WO2013039165A1 - 抗ヒトノロウイルスgii抗体 - Google Patents
抗ヒトノロウイルスgii抗体 Download PDFInfo
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- WO2013039165A1 WO2013039165A1 PCT/JP2012/073511 JP2012073511W WO2013039165A1 WO 2013039165 A1 WO2013039165 A1 WO 2013039165A1 JP 2012073511 W JP2012073511 W JP 2012073511W WO 2013039165 A1 WO2013039165 A1 WO 2013039165A1
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- gii
- amino acid
- norovirus
- antibody
- strain
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- 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
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
Definitions
- the present invention relates to an antibody against human norovirus GII. More specifically, the present invention relates to an anti-human norovirus GII antibody for detecting human norovirus GII in a specimen by an immunoassay.
- Human norovirus orally infects humans and proliferates from the duodenum to the upper small intestine, causing infectious gastroenteritis. Causes small intestinal epithelial cells to fall off the duodenum, causing symptoms such as vomiting, diarrhea, and abdominal pain.
- the incubation period from infection to onset is 12 to 72 hours (on average 1 to 2 days), and virus discharge to the stool continues for about 1 to 3 weeks even after symptoms have subsided, with more than 7 weeks being reported. ing. About 70% of reported food poisoning patients have norovirus infection.
- Norovirus is a virus that does not have an envelope with a plus single-stranded RNA of about 7,500 bases in the genome.
- ORFs protein coding regions
- ORF1 is a nonstructural protein involved in viral replication
- ORF2 is a capsid structural protein (VP1)
- VP3 is a minor structural protein (VP2) It is reported that it is coding.
- Noroviruses are classified into five groups, genogroups I to V (GI to GV), based on the similarity of capsid gene sequences. Among these, GI, GII, and GIV are the mainstream of human infections.
- Genogroup I and Genogroup II (GII) are particularly rich in genetic diversity, and various viruses with different evolutionary strains are detected from human-derived samples.
- Genogroup I has 14 or more types.
- Genotype and genogroup II are classified into 17 or more genotypes.
- Norovirus is detected by detecting the capsid structure protein using an antibody by an enzyme immunoassay (EIA) method (see Non-Patent Document 1) or an immunochromatography method (see Non-Patent Document 2). Therefore, in order to accurately detect human norovirus antigens, antibodies that react with all genotypes are required.
- EIA enzyme immunoassay
- the present invention relates to providing an anti-human norovirus GII antibody that reacts with human noroviruses of almost all genotypes belonging to GII and can collectively detect human norovirus GII.
- the present invention has been studied to obtain antibodies that react in common with human noroviruses of genogroup GII.
- antibodies that bind to specific sites in the P (Protruding) region of capsid proteins of human noroviruses belonging to GII have been found that it binds broadly to human noroviruses of GII and can specifically detect almost all human noroviruses of genotypes (GII / 1 to GII / 17) belonging to the GII.
- the present invention relates to the following 1) to 4). 1) an epitope contained in the amino acid region represented by the following formulas (1) and (2) present in the P domain of the capsid structure protein of human norovirus GII, and the 483rd amino acid of the amino acid sequence represented by SEQ ID NO: 1 or An anti-human Norovirus GII antibody that binds to one or more epitopes consisting of amino acids corresponding thereto.
- the amino acid region represented by formula (1) is the 419th to 424th amino acid region of the amino acid sequence represented by SEQ ID NO: 1 or a region corresponding thereto
- the amino acid sequence represented by formula (2) is the sequence The anti-human Norovirus GII antibody of 1) above, which is the amino acid region from the 516th to the 525th of the amino acid sequence represented by No. 1 or a region corresponding thereto.
- a human norovirus GII detection reagent comprising the antibody according to 1) or 2) above.
- a method for detecting human norovirus GII comprising reacting a specimen suspected of containing human norovirus GII with the antibody of 1) or 2) above, and detecting the virus by an immunoassay.
- the anti-human norovirus antibody of the present invention it is possible to comprehensively detect human noroviruses of the GII group, and it is possible to efficiently detect human genotypes of human norovirus belonging to GII collectively.
- the anti-human Norovirus GII antibody of the present invention can bind to an amino acid region on the P domain that is considered to have few gene mutations, a detection reagent using this antibody constitutes an antibody in accordance with the epidemic type. It has the advantage that there is no need to correct it.
- alphabetic characters in formulas indicating amino acid regions mean single-letter codes of amino acids, and sequences are described in the order from the N-terminus to the C-terminus.
- the anti-human norovirus GII antibody of the present invention is represented by the epitope contained in the amino acid region represented by the following formulas (1) and (2) present in the P domain of the capsid structure protein of human norovirus GII, and SEQ ID NO: 1. It binds to one or more epitopes consisting of the 483rd amino acid of the amino acid sequence or an amino acid corresponding thereto.
- human norovirus GII means a human norovirus belonging to GII (geno group II).
- Human norovirus capsid structural protein (VP1) is known to consist of a shell region (S domain) and a protruding region (P domain). The S domain is thought to govern the assembly of VP1.
- the P domain is further divided into P1 and P2 subdomains.
- the P1 subdomain interacts with the S domain and enhances the physical stability of the capsid.
- the P2 subdomain is located at the outermost part of the virus particle.
- mouse norovirus is a target for neutralizing antibodies.
- amino acid region represented by the formulas (1) and (2) of the present invention, and the 483rd amino acid of the amino acid sequence represented by SEQ ID NO: 1 or the amino acid corresponding thereto is the P domain of the capsid structure protein of human norovirus GII It is considered that there are few gene mutations in the regions and amino acids that are present above and are highly conserved in each genotype, but no antibody that recognizes such regions or amino acids has been known so far.
- P represents proline
- G represents glycine
- E represents glutamic acid
- X 1 represents L (leucine), V (valine), N (asparagine), T (threonine), S (serine), M (methionine) or R (arginine).
- X 2 represents F (phenylalanine), Y (tyrosine), S (serine) or M (methionine), and is preferably F.
- Preferred examples of the amino acid region represented by the formula (1) include the following (1-1) to (1-9).
- F represents phenylalanine
- Y represents tyrosine
- L represents leucine
- P represents proline.
- X 3 represents V (valine) or G (glycine), and is preferably V.
- X 4 represents N (asparagine) or S (serine), and is preferably N.
- X 5 represents Q (glutamine), P (proline) or S (serine), and is preferably Q.
- X 6 represents S (serine), T (threonine) or I (isoleucine), and is preferably S or T.
- X 7 represents A (alanine) or S (serine), and is preferably A.
- X 8 represents M (methionine) or V (valine), and is preferably M.
- amino acid region represented by the formula (2) include the following (2-1) to (2-8).
- the amino acid region represented by the above formula (1) or (2) is present in the P domain of the capsid structure protein of human norovirus GII.
- the formula (1 ) Corresponds to the 419th to 424th amino acid region of the amino acid sequence represented by SEQ ID NO: 1
- the amino acid sequence represented by formula (2) is the amino acid sequence represented by SEQ ID NO: 1.
- the region corresponding to the 419th to 424th amino acid region of the amino acid sequence represented by SEQ ID NO: 1 the region corresponding to the 516th to 525th amino acid region, and the amino acid corresponding to the 483rd amino acid
- the 419th to the 419th amino acid sequence of SEQ ID NO: 1 It means the region or amino acid corresponding to the 424th amino acid region, the 516th to 525th amino acid region, and the 483rd amino acid (see FIG. 1).
- the region corresponding to the 419th to 424th amino acid region of the amino acid sequence represented by SEQ ID NO: 1 becomes the 426th to 431rd amino acid region, and the 516th to 525th amino acids.
- the region corresponding to the amino acid region is the 523rd to 532nd amino acid region, and the amino acid corresponding to the 483rd amino acid is the 490th amino acid.
- the anti-human Norovirus GII antibody of the present invention binds to an epitope contained in the above amino acid region or an epitope consisting of amino acids.
- the “epitope” is an antigenic determinant and means a structural site that specifically binds to an antibody.
- the epitope in the present invention may be a discontinuous amino acid in the region other than the continuous amino acid in a part of the amino acid region. “Binding” also refers to the interaction between a ligand and a substrate, and can be distinguished from background or non-specific or specific interactions.
- the anti-human Norovirus GII antibody of the present invention corresponds to an epitope contained in at least the amino acid region represented by the above formula (1) or (2), the 483rd amino acid of the amino acid sequence represented by SEQ ID NO: 1, or the like Any substance that binds to an epitope consisting of amino acids may be used, and those that bind to all of these epitopes are preferred.
- the epitope contained in the amino acid region represented by the formula (1) is preferably “X 1 ”
- the epitope contained in the amino acid region represented by the formula (2) is preferably “ X 4 ”and / or“ YX 6 -L ”.
- the 420th L of the amino acid sequence represented by SEQ ID NO: 1 and the formula (2-1) among the amino acid regions represented, those having one or more selected from 517th N, 520th to 522th YSL and 483th E as epitopes are preferred.
- the 427th V of the amino acid sequence represented by SEQ ID NO: 1 in the amino acid region represented by the formula (1-2), the formula (2-2) Among the amino acid regions represented, those having one or more selected from 524th N, 527th to 529th YSL and 490th E as epitopes are preferred.
- the anti-human norovirus GII antibody has 485 strain (GII / 1), NG1 strain (GII / 2), 809 strain (GII / 3), 18-3 strain (GII / 3), 336 Strain (GII / 3), 104 strain (GII / 4), 754 strain (GII / 5), 7k strain (GII / 6), 445 strain (GII / 6), 10-25 strain (GII / 7), U25 GII / 1 of strain (GII / 8), 876 strain (GII / 12), NG15 strain (GII / 13), 47 strain (GII / 14), Kamo strain (GII / 15), Alpha strain (GII / 17) It has the reactivity that it can bind to almost all of the GII gene group classified as ⁇ GII / 17 and does not bind to the GI gene group norovirus.
- the animal species from which the anti-human norovirus GII antibody of the present invention is derived is not particularly limited, but in terms of ease of antibody production, rats are preferred.
- the anti-human norovirus GII antibody of the present invention comprises IgG, IgA, IgY, IgD, IgM, IgE or a part of one or more thereof, such as a heavy chain, light chain, Fc or F (ab) part. Can be of any required type.
- the anti-human norovirus GII antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means.
- Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques.
- An anti-human norovirus antibody-producing hybridoma can be basically produced using a known technique as follows. That is, a recombinant GII norovirus-like hollow particle (VLP) is used as a sensitizing antigen and immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. And can be prepared by screening monoclonal antibody-producing cells by an ordinary screening method.
- VLP GII norovirus-like hollow particle
- Recombinant norovirus GII VLP inserts the capsid gene sequence of human norovirus GII into a transfer vector, and simultaneously transfects baculovirus DNA and the aforementioned plasmid into Sf9 cells to produce a recombinant virus. And propagate to obtain seed virus. Then, by expressing the protein in Tn5 cells, the desired recombinant norovirus GII VLP can be purified from the cells or culture supernatant by a known method.
- the mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters and the like are used.
- Immunization of animals with a sensitizing antigen is performed according to a known method.
- the sensitizing antigen is injected by intraperitoneal or subcutaneous injection of a mammal.
- the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, etc., and then mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary.
- an ordinary adjuvant for example, Freund's complete adjuvant
- a polyclonal antibody After immunizing in this manner and confirming that the desired antibody level rises in the serum, a polyclonal antibody can be obtained by collecting blood from the mammal and purifying the serum component.
- an affinity column or the like on which a sensitizing antigen is immobilized can be used.
- immune cells are taken out from a mammal whose antibody level has increased and cell fusion is performed.
- Spleen cells are particularly preferred as preferred immune cells for cell fusion.
- myeloma cells of mammals as the other parent cells to be fused with the immune cells, various known cell lines such as P3X63, NS-1, MPC-11, SP2 / 0 and the like are appropriately used.
- the cell fusion between the immune cells and myeloma cells can be performed according to a known method, for example, the method of Keller et al. (Kohler et al., Nature, Vol. 256, p495-497 (1975)). That is, in the presence of a cell fusion promoter such as polyethylene glycol (PEG having an average molecular weight of 1000 to 6000, concentration of 30 to 60%), Sendai virus (HVJ) and the like, an auxiliary agent such as dimethyl sulfoxide is optionally added, and RPMI1640 culture solution Fusion cells (hybridomas) are formed by mixing immune cells and myeloma cells in a nutrient culture solution such as a MEM culture solution.
- a cell fusion promoter such as polyethylene glycol (PEG having an average molecular weight of 1000 to 6000, concentration of 30 to 60%)
- Sendai virus (HVJ) and the like an auxiliary agent such as dimethyl sulfoxide is optional
- the hybridoma formed by fusion is cultured for 1 to 7 days in a selective medium such as a medium containing hypoxanthine, thymidine and aminopterin (HAT medium), and separated from unfused cells.
- a selective medium such as a medium containing hypoxanthine, thymidine and aminopterin (HAT medium)
- HAT medium a medium containing hypoxanthine, thymidine and aminopterin
- the resulting hybridoma is further selected according to the antibody it produces.
- the selected hybridoma is single-cloned according to a known limiting dilution method and established as a monoclonal antibody-producing hybridoma.
- a known method can be used as a method for detecting the activity of the antibody produced by the hybridoma.
- an ELISA method, an agglutination reaction method, and a radioimmunoassay method can be mentioned.
- the hybridoma is cultured according to a usual method and obtained as a culture supernatant, or the hybridoma is administered to a mammal compatible therewith to proliferate, A method of obtaining ascites is employed.
- Antibody purification can be performed using known purification means such as salting out, gel filtration, ion exchange chromatography, or affinity chromatography.
- human norovirus GII in a specimen can be specifically measured and detected.
- the immunological measurement method is not particularly limited, but a sandwich method using an anti-norovirus GII antibody and a labeled anti-norovirus GII antibody is preferable, and a method using an immobilized anti-norovirus GII antibody and a labeled anti-norovirus GII antibody is more preferable.
- immobilized anti-norovirus GII antibody those immobilized on an insoluble support such as polystyrene plate, latex particle, magnetic particle, glass fiber membrane, nylon membrane, nitrocellulose membrane, cellulose acetate membrane and the like are preferable.
- labeled anti-human norovirus GII antibodies include known labels, such as radioactive isotopes (eg, 32 P, 35 S, 3 H), enzymes (eg, peroxidase, alkaline phosphatase, luciferase), proteins (eg, Avidin), low molecular weight compounds (eg biotin), fluorescent materials (eg FITC), chemiluminescent materials (eg acridinium), latex particles (eg colored latex particles, fluorescent latex particles), metals (eg gold, silver) , Noble metals such as platinum) colloidal particles, carbon atoms, and the like.
- radioactive isotopes eg, 32 P, 35 S, 3 H
- enzymes eg, peroxidase, alkaline phosphatase, luciferase
- proteins eg, Avidin
- low molecular weight compounds eg biotin
- fluorescent materials eg FITC
- chemiluminescent materials e
- Detection of Norovirus GII in the sample is performed by reacting Norovirus in the sample with the immobilized anti-Norovirus GII antibody.
- the sample-containing solution and the immobilized anti-Norovirus GII antibody are reacted.
- the reaction can be performed by reacting the labeled antibody or by reacting the immobilized anti-norovirus GII antibody and the labeled antibody simultaneously.
- the amount of Norovirus GII in the sample can be measured by measuring the amount of label in the complex formed by the Norovirus, the immobilized anti-Norovirus GII antibody and the labeled antibody in the sample.
- the amount of labeling can be measured by means according to the type of the label. For example, when an enzyme or avidin is used as a label, a substrate is added after the reaction, and the enzyme activity is measured. In addition, when fluorescence (including fluorescent latex particles) or a chemiluminescent substance is used as a label, the signal is measured under conditions where quenching does not occur. For colored latex particles, metal colloid particles, carbon particles, and the like, signals are measured visually or by reflected light. As the detection method of norovirus GII of the present invention, ELISA and immunochromatography are more preferable.
- the detection reagent (kit) containing the anti-human norovirus GII antibody of the present invention is composed of, for example, a specimen diluent, a labeled anti-norovirus GII antibody, a reaction substrate and the like in addition to the immobilized anti-human norovirus GII antibody of the present invention. It is preferable.
- Example 1 Acquisition of Anti-Norovirus GII Monoclonal Antibody The antibody used in the method of the present invention was prepared by the following method. 50 ⁇ g of GII norovirus-like hollow particles (VLP) were immunized several times with the adjuvant into the mouse abdominal cavity, and it was confirmed that the serum titer was increased. On the third day after booster immunization (intravenous), the spleen was removed to obtain splenocytes. This and mouse myeloma cells were fused in the presence of polyethylene glycol 3500 (10: 1 cells) to prepare hybridoma cells. The cells were cultured for 1 week at 37 ° C.
- VLP GII norovirus-like hollow particles
- VLP Norovirus-like hollow particles
- VLP Norovirus-like hollow particle
- PBS PBS
- pH 7.3 pH 7.3
- Norovirus virus-like hollow particles (VLP) were immobilized on a microtiter plate at 0.05 ⁇ g / well at 4 ° C. for 12 hours.
- the Norovirus-like hollow particle (VLP) / PBS, pH 7.3 solution that had been added to the wells was removed by decantation, and 145 mM NaCl, 3.6 mM Na 2 HPO 4 , 1.4 was added to the wells of the microtiter plate.
- the hybridoma culture supernatant was added at 100 ⁇ L / well to the well of the above-mentioned Norovirus-like hollow particle (VLP) solid-phased microtiter plate, and heated at 37 ° C. for 1 hour. Thereafter, the culture supernatant added to the well is removed by decantation, PBS-T is added to the well of the microtiter plate at 200 ⁇ L / well, and PBS-T is removed by decantation. Washed. This washing process was performed a total of 3 times.
- VLP Norovirus-like hollow particle
- Peroxidase-Conjugated Goat Anti-Mouse Immunoglobulins (manufactured by DAKO) was added at 100 ⁇ L / well (2000-fold dilution: 0.55 ⁇ g / mL) and heated at 37 ° C. for 1 hour.
- DAKO Peroxidase-Conjugated Goat Anti-Mouse Immunoglobulins
- 145 mM NaCl, 3.6 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 0.05% (v./v.) Tween 20, 0.5% (wt./v.) BSA (hereinafter, Enzyme-labeled antibody diluent) was used.
- reaction stop solution a 313 mM H 2 SO 4 solution (hereinafter referred to as reaction stop solution) was added at 100 ⁇ L / well to the substrate reaction solution in the well to stop the enzyme reaction in the well.
- VLP solid-phased norovirus-like hollow particles
- a monoclonal antibody capable of binding to the amino acid region on the P domain is produced by confirming the binding state of the monoclonal antibody and Norovirus in the same manner as described in Example 3 below. Can be selected with high reproducibility.
- the P domain protein of Norovirus-like hollow particles (VLP) is immunized to bind to the amino acid region on the P domain with good reproducibility.
- a hybridoma producing a monoclonal antibody that can be obtained can be obtained with good reproducibility.
- the method for obtaining the P domain protein is as described in Example 3.
- amino acid region represented by the formulas (1) and (2) of the present invention and the 483rd amino acid of the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence corresponding thereto are selected to comprise these amino acid sequences.
- a hybridoma producing a monoclonal antibody capable of binding to an amino acid region on the P domain can be obtained with good reproducibility.
- the polypeptide can be used as an antigen for human norovirus GII.
- This ascites supernatant (10 mL) was subjected to ammonium sulfate fractionation (final concentration 50% saturated ammonium sulfate) for 1 hour in an ice bath, and the precipitated immunoglobulin fraction was suspended and dissolved in PBS.
- This ammonium sulfate fractionation operation was performed twice in total to obtain a crude immunoglobulin fraction from ascites.
- An equal amount of 20 mM sodium phosphate, pH 7.0 (hereinafter referred to as 20 mM NaPB, pH 7.0) was mixed with the obtained immunoglobulin crude fraction (10 mL), and a protein G column (HiTrap Protein G HP, Affinity purification was performed using 5 mL: manufactured by Amersham BioSciences.
- the dialysis membrane used for the dialysis operation was a cellulose tube for dialysis (manufactured by Viskase Companies).
- the IgG elution fraction thus obtained was used as a purified product of anti-Norovirus GII monoclonal antibody (5B-18-3M), which was stored at 4 ° C. and used for the operations described later.
- the above-mentioned protein G column was connected to the BioLogic LP system (manufactured by Bio Rad Laboratories), and the flow rate was consistently 1 mL / min.
- Example 2 Reactivity of Anti-Norovirus GII Monoclonal Antibody Using the anti-norovirus GII monoclonal antibody (5B-18-3M) obtained in Example 1, a norovirus GII detection reagent using immunochromatography was prepared by the following procedure. . A solution containing 0.36 to 1.45 mg / mL of an anti-norovirus GII monoclonal antibody (5B-18-3M) on a nitrocellulose membrane sheet was applied linearly in an amount of 0.25 to 1.00 ⁇ L / 5 mm to obtain a test line. The control line was coated with anti-mouse globulin antibody as described above.
- a solution containing 0.04 to 0.15 w / v% latex bound with anti-norovirus GII monoclonal antibody (5B-18-3M) was designated as antibody-bound latex solution (5B-18-3M).
- a latex solution was impregnated into a conjugate pad to prepare a dried product.
- the sample pad, the membrane, the conjugate pad and the absorbent pad were attached so that the adjacent members partially overlap in this order, then covered with a plastic laminate, cut to a width of 5 mm, Make a test strip.
- the reaction of the reagent prepared above with each genotype of Norovirus was confirmed by the following procedure.
- the recombinant antigen of each genotype of Norovirus was suspended in the diluent, and 75 ⁇ L of the suspended antigen was dropped onto the sample strip sample pad prepared above. After standing at 15-30 ° C. for 15 minutes, the presence or absence of a line was confirmed. The results are shown in Table 1.
- the 124 strain means Hu / NV / GI / Aichi124-89 / 89 / JP (GeneBank accession number: AB031013) strain
- the 258 strain means Hu / NV / GI / Funabashi258 / 96 / JP (GeneBank accession number: AB078335) strain
- 645 strain means Hu / NV / GI / Kashiwa645 / 99 / JP (GeneBank accession number: BD011871) strain
- CV strain means Hu / NV / GI / Chiba407.
- strain means Hu / NV / GI / 8/99 / JP (GeneBank accession number: AB058547) strain means 485 strain means Hu / NV / GII / Noda485 / 00 / JP (GeneBank accession number: unregistered) strain NG1 strain means Hu / NV / GII / NG1 / 02 / JP (GeneBank accession number: AB195225) strain, and 809 strain means Hu / NV / GII / Sanbu809 / 98 / JP (GeneBank accession number: BD011876), 18-3 strain means Hu / NV / GII / Matsudo18 /
- anti-norovirus GII monoclonal antibody is 485 (GII / 1), NG1 (GII / 2), 809 (GII / 3), 18-3 (GII / 3) ), 336 (GII / 3), 104 (GII / 4), 754 (GII / 5), 7k (GII / 6), 445 (GII / 6), 10-25 (GII / 7) ), U25 strain (GII / 8), 876 strain (GII / 12), NG15 strain (GII / 13), 47 strain (GII / 14), Kamo strain (GII / 15), Alph strain (GII / 17) It was shown that it can bind to almost all of the GII gene groups classified into GII / 1 to GII / 17 and does not bind to the GI gene group norovirus.
- Example 3 X-ray crystal structure analysis
- an X-ray crystal structure analysis was performed according to the following procedure.
- coli BL21 cells (Invitrogen) and expression was induced with IPTG (1 mM) at 22 ° C. for 18 hours. His-tagged fusion P region protein was purified on a Ni column (Qiagen) and treated with HRV-3C protease (Novagen) at 4 ° C. overnight. Thereafter, the P region was purified by passing the treatment solution again through a Ni column. The P region was further purified by molecular sieve chromatography on a Superdex 200 column (GE), concentrated to 2-10 mg / ml and then GFB (0.35 M NaCl, 2.5 mM Tris pH 7.0, 0.02% NaN prior to crystallization). 3 ) Saved to.
- GE Superdex 200 column
- Fab fragment of anti-norovirus GII monoclonal antibody 5B-18-3M
- About 60 mg of purified 5B-18-3M IgG was used to prepare Fab.
- 5B-18-3M IgG was reduced with 100 mM dTT for 1 hour at 37 ° C.
- Reduced 5B-18-3M IgG is dialyzed with dialysis cassette at 4 ° C for 1 hour, substituted with GFB containing 20 mM HEPES (pH 7.7), and alkylated with GFB containing 2 mM iodoacetamide for 48 hours at 4 ° C. did.
- 5B-18-3M IgG was concentrated to 5 mg / mL and digested with papain using a kit (pierce, Rockford, USA). Digested 5B-18-3M IgG was purified only by Fab on a protein A column, and Fab was further purified by molecular sieve chromatography on Superdex 200 column (GE), concentrated to 5 mg / mL, and stored in GFB. The purified P region of Norovirus GII.10 and 5B-18-3M Fab were mixed at 1: 1, reacted at 25 ° C. for 1 hour, and finally purified by molecular sieve chromatography.
- PDB Protein Data Bank
- PDB code 1WEJ As a search model for Fab of anti-Norovirus GII monoclonal antibody
- PDB code 2OBR as a search model for Norovirus capsid P region
- PHASER structure analysis software by molecular replacement.
- a three-dimensional structure was constructed from the data and the amino acid sequence of the anti-Norovirus GII monoclonal antibody (5B-18-3M) and Norovirus GII.10 capsid protein P region. After that, the three-dimensional structure is adjusted with the manual model built in the model building software COOT, corrected with the TLS refinement of the refinement program REFMAC and the automatic structure determination software PHENIX, and superposition and mean square are performed by CCP4. Deviation (RMSD) was calculated and drawn on the molecular graphics tool PyMOL.
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Abstract
Description
したがって、遺伝子型の異なる多数のノロウイルスGIIを一括して検出できる抗体の創生が望まれていた。
1)ヒトノロウイルスGIIのカプシド構造タンパクのPドメインに存在する下記式(1)及び(2)で表されるアミノ酸領域に含まれるエピトープ、並びに配列番号1で示されるアミノ酸配列の483番目のアミノ酸又はこれに相当するアミノ酸からなるエピトープの1以上に結合する抗ヒトノロウイルスGII抗体。
P-X1-X2-P-G-E ・・・(1)
X3-X4-X5-F-Y-X6-L-X7-P-X8 ・・・(2)
〔式中、X1はL、V、N、T、S、M又はRを示し、X2はF、Y又はMを示し、X3はV又はGを示し、X4はN又はSを示し、X5はQ、P又はSを示し、X6はS、T又はIを示し、X7はA又はSを示し、X8はM又はVを示す。〕
2)式(1)で表されるアミノ酸領域が配列番号1で示されるアミノ酸配列の419番目~424番目のアミノ酸領域又はこれに相当する領域であり式(2)で表されるアミノ酸配列が配列番号1で示されるアミノ酸配列の516番目~525番目のアミノ酸領域又はこれに相当する領域である上記1)の抗ヒトノロウイルスGII抗体。
3)上記1)又は2)に記載の抗体を含むヒトノロウイルスGII検出試薬。
4)ヒトノロウイルスGIIを含む疑いのある検体を上記1)又は2)の抗体と反応させ、免疫学的測定法により当該ウイルスを検出することを特徴とするヒトノロウイルスGIIの検出方法。
本発明の抗ヒトノロウイルスGII抗体は、ヒトノロウイルスGIIのカプシド構造タンパクのPドメインに存在する下記式(1)及び(2)で表されるアミノ酸領域に含まれるエピトープ、並びに配列番号1で示されるアミノ酸配列の483番目のアミノ酸又はこれに相当するアミノ酸からなるエピトープの1以上に結合するものである。
P-X1-X2-P-G-E ・・・(1)
X3-X4-X5-F-Y-X6-L-X7-P-X8 ・・・(2)
〔式中、X1はL、V、N、T、S、M又はRを示し、X2はF、Y又はMを示し、X3はV又はGを示し、X4はN又はSを示し、X5はQ、P又はSを示し、X6はS、T又はIを示し、X7はA又はSを示し、X8はM又はVを示す。〕
ヒトノロウイルスのカプシド構造タンパク(VP1)は、shell領域(Sドメイン)と突出領域(Pドメイン)から成ることが知られている。Sドメインは、VP1のアッセンブリーを司ると考えられている。一方、Pドメインは、更にP1とP2サブドメインに分かれ、P1サブドメインは、Sドメインと相互作用し、カプシドの物理的安定性を増強すること、P2サブドメインは,ウイルス粒子の最外郭に位置し、マウスノロウイルスでは中和抗体の標的となることが報告されている。
本発明の式(1)及(2)で表されるアミノ酸領域、並びに配列番号1で示されるアミノ酸配列の483番目のアミノ酸又はこれに相当するアミノ酸は、ヒトノロウイルスGIIのカプシド構造タンパクのPドメイン上に存在し、各遺伝子型で保存性の高い領域及びアミノ酸で遺伝子変異が少ないと考えられるが、これまでに斯かる領域又はアミノ酸を認識する抗体は知られていない。
X1はL(ロイシン)、V(バリン)、N(アスパラギン)、T(スレオニン)、S(セリン)、M(メチオニン)又はR(アルギニン)を示す。
X2はF(フェニルアラニン)、Y(チロシン)、S(セリン)又はM(メチオニン)を示すが、Fであるのが好ましい。
(1-1)P-L-F-P-G-E
(1-2)P-V-F-P-G-E
(1-3)P-N-F-P-G-E
(1-4)P-T-F-P-G-E
(1-5)P-S-F-P-G-E
(1-6)P-T-Y-P-G-E
(1-7)P-M-F-P-G-E
(1-8)R-L-S-L-V-S
(1-9)P-R-M-P-G-E
X3はV(バリン)又はG(グリシン)を示すが、Vであるのが好ましい。
X4はN(アスパラギン)又はS(セリン)を示すが、Nであるのが好ましい。
X5はQ(グルタミン)、P(プロリン)又はS(セリン)を示すが、Qであるのが好ましい。
X6はS(セリン)、T(スレオニン)又はI(イソロイシン)を示すが、S又はTであるのが好ましい。
X7はA(アラニン)又はS(セリン)を示すが、Aであるのが好ましい。
X8はM(メチオニン)又はV(バリン)を示すが、Mであるのが好ましい。
(2-1)V-N-Q-F-Y-S-L-A-P-M
(2-2)V-N-P-F-Y-T-L-A-P-M
(2-3)V-N-Q-F-Y-T-L-A-P-M
(2-4)V-N-Q-F-Y-T-L-A-P-V
(2-5)V-N-Q-F-Y-S-L-A-P-M
(2-6)G-N-Q-F-Y-T-L-A-P-M
(2-7)V-N-Q-F-Y-S-L-A-P-V
(2-8)V-S-S-F-Y-I-L-S-P-V
本発明において、配列番号1で示されるアミノ酸配列の419番目~424番目のアミノ酸領域に相当する領域、516番目~525番目のアミノ酸領域に相当する領域、及び483番目のアミノ酸に相当するアミノ酸とは、例えば、GII/1型の485株のVP1アミノ酸配列をもとに、遺伝子情報処理ソフトウェアGENATYXを用い各遺伝子型とのアライメントをとった場合に、配列番号1で示されるアミノ酸配列の419番目~424番目のアミノ酸領域、516番目~525番目のアミノ酸領域、及び483番目のアミノ酸に対応する領域又はアミノ酸を意味する(図1参照)。VP1のアミノ酸配列をこのような方法で整列させることにより、アミノ酸配列中にある欠失にかかわらず、各ヒトノロウイルスGIIのPドメインにおけるアミノ酸配列中の特定領域を決めることが可能である。相同する領域は、三次元構造中で同じ領域に存在すると考えられ、ヒトノロウイルスGIIについて同様のエピトープを有するものと推定できる。
ここで、「エピトープ」とは、抗原決定基であり、抗体と特異的に結合する構造部位を意味する。本発明におけるエピトープは、上記のアミノ酸領域の一部の連続したアミノ酸の他、該領域の不連続なアミノ酸であってもよい。
また、「結合」とは、リガンドと基質間の相互作用を意味し、バックグラウンドまたは非特異的或いは特異的相互作用とは区別され得る。
式(1)で表されるアミノ酸領域に含まれるエピトープとしては、好適には、「X1」であり、式(2)で表されるアミノ酸領域に含まれるエピトープとしては、好適には、「X4」及び/又は「Y-X6-L」が挙げられる。
また、GII/1型のNG1株の例においては、式(1-2)で表されるアミノ酸領域のうち、配列番号1で示されるアミノ酸配列の427番目のV、式(2-2)で表されるアミノ酸領域のうち524番目のN、527番目~529番目のY-S-L及び490番目のEから選ばれる1以上をエピトープとするものが好適に挙げられる。
また、本発明の抗ヒトノロウイルスGII抗体は、IgG、IgA、IgY、IgD、IgM、IgEまたはそれらの1つ以上の一部、例えば、重鎖、軽鎖、FcまたはF(ab)一部のような任意の要求された類型であることができる。
前記免疫細胞と融合される他方の親細胞としての哺乳動物のミエローマ細胞は、すでに、公知の種々の細胞株、例えばP3X63、NS-1、MPC-11、SP2/0等が適宜使用される。
ハイブリドーマの産生する抗体の活性を検出する方法は、公知の方法を使用することができる。例えばELISA法、凝集反応法、ラジオイムノアッセイ法が挙げられる。
免疫学的測定法としては、特に制限されないが、抗ノロウイルスGII抗体と標識抗ノロウイルスGII抗体を用いたサンドイッチ法が好ましく、固定化抗ノロウイルスGII抗体と標識抗ノロウイルスGII抗体を用いる方法がさらに好ましい。
固定化抗ノロウイルスGII抗体としては、ポリスチレンプレート、ラテックス粒子、磁性粒子、ガラス繊維膜、ナイロン膜、ニトロセルロース膜、酢酸セルロース膜等の不溶性支持体に固定化したものが好ましい。
また、標識抗ヒトノロウイルスGII抗体としては、公知の標識体、例えば、放射性同位体(例えば、32P、35S、3H)、酵素(例えば、ペルオキシダーゼ、アルカリフォスファターゼ、ルシフェラーゼ)、タンパク(例えば、アビジン)、低分子化合物(例えば、ビオチン)、蛍光物質(例えば、FITC)、化学発光物質(例えば、アクリジニウム) 、ラテックス粒子(例えば、着色ラテックス粒子、蛍光ラテックス粒子)、金属(例えば、金、銀、白金等の貴金属)コロイド粒子、炭素原子等を用いることができる。
本発明のノロウイルスGIIの検出方法としてはELISA及びイムノクロマトグラフ法がより好ましい。
本発明の方法に使用する抗体は以下の方法で作成した。
GIIノロウイルス様中空粒子(VLP)50μgをマウス腹腔にアジュバントと共に数回免疫し、その血清力価が上昇したことを確認した。追加免疫(静脈内)後3日目に脾臓を取り出し、脾細胞を得た。これとマウスミエローマ細胞をポリエチレングリコール3500の存在下(10:1細胞)で融合させ、ハイブリドーマ細胞を作製した。この細胞を1週間CO2気下37℃で培養し、その培養上清中の抗ノロウイルス抗体の有無を調べた。そこで抗体産生を認めた陽性ウェル中の細胞を限界希釈法により希釈し2週間培養し、同様に培養上清の抗ノロウイルス抗体の有無を調べた。更にその後、抗体産生を認めた陽性ウェル中の細胞を再度限界希釈し、同様の培養を行った。この段階で抗ノロウイルス抗体を産生している細胞を、フラスコにて培養し、その一部をジメチルスルホキシド(DMSO) 10%含有ウシ胎児血清(FCS)にサスペンドし(5×106個/mL)、液体窒素中に保存した。
次に、これらの細胞を25mLのフラスコで培養し、更に75mLのフラスコで培養した。この細胞をプリスタン処理マウス腹腔中に注射し、腹水を採取した。
モノクローナル化ハイブリドーマ細胞の選択においては、下記実施例3に記載の方法と同様の方法でモノクローナル抗体とノロウイルスとの結合状態を確認することにより、Pドメイン上のアミノ酸領域に結合し得るモノクローナル抗体を産生するハイブリドーマを再現性良く選択することができる。
上記実施例1の方法において、GIIノロウイルス様中空粒子(VLP)を免疫する代わりに、ノロウイルス様中空粒子(VLP)のPドメインタンパク質を免疫する事により、再現性良くPドメイン上のアミノ酸領域に結合し得るモノクローナル抗体を産生するハイブリドーマを再現性良く取得することができる。Pドメインタンパク質の取得方法は実施例3に記載のある通りである。
また、本発明の式(1)及び(2)で表されるアミノ酸領域、並びに配列番号1で示されるアミノ酸配列の483番目のアミノ酸又はこれらに相当するアミノ酸配列を選択してこれらアミノ酸配列からなるポリペプチドを製造し、該ポリペプチドを免疫する事により、再現性良くPドメイン上のアミノ酸領域に結合し得るモノクローナル抗体を産生するハイブリドーマを再現性良く取得することができる。
該ポリペプチドはヒトノロウイルスGIIの抗原として使用することができる。
得られた腹水(10mL)と混濁血清処理剤(FRIGEN(登録商標)II:協和純薬工業社製)を、腹水1.5容に対してFRIGEN(登録商標)IIを1容の比率で混和し、1~2分攪拌振とうすることで、腹水からの脱脂を行った。遠心機で3000rpm(1930×g)、10分間遠心分離を行い、清澄化された腹水遠心上清(10mL)を分取した。この腹水遠心上清(10mL)に硫安分画処理(終濃度50%飽和硫安)を氷浴中で1時間施し、沈降したイムノグロブリン画分をPBSで懸濁溶解させた。この硫安分画操作を計2回行い、腹水からのイムノグロブリン粗画分を得た。得られたイムノグロブリン粗画分(10mL)に対して等量の20mMリン酸ナトリウム, pH7.0(以下、20mM NaPB,pH7.0と称す)を混合し、プロテインGカラム(HiTrap Protein G HP,5mL:Amersham BioSciences社製)を用いてアフィニティー精製を行った。サンプルをプロテインGカラムに吸着後、20mM NaPB,pH7.0(50mL)をプロテインGカラム内に通し、サンプル中のIgG以外の夾雑物を洗浄除去した。その後、プロテインGカラムにアフィニティー吸着したIgGは、0.1M グリシン-HCl,pH2.7で溶出させ、カラムからの溶出直後の溶出画分を1M Tris(hydroxymethyl)aminomethane-HCl,pH9.0(以下、Tris(hydroxymethyl)aminomethaneをTrisと略す)で中和し回収した。中和後、アフィニティー精製物に対して500倍容のPBSで4℃、6時間の透析を行い、本透析は計2回行った。本透析操作に用いた透析膜は透析用セルロースチューブ(Viskase Companies社製)で行った。そこで得られたIgG溶出画分を、抗ノロウイルスGIIモノクローナル抗体(5B-18-3M)の精製物とし、4℃での保存ならびに後述する操作に用いることとした。尚、本精製には、BioLogic LPシステム(Bio Rad Laboratories社製)に上述のプロテインGカラムを接続し、流速は1mL/minで一貫して行った。
実施例1で得られた抗ノロウイルスGIIモノクローナル抗体(5B-18-3M)を用いて、以下の手順でイムノクロマト法を利用したノロウイルスGII検出試薬を作成した。
ニトロセルロースメンブレンシートに抗ノロウイルスGIIモノクローナル抗体(5B-18-3M)を0.36~1.45 mg/mL含有する溶液を0.25~1.00 μL/5mmの量で、線状に塗布しテストラインとした。コントロールラインは抗マウスグロブリン抗体を上記同様に塗布した。
抗ノロウイルスGIIモノクローナル抗体(5B-18-3M)を結合させたラテックスを0.04~0.15w/v%含有する溶液を抗体結合ラテックス溶液(5B-18-3M)とした。ラテックス溶液をコンジュゲートパッドに含浸させ、乾燥したものを作成した。
プラスチック製バッキングシート上に、サンプルパッド、上記メンブレン、コンジュゲートパッド及び吸収パッドをこの順で隣り合う部材が一部重なるように貼り付けた後に、プラスチックラミネートで被覆し、5mm幅に切断して、テストストリップを作製する。
上記で作成した試薬を、下記の手順でノロウイルスの各遺伝子型との反応を確認した。
ノロウイルスの各遺伝子型の組換え抗原を希釈液に浮遊し、浮遊した抗原75μLを上記で作成したテストストリップのサンプルパッドに滴下した。15~30℃で15分間静置した後、ラインの有無を確認した。結果を表1に示す。尚、124株とは、Hu/NV/GI/Aichi124-89/89/JP(GeneBank accession number: AB031013)株を意味し、258株とは、Hu/NV/GI/Funabashi258/96/JP(GeneBank accession number: AB078335)株を意味し、645株とは、Hu/NV/GI/Kashiwa645/99/JP(GeneBank accession number: BD011871)株を意味し、CV株とは、Hu/NV/GI/Chiba407/87/JP(GeneBank accession number: AB042808)株を意味し、W18株とは、Hu/NV/GI/WUG1/00/JP(GeneBank accession number: AB081723)株を意味し、#8株とは、Hu/NV/GI/8/99/JP(GeneBank accession number: AB058547)株を意味し、485株とは、Hu/NV/GII/Noda485/00/JP(GeneBank accession number: 未登録)株を意味し、NG1株とは、Hu/NV/GII/NG1/02/JP(GeneBank accession number: AB195225)株を意味し、809株とはHu/NV/GII/Sanbu809/98/JP(GeneBank accession number: BD011876)を意味し、18-3株とは、Hu/NV/GII/Matsudo18/00/JP(GeneBank accession number: 未登録)株を意味し、336株とは、Hu/NV/GII/Kashiwa336/00/JP(GeneBank accession number: 未登録)株を意味し、104株とは、Hu/NV/GII/Narita104/97/JP(GeneBank accession number: 未登録)株を意味し、754株とは、Hu/NV/GII/Ichikawa754/98/JP(GeneBank accession number: BD011877)株を意味し、7k株とは、Hu/NV/GII/Ueno7k/94/JP(GeneBank accession number: AB078337)株を意味し、445株とは、Hu/NV/GII/Sanbu445/00/JP(GeneBank accession number: 未登録)株を意味し、10-25株とは、Hu/NV/GII/Osaka10-25/99/JP(GeneBank accession number: BD011881)株を意味し、U25株とは、Hu/NV/GII/SaitamaU25/**/JP(GeneBank accession number: AB039780)株を意味し、1876株とは、Hu/NV/GII/Chitta/Aichi76-96/96/JP(GeneBank accession number: AB032758)株を意味し、NG15株とは、Hu/NV/GII/NG15/03/JP(GeneBank accession number: 未登録)株を意味し、47株とは、Hu/NV/GII/Kashiwa47/97/JP(GeneBank accession number: AB078334)株を意味し、Kamo株とは、Hu/NV/GII/Kamo8/03/JP(GeneBank accession number: 未登録)株を意味し、Alph株とは、Hu/NV/GII/Alph23/**/JP(GeneBank accession number: 未登録)株を意味する。
抗ノロウイルスGIIモノクローナル抗体(5B-18-3M)とノロウイルスとの結合状態を把握するため、以下の手順でX線結晶構造解析を行った。
(1)サンプルの調製
<ノロウイルスP領域のタンパク質発現、精製と結晶化>
ノロウイルスVietnam026 GII.10のP領域の全長に近い(アミノ酸残基224-538)P領域(アミノ酸の長さ314)を、E.coliでの発現のためにデザインし、BamHIとNotI(New England Biolabs)制限酵素サイトで切断したpMal-c2xベクターに挿入し、発現クローンを作製した。これをE.coli BL21細胞(Invitrogen)にトランスフォームし、発現は、IPTG(1mM)で22℃で18時間誘導した。His-tagged融合P領域タンパク質は、Niカラム(Qiagen)で精製し、HRV-3Cプロテアーゼ(Novagen)により、4℃で一晩処理した。その後、再度その処理液をNiカラムに通すことで、P領域を精製した。
P領域はSuperdex 200カラム(GE)で分子ふるいクロマトグラフィーにより、さらに精製し、2-10mg/mlまで濃縮した後、結晶化の前にGFB(0.35M NaCl、2.5mMトリスpH 7.0、0.02% NaN3)に保存した。
精製した5B-18-3M IgGを約60 mg使用し、Fabの調整をした。5B-18-3M IgG は、100mM dTTで37℃で1時間還元した。還元した5B-18-3M IgGは、透析カセットで4℃で1時間透析を行い、20mM HEPES(pH 7.7)を含むGFBにBuffer置換し、4℃で48時間2mMヨードアセトアミドを含むGFBでアルキル化した。その後、カセットをヨードアセトアミドを含まない新しいGFBに移し、4℃で1h Buffer置換を行った。5B-18-3M IgGは、5mg/mLに濃縮し、キット(pierce,Rockford, USA)を用いてパパインで消化した。消化した5B-18-3M IgGは、プロテインAカラムでFabのみ精製し、Fabは、さらにSuperdex 200カラム(GE)で分子ふるいクロマトグラフィーにより精製し、5mg/mLに濃縮した後GFBに保存した。
精製したそれらの、ノロウイルス GII.10のP領域と、5B-18-3M Fabは、1:1で混合し25℃で1時間反応させ、最終的に分子ふるいクロマトグラフィーにより精製した。
上記、ノロウイルスP領域と抗ノロウイルスGIIモノクローナル抗体(5B-18-3M)のFab の複合体はハンプトンリサーチ社の試薬とhanging-drop vapor diffusion法を用いるのとは少し異なる状況下で結晶化させた。
この研究のために、P領域-Fab複合体の結晶は、GII.10 P領域-Fab複合体とPEG400(40% v/v)、PEG3350(5% w/v)、0.1M酢酸(pH5.5)を含むGFBを1:1の比率で混合し、成長させた。データ収集の前に結晶は、30%のエチレングリコールにGFBを含む凍結防止剤へ移した。
抗ノロウイルスGIIモノクローナル抗体(5B-18-3M)とノロウイルスGII.10キャプシドタンパクP領域との複合体の結晶のX線回析データは、アルゴンヌ国立研究所(Argonne, IL)のビームライン、Southeast Regional Collaborative Access Team (SER-CAT) 22-IDと22-BMを使用して作成された。回析データは蛋白・低分子データ処理ソフトウェアHKL2000とプログラムパッケージXDSによって処理した。構造は、PDB( Protein Data Bank) コード1WEJを抗ノロウイルスGIIモノクローナル抗体のFabのサーチモデルとし、PDB コード2OBRをノロウイルスキャプシドP領域のサーチモデルとして、分子置換による構造解析ソフトPHASERを用いて、回析データと抗ノロウイルスGIIモノクローナル抗体(5B-18-3M)とノロウイルスGII.10キャプシドタンパクP領域アミノ酸配列より立体構造を構築した。
その後、モデル構築ソフトCOOT内に構築されているマニュアルモデルで立体構造を調整し、精密化プログラムREFMACのTLS refinementと自動構造決定ソフトウェアPHENIXで修正を行い、CCP4によって、重ね合わせ(superposition)と平均二乗偏差(RMSD)が計算され、分子グラフィックスツールPyMOLに描画した。
これにより得られた、図2の構造よりGII.10キャプシドタンパクP領域に抗ノロウイルスGIIモノクローナル抗体(5B-18-3M)が結合している部位のノロウイルスGII.10キャプシドタンパクP領域のアミノ酸配列の同定を行ったところ、3箇所の部位が明らかとなった。その後、各遺伝子型のアミノ酸配列の相同性比較を行った結果、図1に示すように、各遺伝子間で非常に保存された3ヶ所の領域であるという結果が得られた。
Claims (4)
- ヒトノロウイルスGIIのカプシド構造タンパクのPドメインに存在する下記式(1)及び(2)で表されるアミノ酸領域に含まれるエピトープ、並びに配列番号1で示されるアミノ酸配列の483番目のアミノ酸又はこれに相当するアミノ酸からなるエピトープの1以上に結合する抗ヒトノロウイルスGII抗体。
P-X1-X2-P-G-E ・・・(1)
X3-X4-X5-F-Y-X6-L-X7-P-X8 ・・・(2)
〔式中、X1はL、V、N、T、S、M又はRを示し、X2はF、Y又はMを示し、X3はV又はGを示し、X4はN又はSを示し、X5はQ、P又はSを示し、X6はS、T又はIを示し、X7はA又はSを示し、X8はM又はVを示す。〕 - 式(1)で表されるアミノ酸領域が配列番号1で示されるアミノ酸配列の419番目~424番目のアミノ酸領域又はこれに相当する領域であり式(2)で表されるアミノ酸配列が配列番号1で示されるアミノ酸配列の516番目~525番目のアミノ酸領域又はこれに相当する領域である請求項1記載の抗ヒトノロウイルスGII抗体。
- 請求項1又は2に記載の抗体を含むヒトノロウイルスGII検出試薬。
- ヒトノロウイルスGIIを含む疑いのある検体を請求項1又は2に記載の抗体と反応させ、免疫学的測定法により当該ウイルスを検出することを特徴とするヒトノロウイルスGIIの検出方法。
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WO2022244860A1 (ja) | 2021-05-20 | 2022-11-24 | デンカ株式会社 | 抗ノロウイルス抗体 |
WO2022244862A1 (ja) * | 2021-05-20 | 2022-11-24 | デンカ株式会社 | ノロウイルスの免疫測定方法及び免疫測定器具 |
WO2022244861A1 (ja) | 2021-05-20 | 2022-11-24 | デンカ株式会社 | 抗ノロウイルス抗体 |
KR20240009995A (ko) | 2021-05-20 | 2024-01-23 | 덴카 주식회사 | 항노로바이러스 항체 |
KR20240009996A (ko) | 2021-05-20 | 2024-01-23 | 덴카 주식회사 | 항노로바이러스 항체 |
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EP2757111A1 (en) | 2014-07-23 |
EP2757111A4 (en) | 2015-04-22 |
KR20140068067A (ko) | 2014-06-05 |
US9244072B2 (en) | 2016-01-26 |
KR102000479B1 (ko) | 2019-07-16 |
EP2757111B1 (en) | 2017-11-08 |
JPWO2013039165A1 (ja) | 2015-03-26 |
JP6105474B2 (ja) | 2017-03-29 |
US20140349277A1 (en) | 2014-11-27 |
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