each well and allowed to react at room temperature for 1 hour Thereafter, the plate was washed 5 times with 0.35 ml of washing solution, and further peroxidase-labeled monoclonal antibody (5C3) 100 1 was added and reacted at room temperature for 30 minutes.
- 5C3 peroxidase-labeled monoclonal antibody
- the plate was washed 6 times with 0.35 ml of the above-described washing solution, and the substrate (ortho-phenylene, hereinafter referred to as OPD) solution 100 1 was added and reacted at room temperature for 30 minutes, and then a 2N sulfuric acid solution was added.
- OPD substrate
- the absorbance at a wavelength of 492 nm (OD492) was measured using the absorbance at a wavelength of 630 nm as a control.
- HBs antigen and anti-HBs antibody were measured by AB BOTT's CLIA method, and HBN-DNA was measured by Gen-Probe's TMA method.
- a BBOTT's CLIA method is also positive for HBs antibody and is considered to be serum from HBV-infected patients. available.
- ABBOTT's HBsAgCLIA method which is a conventional method for measuring HBs antigen, has been determined to be No. 2, No. 3, and No. 5!
- HBs antigens could be detected in the No. 2 and No. 5 specimens.
- the No. 3 specimen that was determined to be antigen-negative by both the measurement method of the present invention and the ABBTT HBsAgCLIA measurement method can be detected by the HBcrAg measurement method. Simultaneous measurement of HBs antigen and HBcr antigen is not possible. It is useful for more reliable detection of HBV antigen.
- HBV antigen-negative sample or HBV antigen-positive sample containing anti-HBs antibody # 990493, # 990640, # 990650
- 50 ⁇ L of HBV antigen-negative sample or HBV antigen-positive sample containing anti-HBs antibody # 990493, # 990640, # 990650
- 50 ⁇ L of various concentrations of aqueous hydrochloric acid 50 ⁇ L of various concentrations of aqueous hydrochloric acid
- the anti-HBs antigen monoclonal antibody 6G6 was diluted with 10 mM phosphate buffer (pH 7.3) containing 0.15 M NaCl to a final concentration of 6 ⁇ gZml, and a 96-well microphone mouthplate was obtained. (Nunk Co., Ltd.) 100 ⁇ l was dispensed per well. Incubate at 4 ° C.
- HBs antigen positive specimens (# 990493, # 990640, # 990650) including anti-HBs antibodies are Strong force that could hardly detect HBs antigen activity even after incubation for 10 minutes at room temperature in a solution containing no hydrochloric acid. HBs antigen activity was observed from 0.05N of hydrochloric acid concentration at the time of treatment. The peak was reached (Table 5).
- HBV antigen negative specimen or HBs antigen positive specimen (# 990493, # 990640, # 9906 50)
- the hydrochloric acid concentration and surfactant concentration shown in the table are expressed as the concentration at the time of treatment after mixing the sample and the treatment agent.
- An amphoteric surfactant or a cationic surfactant having a single-chain alkyl group and a tertiary amine or quaternary ammonium salt in the same molecule as the acidic agent Increased measurement sensitivity was observed by adding.
- the acidifying agent was removed from the acidifying agent and the surfactant treatment agent, and the treatment was performed only with the effective surfactant, but the measurement sensitivity was greatly reduced. Therefore, the increase in measurement sensitivity is due to the inactivation of anti-HBs antibody, which is an inhibitor when HBs antigen is detected by an acidifying agent, and the addition of a surfactant to the lipid bilayer membrane of the HBs antigen in the sample.
- the epitopes that existed inside of were suggested, and the reactivity with 6G6 was thought to be greatly improved.
- HBV negative sample HBV exposure sample Healthy subject serum # 990493 # 990640 # 990650 Concentration (
- Tetradecyltrimethylammmonium Chl rid 0.5 0.029 0.924 0.261 0.357
- HBV Fertility Sample HBV Fertility Sample Healthy Blood # 990493 # 990640 # 990650 Intensity (%)
- HBV negative sample HBV fertile sanre Healthy blood sample # 990493 # 990640 # 990650 Concentration (%)
- Triton X-100 0.5 0.01 1 0.545 at 17 0.284
- Triton X-114 0.5 0.006 0.470 0.t t2 0.283
- HBV negative specimen or HBs antigen positive specimen (# 990493, # 990640, # 990650) In 30 / L, dissolve the protein denaturant urea or guanidine hydrochloride in 1. ON hydrochloric acid aqueous solution, After adding XL and incubating at room temperature for 10 minutes, 50 were used as measurement samples and examined by the method described in 1) above. Table 10 shows the immunoreactivity of each HBs antigen positive sample. The hydrochloric acid concentration and the protein denaturant concentration shown in Table 10 are expressed as the concentration at the time of treatment after mixing the sample and the treatment agent.
- HBV antigen-negative specimen Normal plasma
- 3 HBs antigen-positive specimens # 9904 93, # 990640, # 990650
- 30 This, 1. Solution containing hydrochloric acid
- This reducing agent dithiothreitol, 2-mercapto Add 30 L of a mixed solution of ethylamine hydrochloride and 2-jetylaminoethane hydrochloride, and incubate at room temperature for 10 minutes. Using 50 ⁇ L of the sample as the measurement sample, 1) (Table 11).
- the reducing agent concentration used here was expressed as the concentration at the time of treatment of the specimen. In HBV antigen-negative specimens, there was little change in the signal even when a reducing agent was added. In the Bs antigen positive specimen, dithiothreitol concentration was 1 to 5 mM, and an increase of 30% or more was observed in one specimen (# 990640).
- HBV antigen negative sample or HBV antigen positive sample containing anti-HBs antibody (# 990493, # 990640, # 990650) Add 50 ⁇ L of various concentrations of aqueous sodium hydroxide solution and incubate for 10 minutes at room temperature Using the 50 L sample as the measurement sample, the following measurement method was used.
- the anti-HBs antigen monoclonal antibody 6G6 was diluted with 10 mM phosphate buffer ( ⁇ 7.3) containing 0.15 M NaCl to a final concentration of 6 gZml, and a 96-well microphone mouthplate ( Nunk) 100 ⁇ l was dispensed per well. Incubate at 4 ° C.
- HBsl 24, a monoclonal antibody labeled with piotin, was expressed and purified by the method according to Example 1 using an antigen consisting of amino acids 1 to 226 which is the full length of the HBs antigen, and the recombinant antigen Is a monoclonal antibody obtained by immunizing mice. It was confirmed that this antibody binds to the above recombinant HBs antigen.
- the antibody did not react with any synthetic peptide. Therefore, it is considered that HBsl24 recognizes a structural epitope that is not a continuous epitope of the amino acid sequence of the HBs antigen.
- HBV-positive specimens containing anti-HBs antibodies (# 990493, # 990640, # 990650) cannot detect HBs antigen activity even when incubated for 10 minutes at room temperature in a solution that does not contain sodium hydroxide. Increased signal for HBs antigen was observed at sodium hydroxide concentrations of 0.25N to 1N during treatment (Table 12).
- HBV antigen negative sample or HBs antigen positive sample (# 990493, # 990640, # 9906 50) 30 / zL dissolved in various aqueous solutions of 1. ON sodium hydroxide and sodium hydroxide. After incubation at room temperature for 10 minutes, 50 ⁇ L of the sample was used as a measurement sample and examined by the method described in 5) (Tables 13 to 17). In addition, the sodium hydroxide concentration and the surfactant concentration shown in the table are expressed as the concentration at the time of treatment after mixing the sample and the treatment agent.
- nonionic surfactants such as TritonX100, Tween 20, and Bridj 35, and surfactants having a steroid skeleton such as CHA PS were also observed to have an additive effect.
- An increase in measurement sensitivity was observed by adding a surfactant. Except for the alkaline agent and the treating agent of the alkaline agent and the boundary H surfactant, the treatment was carried out only with the effective surfactant, but no increase in the measurement sensitivity was observed.
- Anti-HBs antibody which is an inhibitor of HBs antigen measurement, is inactivated by the combination of alkaline agent and surfactant, and an epitope present inside the lipid bilayer of HBs antigen in the specimen is presented. The reactivity was considered to be greatly improved.
- HBV negative sample HBV positive sample Healthy human serum # 990493 # 990640 # 990650 Concentration (%)
- N-Lauroylsaroosine sodium salt 0.5 0.008 0.254 0.280 0.143
- Triton X-100 0.5 0.021 0.305 0.174 0.108
- Triton X- 114 0.5 0.023 0.293 0.128 0.1 1 1
- HBs antigen negative sample or HBs antigen positive sample (# 990493, # 990640, # 99065 0) 30 and the protein denaturant urea or guanidine hydrochloride is dissolved in 1. ON sodium hydroxide aqueous solution. Then, 30 L was added and incubated at room temperature for 10 minutes. The 50 // L was used as a measurement sample and examined by the method described in 5) above.
- Table 18 shows the immunoreactivity of each HBs antigen positive specimen. The sodium hydroxide concentration and the protein denaturant concentration shown in Table 18 are expressed as the concentration at the time of treatment after mixing the sample and the treatment agent.
- HBV antigen negative sample or 3 HBs antigen positive samples (# 990493, # 990640, # 990650) 30 / z L, 1. ON a solution containing sodium hydroxide and dithiothreitol, 2-mercapto Add 30 ⁇ L of a mixed solution of ethylamine hydrochloride, jetylaminoethanethiol hydrochloride, 2-mercaptoethanol, and tri (2-carboxyethyl) phosphine hydrochloride, and add 10 mL at room temperature. Incubation was performed for 5 minutes, and 50 ⁇ L of the sample was used as a measurement sample and examined using the method described in 5) (Table 19).
- the reducing agent concentration used here was expressed as the concentration at the time of processing of the specimen.
- HBV antigen-negative specimens there was almost no change in the signal even when a reducing agent was added.
- Bs antigen-positive specimens 3-mercaptoethylamine hydrochloride and jetylaminoethanethiol hydrochloride were used in all three cases.
- the concentration of salt, 2-mercaptoethanol is about 2 to 3 times at 20 mM. An increase in nulls was observed. More effective was tri (2-carboxyethyl) phosphine hydrochloride, which showed a 1.5-fold increase in signal at a concentration of 2 mM and a 15-fold increase in signal at lOmM.
- FIG. 1 schematically shows the secondary structure of HBs antigen
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CN2005800314440A CN101023098B (zh) | 2004-09-22 | 2005-09-21 | 乙肝病毒s抗原的检测方法 |
US11/663,517 US8679762B2 (en) | 2004-09-22 | 2005-09-21 | Method of detecting hepatitis B virus s antigen |
JP2006536401A JP4430677B2 (ja) | 2004-09-22 | 2005-09-21 | B型肝炎ウイルスs抗原の検出法 |
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JP2007278902A (ja) * | 2006-04-07 | 2007-10-25 | Abbott Japan Co Ltd | パルボウイルスb19抗原測定方法 |
WO2008053901A1 (en) | 2006-10-30 | 2008-05-08 | Advanced Life Science Institute, Inc. | METHOD FOR ANALYSIS OF HEPATITIS B VIRUS s ANTIGEN |
JP2011112631A (ja) * | 2009-11-30 | 2011-06-09 | Sysmex Corp | C型肝炎ウイルスのコア蛋白検出のための試料の前処理方法及び前処理用試薬キット |
JP5332011B2 (ja) * | 2006-10-30 | 2013-11-06 | 株式会社先端生命科学研究所 | B型肝炎ウイルスの高感度免疫学的分析方法及び免疫学的分析用試薬 |
WO2014115878A1 (ja) | 2013-01-28 | 2014-07-31 | シスメックス株式会社 | HBs抗原を検出するための試料の前処理方法およびその利用 |
JP2021505906A (ja) * | 2017-12-04 | 2021-02-18 | シァメン・イノドックス・バイオテック・カンパニー・リミテッド | HBsAgの定量検出のためのキット及び方法 |
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CN102094002A (zh) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | 乙型肝炎病毒dna提取试剂及提取方法 |
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EP2088431A1 (en) * | 2006-10-30 | 2009-08-12 | Advanced Life Science Institute, Inc. | METHOD FOR ANALYSIS OF HEPATITIS B VIRUS s ANTIGEN |
EP2088431A4 (en) * | 2006-10-30 | 2010-07-21 | Advanced Life Science Inst Inc | METHOD OF ANALYZING ANTIGEN S OF HEPATITIS B VIRUS |
JP5332011B2 (ja) * | 2006-10-30 | 2013-11-06 | 株式会社先端生命科学研究所 | B型肝炎ウイルスの高感度免疫学的分析方法及び免疫学的分析用試薬 |
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JP2011112631A (ja) * | 2009-11-30 | 2011-06-09 | Sysmex Corp | C型肝炎ウイルスのコア蛋白検出のための試料の前処理方法及び前処理用試薬キット |
WO2014115878A1 (ja) | 2013-01-28 | 2014-07-31 | シスメックス株式会社 | HBs抗原を検出するための試料の前処理方法およびその利用 |
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 |
US10352937B2 (en) | 2013-01-28 | 2019-07-16 | Sysmex Corporation | Pretreatment method of sample for detecting HBs antigen and use thereof |
JP2021505906A (ja) * | 2017-12-04 | 2021-02-18 | シァメン・イノドックス・バイオテック・カンパニー・リミテッド | HBsAgの定量検出のためのキット及び方法 |
JP7194746B2 (ja) | 2017-12-04 | 2022-12-22 | シァメン・イノドックス・バイオテック・カンパニー・リミテッド | HBsAgの定量検出のためのキット及び方法 |
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