WO2013147233A1 - 抗体検出用試薬の製造方法、及びその用途 - Google Patents
抗体検出用試薬の製造方法、及びその用途 Download PDFInfo
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- WO2013147233A1 WO2013147233A1 PCT/JP2013/059692 JP2013059692W WO2013147233A1 WO 2013147233 A1 WO2013147233 A1 WO 2013147233A1 JP 2013059692 W JP2013059692 W JP 2013059692W WO 2013147233 A1 WO2013147233 A1 WO 2013147233A1
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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- G—PHYSICS
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
- G01N2333/4704—Inhibitors; Supressors
Definitions
- the present invention relates to a method for producing an antibody detection reagent.
- the present invention also relates to an antibody detection reagent produced by the same method and its use.
- ELISA Enzyme-linked immunosorbent assay
- Patent Document 1 or 2 describes a method for detecting an antibody contained in the blood of a cancer patient. This method detects antigen epitope peptide-specific antibodies contained in the blood of the subject by immobilizing the antigen epitope peptide (antigen peptide consisting of several amino acids) on the bead and bringing the bead into contact with the blood component of the subject. .
- the epitope portion to which the antibody binds varies depending on the type of HLA, in order to use the method described in Patent Document 1 or 2, the subject's HLA type is examined, and the epitope peptide that matches the subject's HLA type It is necessary to clear various conditions such as being identified.
- a detection reagent for an antibody against a specific antigen protein When preparing a detection reagent for an antibody against a specific antigen protein, to prepare a reagent that includes all epitope parts derived from one type of antigen protein on the surface of one type of bead, and detects it with high sensitivity and stability. It is preferable to obtain an antigen having a water-soluble and flexible structure. However, many denatured proteins, poorly soluble proteins such as membrane proteins, and proteins with unstable physical properties are likely to aggregate. Therefore, conventionally, partial peptides that can exhibit stable physical properties have been often used.
- the protein When using partial peptides, it is necessary to synthesize a wide variety of overlapping peptides in order to cover all epitopes, to prepare various types of beads, and to provide seamless epitope peptides Is practically difficult. Fortunately, even when full-length Native structure antigens can be obtained, the protein generally forms a higher-order structure that embeds a hydrophobic portion inside, so that epitopes that react with antibodies are not necessarily exposed. There is a case.
- the thiol groups contained in the protein may form disulfide bonds with time, which may affect antibody detection. is there.
- Patent Document 3 describes a method for detecting an anti-HCV antibody contained in the serum of a subject using a long-chain polypeptide of human hepatitis C virus (HCV).
- HCV human hepatitis C virus
- Patent Document 3 describes that a disulfide bond generated within a protein or between proteins with the passage of time decreases the detection sensitivity of an antibody.
- the detection sensitivity of the antibody is improved by releasing a disulfide bond within or between proteins using a reducing agent before or during detection. That is, even a solubilized protein may precipitate over time, and some means must be taken to eliminate it.
- TAPS-Sulfonate trimethylammoniopropylmethanethiosulfonate, hereinafter referred to as TAPS
- TAPS binds to a thiol group of a protein and can reversibly cationize the protein (see, for example, Non-Patent Documents 1 and 2).
- Cationized protein has improved water solubility.
- TAPS is a reversible reaction
- an antibody binds to a protein modified with a sugar chain, there is no report as to whether the antibody can bind to a protein bound with an artificially synthesized compound such as TAPS.
- Patent No. 3960614 (Immunodia) JP 2005-098877 (Hitachi Software) Patent No. 3225468 (Dynabot)
- the present invention has been made in view of the above circumstances, and is a method for efficiently producing a reagent for detecting an antibody that specifically binds to a poorly soluble antigen protein present in a liquid sample, and is produced by the production method.
- the present invention provides an antibody detection reagent and its use.
- the present invention aims to provide the following means.
- a method for producing an antibody detection reagent comprising an antigen protein and a carrier comprising the steps of cationizing and solubilizing the antigen protein, and binding the cationized antigen protein to the carrier
- a method for producing an antibody detection reagent (2) The method for producing a reagent for antibody detection according to (1), wherein the antigen protein is a full-length protein, (3) The method for producing a reagent for antibody detection according to (1) or (2), wherein the antigen protein is a membrane protein, (4) The method for producing an antibody detection reagent according to any one of (1) to (3), wherein the antigen protein is a cancer antigen protein; (5) The antibody detection reagent according to any one of (1) to (4), wherein the cationization is performed by binding a cationizing agent to a thiol group of an antigen protein. Production method.
- the cationizing agent is selected from any one of a thiosulfonate compound, a mixed disulfide compound, a pyridyl sulfide cationizing agent, an alkyl halide cationizing agent, or a mixture thereof.
- the thiosulfonate compound is a compound of the following formula (6)
- a method for producing an antibody detection reagent (wherein R 1 represents a linear alkylene group having 2 to 20 carbon atoms, and R 2 represents an alkyl group having 1 to 3 carbon atoms) , N is an integer from 1 to 3), (8)
- the compound is TAPS-Sulfonate wherein R 1 is — (CH 2 ) 3 —, R 2 is CH 3 —, and n is 1.
- the compound is TAP3S-Sulfonate wherein R 1 is — (CH 2 ) 3 —, R 2 is CH 3 —, and n is 3.
- a method for detecting an antigen-specific antibody contained in a sample comprising the step of contacting the sample with the antibody detection reagent according to (12), and adding a labeled secondary antibody that binds to the antibody
- An antibody detection method comprising: a step of binding to an antibody; a step of recovering an antibody detection reagent; and a step of detecting an antibody detection reagent bound to the antibody,
- an antibody detection reagent for detecting an antibody against an antigen protein can be produced with the above-described configuration, and completed the present invention.
- the antigen protein can be efficiently solubilized and recovered by using a cationizing agent.
- a reagent for antibody detection in which a large number of antigen protein molecules are bound to the carrier. Can be manufactured efficiently.
- antibody detection reagents are more stable than reagents produced by conventional methods and can be stored for a long time.
- a hardly soluble antigen protein can be used as an antigen for detecting an antibody. Since a protein is used, even if the epitope portion that can be recognized by HLA is different, it can be detected. Therefore, compared to the case where the epitope peptide is immobilized on the beads, it is not necessary to produce a plurality of reagents according to the type of HLA of the subject, and it can be said that the production efficiency is high.
- the cationizing agent is bonded to the thiol group of the antigen protein, it is possible to suppress a disulfide bond within the protein or between the proteins that occurs with time. Therefore, the effect which suppresses aggregation of the reagents for antibody detection by the disulfide bond between proteins with time passage is anticipated. Therefore, even when a poorly soluble antigen protein is bound to a carrier, or after long-term storage at room temperature, 4 ° C. or ⁇ 20 ° C., the antibody detection reagent is more effective than the conventional method. Function can be maintained.
- the artificially synthesized compound is bound to the antigen protein, thereby suppressing the binding between the antigen protein and the antibody.
- the inventors have also found that antibodies can be detected.
- the antibody detection reagent can be efficiently produced by using the production method of the present invention.
- the antibody detection reagent produced by the production method of the present invention can detect an antibody against an antigenic protein present in a liquid sample, for example, it can be removed from a serum sample regardless of the HLA type of the cancer patient. It becomes possible to detect an antibody specific for an antigen protein.
- TAPS-Sulfonate and TAP-Br have a low molecular weight, so that steric hindrance that inhibits the reaction between the protein and the antibody can be minimized while maintaining high solubilizing ability.
- it since it has a low molecular weight, it is easy to bind a plurality of molecules to a protein, and all SH groups contained in the protein can be cationized. Therefore, aggregation of beads when stored for a long time can be suppressed. From these characteristics, the method of the present invention using TAPS-Sulfonate and TAP-Br is more effective than the conventional method.
- FIG. 1 is an explanatory diagram regarding protein cationization.
- FIG. 2 is a diagram showing the result of analysis by SDS-PAGE after cationization of WT-1 with TAPS.
- FIG. 3 shows the results of analysis by SDS-PAGE after cationization of MAGE-A4 with TAPS.
- FIG. 4 is a diagram showing the results of purifying TAPS-modified WT-1 by HPLC.
- FIG. 5 is a diagram showing the results of purification of TAPS-modified MAGE-A4 by HPLC.
- FIG. 6 is a diagram showing the results of analysis by SDS-PAGE after purification of TAPS-modified WT-1 and MAGE-A4 by HPLC.
- Min represents the elution time when purification is performed by HPLC (FIGS.
- FIG. 7 is a diagram showing the results of analyzing the prepared antigen protein by SDS-PAGE. Each lane was filled with 2 ⁇ g of antigen protein.
- FIG. 8 is a diagram showing the results of detecting antibodies against 6 types of cancer antigen proteins contained in the serum of cancer patients using the antibody detection reagent produced by the production method of the present invention.
- FIG. 9 is a view showing the results of detecting antibodies against three types of cancer antigen proteins contained in the serum of cancer patients using the antibody detection reagent produced by the production method of the present invention.
- FIG. 10 is a diagram showing the results of detecting antibodies against three types of cancer antigen proteins contained in the serum of cancer patients using the antibody detection reagent produced by the production method of the present invention.
- FIG. 11 is a diagram showing the results of reverse phase HPLC (high performance liquid chromatography) for TAPS, TAP3S, Native, and reduced lysozyme.
- FIG. 12 shows bead aggregates contained in a suspension in which beads immobilized with TAPS, TAP3S, Native and reduced MAGE-A4 were stored at 4 ° C. for 5 days, 37 ° C. for 10 days or 41 days. It is a figure which shows a ratio.
- FIG. 11 is a diagram showing the results of reverse phase HPLC (high performance liquid chromatography) for TAPS, TAP3S, Native, and reduced lysozyme.
- FIG. 12 shows bead aggregates contained in a suspension in which beads immobilized with TAPS, TAP3S, Native and reduced MAGE-A4 were stored at 4
- FIG. 13 shows a stock solution in which TAPS-modified MAGE-A4 immobilized beads (lane 2), TAP3S-modified MAGE-A4 immobilized beads (lane 3), and Native MAGE-A4 immobilized beads (lane 4) were stored for 104 days. It is a figure which shows the result of having performed western blotting. In lane 1, 100 ng of MAGE-A4 protein was run as a control.
- FIG. 14 shows an image in which proteins bound to the surface of Bio-Plex COOH beads, TAPS-modified MAGE-A4 immobilized beads, TAP3S-modified MAGE-A4 immobilized beads, or Native MAGE-A4 immobilized beads stored for 104 days are detected. It is. The left is an image taken with a confocal laser fluorescence microscope, and the right is a differential interference image (bright field) taken simultaneously.
- the production method of the present invention is a method for producing an antibody detection reagent comprising a step of cationizing and solubilizing an antigen protein and a step of binding the cationized antigen protein to a carrier.
- an antigen protein may be a hardly soluble protein.
- sparingly soluble is used to explain the characteristics of a protein and does not dissolve in a liquid to form a uniform mixed solution. In particular, it cannot or cannot be dissolved in water or a physiological solvent. The characteristic that it is difficult.
- a sparingly soluble protein of the type described herein can be obtained after centrifugation for 1 hour at 15,100 ⁇ g when the protein is in water, water and salts, water and physiological solvents that do not denature the protein, It can be defined as a poorly soluble protein when substantially the majority is recovered in the precipitated fraction.
- protein as used herein includes peptides, polypeptides, etc., and is not limited to naturally occurring proteins existing in nature. Recombinant proteins derived from cells transformed by gene transfer or the like, cell-free proteins in vitro Proteins expressed using an expression system and synthetic proteins produced by organic synthetic chemistry are also included. Furthermore, some or all of the amino acids constituting the protein may be those to which functional groups such as acetylation, phosphorylation, and methylation have been added, or those modified with sugars, proteins, lipids, and the like.
- the “slightly soluble protein” as used herein means that it does not dissolve or hardly dissolves at room temperature in water or a physiological solvent, for example, even if dissolved by using a denaturing agent, it is replaced with a physiological solvent. It refers to the protein that causes precipitation.
- the natural type is originally a soluble protein, when expressed using a heterologous organism as a recombinant protein (for example, when an expression system in E. coli is constructed using a gene recombination technique), When the target protein is recovered as an inclusion body (inclusion body), the target protein is also referred to as a hardly soluble protein.
- examples of poorly soluble proteins include, but are not limited to, full-length proteins, membrane proteins, cancer antigen proteins, and the like.
- “solubilization” refers to dissolving a protein in a physiological solvent while maintaining the amino acid sequence.
- the solution containing the protein dissolved by the denaturing agent is replaced with the physiological solvent and centrifuged. In this case, it means that the amount of the protein recovered in the supernatant after centrifugation is increased.
- the full-length protein means not only a natural protein that has been confirmed to exist in the living body but also a protein having the largest open reading frame (ORF) predicted from the genomic sequence.
- ORF open reading frame
- the amino acid sequence of such a full-length protein is obtained from a database such as The ORFeome Collaboration (http://www.orfeomecollaboration.org/), GeneCard (registered trademark) (http://www.genecards.org/), or the like. It is possible, but not limited to this.
- Membrane protein is a protein having a transmembrane structure and present on the surface of cell membrane, nuclear membrane and other intracellular organelles. A hydrophilic part in contact with the inside or outside of the cell and a hydrophobic part buried in the cell membrane are present in one protein molecule. Therefore, when it is obtained by expression in Escherichia coli or the like, it is a protein that is difficult to form a three-dimensional structure in an aqueous solution and easily forms inclusion bodies.
- Cancer antigen protein refers to an antigen protein that is expressed in a tumor and induces an immune response or an antigen protein that can serve as an indicator of the presence of a tumor.
- proteins whose expression increases when cells become cancerous proteins whose amino acids are partly mutated when cells become cancerous, and the like.
- the production method of the present invention is characterized by cationizing a protein. More preferably, it is characterized by cationizing a hardly soluble protein.
- Protein cationization means adding an excessive positive charge to the protein. Cationic proteins have improved water solubility due to charge repulsion. In order to cationize a protein, a cationizing agent is bound to the protein.
- Carrier refers to a substance that binds the antigen protein to a solid surface. Specific examples include glass, nylon membrane, semiconductor wafer, and microbeads, but are not limited thereto.
- the binding of the antigen protein to the carrier means that the “antigen protein” is directly immobilized on the surface of the carrier using a known technique, or through a bond such as biotin-avidin or via a linker molecule. It may be fixed indirectly.
- sample refers to an antibody (including subtypes such as IgG, IgA, IgM, IgD, and IgE) that is detected by the antibody detection reagent according to the present invention, and a fragment that maintains its activity (eg, Fab fragment, F ( ab ′) a sample containing 2 fragments and the like.
- a fragment that maintains its activity eg, Fab fragment, F ( ab ′) a sample containing 2 fragments and the like.
- Body fluid refers to a liquid sample that can be collected from a living body.
- Peripheral blood, bone marrow fluid, umbilical cord blood, pleural effusion, ascites, urine, and the like fall under this, and samples obtained by treating these with a method well known to those skilled in the art (for example, plasma and serum obtained by centrifuging peripheral blood from the supernatant) This also applies to this.
- “Secondary antibody” refers to an antibody (including subtypes such as IgG, IgA, IgM, IgD, IgE) to be detected by the antibody detection reagent according to the present invention, and a fragment (for example, Fab) that maintains its activity. Fragments, F (ab ′) 2 fragments and the like, and active fragments thereof.
- “Labeled secondary antibody” refers to a secondary antibody bound to a label such as a radioisotope, a luminescent agent, a fluorophore, or the like.
- an enzyme such as luciferase or peroxidase, or a protein such as biotin or green fluorescent protein (GFP) can be used as such a “label”.
- a protein such as biotin or green fluorescent protein (GFP)
- GFP green fluorescent protein
- the “labeled secondary antibody” can also be produced by genetic engineering as a single recombinant protein in the form of a fusion protein.
- cationizing agent used in the production method of the present invention various compounds can be used as long as they can add a positive charge to a protein via a disulfide bond (FIG. 1).
- disulfide bond FOG. 1
- thiosulfonate compounds, mixed disulfide compounds, pyridyl disulfide cationizing agents, alkyl halide cationizing agents, and the like can be used.
- the thiosulfonate compound used in the production method of the present invention is a compound represented by the following formula [Chemical Formula 2].
- X is a group having a cation. One or a plurality of groups having a cation of X may be linked.
- R 2 represents a lower alkyl group having 1 to 3 carbon atoms. That is, the thiosulfonate compound used in the production method of the present invention is a thiosulfonate compound having one or more cations derived from X in one molecule.
- Examples of X include a quaternary ammonium group.
- the thiosulfonate compound used in the production method of the present invention is a thiosulfonate compound represented by the following formula [Chemical Formula 3] having one or more cations derived from a quaternary ammonium group in one molecule.
- R 1 is a direct number of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14 , 15, 16, 17, 18, 19 or 20 carbon atoms.
- R 2 represents a lower alkyl group having 1, 2 or 3 carbon atoms
- n represents any of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 Or more preferably an integer of 1, 2, or 3
- the dissociation group R 2 formed after the cationization reaction of the protein is an inactive dissociation group.
- TAPS trimethylammoniopropylmethanethiosulfonate
- TAP3S-Sulfonate TAP3S-Sulfonate
- TAPS is a compound represented by the following formula [Formula 4].
- TAPS has a quaternary amine with a strong positive charge in the molecule, and cationizes the protein by binding to the protein via a cysteine residue. The cationized protein has improved solubility and the SH group is stably protected.
- TAPS is synthesized with reference to, for example, Biotechnol. Appl. Biochem. (1998) 28, 207-213, or purchased as a reagent in the form of a Br salt (formula [Formula 5] below) (molecular weight 292.26). It is possible (Wako Pure Chemicals, Katayama Chemical Industries, etc.).
- TAP3S is a molecular weight compound represented by the following formula [Chem. 6]. TAP3S has three quaternary amines with a strong positive charge in the molecule and can cationize more strongly than TAPS. TAP3S can be synthesized with reference to, for example, International Publication No. 2011/118731.
- Examples of the mixed disulfide compound used in the production method of the present invention include cystamine as shown in the following formula [Chemical Formula 7].
- cystamine As shown in the following formula [Chemical Formula 7].
- NH 3 + is a group having a cation.
- Examples of the pyridyl sulfide-based cationizing agent used in the production method of the present invention include compounds represented by the following formula [Chemical Formula 8].
- X is a group having a cation. One or more groups having a cation of X may be connected.
- a halogenated alkyl cationizing agent capable of adding a positive charge to a protein by S-alkylation
- a halogenated alkyl cationizing agent capable of adding a positive charge to a protein by S-alkylation
- TAP-Br 3-bromopropyl trimethylammonium
- TAP-Br binds irreversibly to the SH group of the protein and improves the protein solubilizing ability.
- TAP-Br (molecular weight 261.00) can be purchased as a reagent, for example, in the form of a Br salt ([Chemical Formula 9]) (for example, SIGMA-ALDRICH).
- TAPS-Sulfonate and TAP-Br have a low molecular weight, so the reaction between the protein and the antibody while maintaining high solubilization ability. The steric hindrance which inhibits can be minimized.
- it since it has a low molecular weight, it is easy to bind a plurality of molecules to a protein, and all SH groups contained in the protein can be cationized. Therefore, aggregation of beads when stored for a long time can be suppressed.
- the gene of the antigen protein is introduced into E. coli and cultured.
- the cultured E. coli is disrupted and the antigenic protein is recovered.
- the antigen protein may form an inclusion body.
- Solubilize the collected inclusion bodies with a denaturant examples include, but are not limited to, urea, guanidine hydrochloride, and a surfactant.
- the antigen protein After denaturing the antigen protein, it is treated with a reducing agent to cleave the SS bond.
- a reducing agent examples include, but are not limited to, dithiothreitol (DTT) and 2-mercaptoethanol (2ME).
- TAPS to the solution containing the denatured antigen protein and let stand at room temperature for 30 minutes.
- the amount of TAPS added is preferably such that TAPS has a molar concentration of 1 to 10 times the molar concentration of the antigen protein and the thiol group contained in the solution. More preferably, it is 1.1 to 1.2 times.
- TAPS antigen protein contained in the supernatant is purified.
- a method such as dialysis or column chromatography can be used. For example, transfer to a dialysis tube and dialyze against pure water to 0.5% acetic acid at 4 ° C to remove the denaturant. Centrifuge the dialyzed liquid at 12,000 rpm, 4 ° C. to room temperature for 15 minutes and collect the supernatant.
- the cationized antigen protein exhibits high water solubility, is easy to prepare, and exhibits extremely high stability under weakly acidic conditions (preferably pH 6 or less, more preferably pH 2 to 5). Therefore, water solubility can be maintained in a state where the epitope of the full-length protein is exposed seamlessly. For this reason, all the epitopes contained in one antigen protein can be immobilized on one kind of bead in a state in which it easily reacts with an antibody.
- the TAPS protein is bound to the carrier.
- magnetic beads non-magnetic beads, microplates, and the like can be used as the carrier.
- beads are used as a carrier will be described.
- the bead Suspend the beads in the binding solution. If the bead has already been modified to assist in binding to the protein, the bead is directly bound to the TAPS protein. In the case where the modification that assists the binding between the bead and the protein is not made, the bead is modified using a substance that does not inhibit the reaction between the TAPS protein and the antibody when detecting the antibody, such as skim milk.
- the time for mixing with beads activated to react with amino groups is preferably 2 to 16 hours.
- ⁇ ⁇ ⁇ Collect the beads, suspend in the washing solution, centrifuge and wash.
- the washing solution include a phosphate buffer.
- the storage buffer include a storage buffer manufactured by Bio-Rad. If the storage buffer is weakly acidic (preferably pH 6 or lower, more preferably pH 2 to 5), the solubility of the TAPS protein is more stably maintained.
- an antibody detection reagent can be produced.
- the antibody detection method as one aspect using the antibody detection reagent (hereinafter referred to as the reagent of the present invention) produced by the production method of the present invention will be described in detail.
- the reagent of the present invention may be an antibody detection reagent comprising a cationized hardly soluble antigen protein and beads.
- a body fluid can be used as the sample.
- peripheral blood As the body fluid, peripheral blood, bone marrow fluid, umbilical cord blood, pleural effusion, ascites, urine and the like can be used.
- Peripheral blood is preferably used in consideration of ease of collection and less burden on the subject. It is preferable to collect an amount that does not burden the subject.
- whole blood collection using a vacuum blood collection tube, a blood collection bag or the like can be used. In collecting blood, heparin or the like may be added to prevent coagulation of blood.
- plasma from the collected blood by centrifugation.
- plasma can be obtained when partial blood collection is performed using a component blood collection device.
- peripheral blood remove blood cell components and blood coagulation components, obtain serum, and use it as a sample.
- ⁇ ⁇ Mix the reagent of the present invention and plasma.
- the mixing time for example, when the reaction is performed at room temperature, it is preferable to leave for about 2 hours, and when the reaction is performed at 4 ° C., it is preferably about overnight.
- the reagent of the present invention is recovered by a centrifugal method or a magnetic device and washed.
- Bind secondary antibody to detect antibody when the subject is a human, the antibody bound to the reagent of the present invention is a human antibody. Therefore, labeling is performed using a labeled anti-human antibody as the secondary antibody.
- a labeled anti-human antibody for example, biotinylated anti-human IgG mouse monoclonal antibody and the like can be mentioned.
- antibodies can be detected from other liquid samples using the reagent of the present invention.
- the following situation is assumed by analyzing the antibody contained in the blood of the subject.
- the subject's allergic reactivity is determined by analyzing the subject's in vivo antibodies.
- the treatment policy is determined and the prognosis is predicted and judged using the found antibody as an index.
- the therapeutic effect is improved by using an antigen in which an antigen-specific antibody is detected in the serum of a patient.
- Predicting the therapeutic effect that is related to immune function such as Abscopal effect by analyzing antibody before and after radiotherapy.
- the above-described effects can be obtained with an antibody detection reagent using an epitope peptide, but the reagent of the present invention is excellent in that a protein can be used. This is because by using the protein, the antibody can be detected without being limited to the HLA type of the subject. Therefore, even an HLA type that is generally regarded as a minor and for which analysis of epitope peptides has not progressed can be analyzed.
- His-tag fusion MAGE-A4 (SEQ ID NOs: 16, 17, antigen protein sequences are SEQ ID NOs: 3, 4) and His-tag fusion WT-1 (SEQ ID NOs: 14, 15, antigen protein sequences) cloned in the pET28b vector E. coli T7express was transformed with two types of plasmid DNAs of SEQ ID NOs: 1, 2), several colonies formed on the plate were added to 10 mL of LB / Kan25 medium, and cultured with shaking for 2 hours.
- the cells were dispersed in a 40 mL lysis buffer (20 mM Tris HCl pH 8.0, 50 mM NaCl, 5 mM MgSO 4 , 0.2% Tween20) using an ultrasonic crusher, and further crushed for 1 minute ⁇ 3 sets. .
- HC Benzonase
- the precipitate (inclusion body) was collected by centrifugation (8 krpm, 10-15 min, room temperature), and the supernatant was removed. 50-100 mL of RO water was added to the recovered precipitate, the precipitate was suspended again using a sonicator, and the precipitate was recovered by centrifugation (8 krpm, 10-15 min, room temperature).
- MAGE-A4 recovered the supernatant by centrifugation (8 krpm, 10-15 min, room temperature).
- the precipitate was dissolved with 5-10 mL of 6 M guanidine 0.1 M Tris-HCl pH 8 + 1 mM EDTA. After deaeration and nitrogen substitution, 30 mM DTT (solid) was added, and the mixture was treated at 37 ° C. for about 1 hour.
- the above affinity purified MAGE-A4 was added to an eggplant-shaped flask, and the water was removed with a freeze dryer, and then dissolved in 3 mL of 6M guanidine, 0.1M Tris-HCl pH 8. After deaeration and nitrogen substitution, 30 mM DTT (solid) was added, and the mixture was treated at 37 ° C. for about 1 hour. 70 mM TAPS-Sulfonate was added to the solution containing MAGE-A4 and treated at 37 ° C. for about 30 minutes.
- FIG. 6 shows the results of SDS-PAGE analysis for TAPS-modified MAGE-A4 and WT-1 after HPLC. From the results of the analysis, it was confirmed that the TAPS protein can be prepared as a highly pure antigen protein by purifying by reverse phase HPLC. MAGE-A4 collects fractions with an elution time of 50 to 51 minutes (lane 50 min in Fig. 6), and WT-1 collects fractions with an elution time of 52 to 53 minutes (lane 52 min in Fig. 6). used.
- FIG. 7 shows the result of SDS-PAGE analysis of the prepared protein.
- Immobilization was performed using 6 types of Bio-Plex COOH beads (1/10 volume (1.25 ⁇ 10 6 beads)) and Bio-Plex amine coupling kit (manufactured by Bio-Rad).
- the antigens used were 11 ⁇ g each, and they were all denatured antigens solubilized by TAPS.
- the solubilized protein was dissolved in a buffer and allowed to stand on ice.
- the container of COOH beads was shaken with a vortex mixer for 30 seconds, and the ultrasonic wave was suspended for 30 seconds.
- the COOH bead suspension was centrifuged at 14,000 ⁇ g for 4 minutes, and the supernatant was removed.
- 80 ⁇ L of bead activation buffer was added to the collected precipitate, and the mixture was well shaken with a vortex mixer for 30 seconds, and ultrasonic waves were suspended for 30 seconds.
- the COOH bead suspension and the TAPS protein suspension were mixed, and PBS was added so that the total amount became 500 ⁇ L.
- the beads were shielded from light so that they were not exposed to light and permeated using a rotary incubator at room temperature for 2 hours.
- the suspension of COOH beads and TAPS protein was centrifuged at 14,000 ⁇ g for 4 minutes, and the supernatant was removed.
- the COOH beads were shielded from light with aluminum foil so that they were not exposed to light, and shaken using a rotary incubator at room temperature for 30 minutes.
- the COOH bead suspension was centrifuged at 14,000 ⁇ g for 4 minutes, and the supernatant was removed.
- the beads immobilized with the TAPS protein thus obtained were suspended in a storage buffer of 150 ⁇ L and stored.
- Table 1 shows the concentration of each bead.
- Donor2 confirmed the reaction to XAGE1b.
- Serum was obtained from 8 patients with renal cell carcinoma (Donor 2 to Donor 9) before and after EP-DC treatment.
- EP-DC treatment a tumor tissue removed from a cancer patient by surgery is prepared as a lysate by freeze-thawing, etc., taken into dendritic cells by electroporation, and a dendritic cell vaccine prepared It is a treatment method administered to a patient.
- the beads were washed with a wash station (Bio-Rad), PE-labeled streptavidin (Vector (Laboratories) was added, light-shielded with aluminum foil, and shaken at room temperature for 10 minutes to react.
- the beads were washed with a wash station (Bio-Rad) and analyzed using a BioPlex (Bio-Rad) measuring device.
- the patient serum used was diluted 400 times, 1600 times, or 6400 times.
- the Donor3 serum had a higher response to WT-1 after the DC vaccine treatment, compared with before and after the DC vaccine treatment.
- the presence of an antibody that recognizes WT-1 in the blood of Donor3 suggests that the tumor tissue of Donor3 may express WT-1. In that case, it is presumed that it is appropriate to perform peptide vaccine or DC vaccine treatment using WT-1.
- lysozyme 15 mg was weighed into a 100 mL pear-shaped flask and dissolved in 2 mL of 6 M guanidine hydrochloride, 0.1 M Tris-HCl, pH 8.5, 2 mM EDTA. The resulting solution was degassed and purged with nitrogen, dithiothreitol (DTT) was added to a final concentration of 30 mM, and the mixture was reacted in a 37 ° C. constant temperature bath for 2 hours.
- DTT dithiothreitol
- each cationization reagent (TAPS-sulfonate (manufactured by Katayama Chemical Co., Ltd.) or TAP3S-sulfonate) having a final concentration of 70 mM was added and further reacted at room temperature for 2 hours. 1/10 amount of acetic acid was added to the resulting solution to stop the reaction, and then dialyzed against Milli-Q water at 4 ° C. for 1 day to obtain TAPS lysozyme and TAP3S lysozyme.
- the TAP3S-sulfonate used was synthesized by the method described in Japanese Patent Application No. 2010-070804.
- Reverse phase HPLC high performance liquid chromatography
- Native lysozyme has a certain degree of hydrophilicity because there are four SS bonds in one molecule and the hydrophobic group is not exposed.
- Reduced lysozyme is a denatured protein in which SS bonds are reduced and has the highest hydrophobicity (low solubility in water).
- the column used was COSMOSIL-Protein-R (manufactured by Nacalai Techs), the solvent used was acetonitrile diluted with 0.1% hydrochloric acid, and the acetonitrile concentration was set from 1% to 50%.
- FIG. 11 shows the result of summarizing the reverse phase HPLC chart.
- the straight line indicates the concentration of acetonitrile.
- TAP3S lysozyme is more hydrophilic than native and reduced lysozyme and is eluted with a low concentration of acetonitrile solvent.
- TAPS-modified lysozyme had all eight SH groups protected and cationized and was highly uniform.
- the difference in the protective ability of SH groups between TAPS and TAP3S depends on the size of the molecule.
- TAP3S which has a large molecule, is It is presumed that the steric hindrance above occurs and a small amount of SH groups that cannot bind to TAP3S remain, so that the SH / SS exchange reaction proceeds slowly thereafter.
- TAPS, TAP3S, and Native MAGE-A4 were prepared, and beads with respective proteins immobilized thereon were prepared.
- the prepared beads were suspended in a storage buffer and stored at 4 ° C. or 37 ° C. After a certain period of time, the degree of aggregated beads among the beads in the suspension was measured with a hemocytometer. The ratio of bead aggregates shown in the results was calculated by the number of aggregates formed of three or more beads / total number of beads ⁇ 100.
- FIG. 12 shows the ratio of bead aggregates contained in the suspension stored at 4 ° C. for 5 days, 37 ° C. for 10 days or 41 days.
- the beads aggregates of TAGS-modified MAGE-A4 were preserved compared to the beads immobilized with Native MAGE-A4. The rate is falling.
- ⁇ His-tag is added to the N-terminal side of MAGE-A4 immobilized on beads. Therefore, free MAGE-A4 protein contained in the stock solution of beads immobilized with MAGE-A4 was detected by Western blotting using an anti-His-tag antibody (OGHis, manufactured by MBL).
- binding proteins were detected for Bio-Plex® COOH beads, TAPS-modified MAGE-A4 immobilized beads, TAP3S-modified MAGE-A4 immobilized beads, or Native® MAGE-A4 immobilized beads stored for 104 days.
- beads prepared by the method for producing an antibody detection reagent disclosed in the present invention have an effect of suppressing the formation of aggregates compared to the preparation of beads using a native protein. Became clear. It was also confirmed that the suppression of aggregate formation was not due to the release of the antigen protein on the bead surface. Furthermore, it was known that TAP3S was superior to TAPS in terms of protein solubilization ability, but TAPS was superior to the solubilization ability of the prepared beads, unlike the solubilization ability. Became clear.
- an antibody detection reagent using an antigen protein can be efficiently produced by using the production method of the present invention. It has also been confirmed that the antibody detection reagent produced by the production method of the present invention has high stability and can efficiently detect antibodies in a liquid sample.
- the reagent of the present invention it is possible to provide an antibody analysis test that is not restricted by the HLA type of the subject. By performing this test, it is considered that, for example, it is possible to determine a treatment policy for a disease involving immunity and to predict / determine a therapeutic effect.
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Abstract
Description
(2)前記抗原タンパク質が、全長タンパク質であることを特徴とする、(1)に記載の抗体検出用試薬の製造方法、
(3)前記抗原タンパク質が、膜タンパク質であることを特徴とする、(1)又は(2)に記載の抗体検出用試薬の製造方法、
(4)前記抗原タンパク質が、がん抗原タンパク質であることを特徴とする、(1)~(3)のいずれか一つに記載の抗体検出用試薬の製造方法、
(5)前記カチオン化が、抗原タンパク質のチオール基にカチオン化剤を結合させることによって行われることを特徴とする、(1)~(4)のいずれか一つに記載の抗体検出用試薬の製造方法。
(7)前記チオスルホナート化合物が、以下の式の化合物である
(6)に記載の抗体検出用試薬の製造方法(式中、R1は、炭素原子数2~20の直鎖アルキレン基を表し、R2は、炭素原子数1~3のアルキル基を表し、nは1~3のいずれかの整数である)、
(8)前記化合物が、R1が-(CH2)3-であり、R2がCH3-であり、nが1で表されるTAPS-Sulfonateであることを特徴とする、(7)に記載の製造方法、
(9)前記化合物が、R1が-(CH2)3-であり、R2がCH3-であり、nが3で表されるTAP3S-Sulfonateであることを特徴とする、(7)に記載の抗体検出用試薬の製造方法、
(10)前記ハロゲン化アルキル系カチオン化剤が、TAP-Brであることを特徴とする、(6)に記載の抗体検出用試薬の製造方法、
(11)前記担体が、磁気ビーズであることを特徴とする、(1)~(10)のいずれか一つに記載の抗体検出用試薬の製造方法。
(14)前記サンプルが、単離された体液であることを特徴とする、(13)に記載の抗体検出方法。
更に、かかる抗体検出用試薬は従来方法で製造された試薬に比べ安定性にすぐれ、長期の保存が可能である。
・被験者の生体内抗体の解析により、被験者のアレルギー反応性を判定する。
・治療の前後において抗体の解析を行い、治療の効果や治療後の経過と相関関係を有する抗体を見出す。見出した抗体を指標として治療方針の決定、予後の予測・判定を行う。
・免疫細胞療法において、患者血清中に抗原特異的抗体が検出された抗原を治療に用いることにより、治療効果の向上を図る。
・放射線療法の前後に、抗体の解析を行うことで、アブスコパル効果等の免疫機能が関与するとされている治療効果の予測を行う。
まず、抗体検出に使用する抗原タンパク質の調製を行った。
TAPS化したMAGE-A4、WT-1に対し逆相HPLCを用いて精製を行った。精製図を図4、5に示す。
COOHビーズの容器をボルテックスミキサーで30秒間振盪し、超音波を30秒かけて、懸濁した。
COOHビーズ懸濁液を14,000×gで4分間遠心し、上清を除去した。
COOHビーズ懸濁液を14,000×gで4分間遠心し、上清を除去した。
2名のがん患者(Donor1及びDonor2)から血液を採取し、血清を取得した。
腎細胞がん患者8名(Donor2~Donor9)から、EP-DC治療の前後で、血清を取得した。
EP-DC治療は、がん患者から手術によって除去した腫瘍組織を凍結融解法などによりライセートとして調製し、エレクトロポレーション法により樹状細胞に取り込ませ、樹状細胞ワクチンを調製したものを、当該患者へ投与する治療方法である。
ニワトリ卵白リゾチーム(配列番号13、以下、単にリゾチームとも記す、キューピー社製)をサンプルとして、カチオン化試薬のSH基保護能の評価を行った。
タンパク質のSH基がすべて保護されていない状態で抗体検出用試薬を調製・保存した場合には、保護されずに残ったSH基がSS結合を形成し、タンパク質の凝集や抗体検出用試薬の凝集が起こることが想定される。そこで、抗体検出用試薬の保存安定性を、以下の手順により確認した。
ナス型フラスコに上記アフィニティー精製後のXAGE1b又はNY-ESO-1を加え、水分を凍結乾燥機で除去したものを3mLの6M グアニジン,0.1M Tris-HCl pH8で溶解した。脱気・窒素置換後に30mMのDTT(固体)を加え、37℃で1時間ほど処理した。XAGE1b又はNY-ESO-1の含まれる溶液に70mM TAP-Brを添加し、37℃で60分ほど処理した。
Claims (14)
- 抗原タンパク質と担体とで構成される抗体検出用試薬の製造方法であって、抗原タンパク質をカチオン化して可溶化する工程と、カチオン化した抗原タンパク質を担体に結合させる工程と、を含む抗体検出用試薬の製造方法。
- 前記抗原タンパク質が、全長タンパク質であることを特徴とする、請求項1に記載の抗体検出用試薬の製造方法。
- 前記抗原タンパク質が、膜タンパク質であることを特徴とする、請求項1又は2に記載の抗体検出用試薬の製造方法。
- 前記抗原タンパク質が、がん抗原タンパク質であることを特徴とする、請求項1乃至3のいずれか一項に記載の抗体検出用試薬の製造方法。
- 前記カチオン化が、抗原タンパク質のチオール基にカチオン化剤を結合させることによって行われることを特徴とする、請求項1乃至4のいずれか一項に記載の抗体検出用試薬の製造方法。
- 前記カチオン化剤が、チオスルホナート化合物、混合ジスルフィド化合物、ピリジルスルフィド系のカチオン化剤、ハロゲン化アルキル系カチオン化剤のいずれか1つ、又はその混合物から選択されることを特徴とする、請求項5に記載の抗体検出用試薬の製造方法。
- 前記化合物が、R1が-(CH2)3-であり、R2がCH3-であり、nが1で表されるTAPS-Sulfonateであることを特徴とする、請求項7に記載の製造方法。
- 前記化合物が、R1が-(CH2)3-であり、R2がCH3-であり、nが3で表されるTAP3S-Sulfonateであることを特徴とする、請求項7に記載の抗体検出用試薬の製造方法。
- 前記ハロゲン化アルキル系カチオン化剤が、TAP-Brであることを特徴とする、請求項6に記載の抗体検出用試薬の製造方法。
- 前記担体が、磁気ビーズであることを特徴とする、請求項1乃至10のいずれか一項に記載の抗体検出用試薬の製造方法。
- 請求項1乃至11の製造方法によって製造された抗体検出用試薬。
- サンプル中に含まれる抗原特異的な抗体の検出方法であって、請求項12に記載の抗体検出用試薬とサンプルとを接触させる工程と、抗体に結合する標識した二次抗体を添加し、抗体と結合させる工程と、抗体検出用試薬を回収する工程と、抗体が結合した抗体検出用試薬を検出する工程とを含む、抗体検出方法。
- 前記サンプルが、単離された体液であることを特徴とする、請求項13に記載の抗体検出方法。
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Also Published As
Publication number | Publication date |
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JPWO2013147233A1 (ja) | 2015-12-14 |
AU2013240942A1 (en) | 2014-11-13 |
SG11201406160QA (en) | 2014-11-27 |
EP2833143A4 (en) | 2015-11-11 |
CN104380106B (zh) | 2017-02-22 |
EP2833143B1 (en) | 2019-04-24 |
TWI582424B (zh) | 2017-05-11 |
US20150064801A1 (en) | 2015-03-05 |
JP6168497B2 (ja) | 2017-07-26 |
US10822384B2 (en) | 2020-11-03 |
AU2013240942B2 (en) | 2018-07-05 |
US20210032302A1 (en) | 2021-02-04 |
CN104380106A (zh) | 2015-02-25 |
TW201400814A (zh) | 2014-01-01 |
EP2833143A1 (en) | 2015-02-04 |
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