WO2011021308A1 - げっ歯類由来IgG抗体に結合性を有する核酸分子、結合剤、検出試薬および検出キット - Google Patents
げっ歯類由来IgG抗体に結合性を有する核酸分子、結合剤、検出試薬および検出キット Download PDFInfo
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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
- G01N33/68—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
- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Definitions
- the present invention relates to a nucleic acid molecule capable of binding to a rodent-derived IgG antibody, a binding agent, a detection reagent, and a detection kit.
- the antigen-antibody reaction is a highly specific reaction, it is applied to the detection of specific proteins.
- an antibody labeled with an enzyme is reacted with a specific antigen (protein or the like) in a sample to form a complex, and the complex is further captured with an antibody immobilized on a bead or the like.
- an antigen protein or the like
- antibodies are also used as pharmaceuticals by utilizing their specificity (see, for example, Patent Document 1).
- antibodies derived from rodents such as mice and rats are used.
- detection kits using rodent-derived antibodies antibodies that specifically bind to rodent-derived antibodies are used.
- problems that it is difficult to produce antibodies and that handling of the antibodies is complicated.
- An object of the present invention is to provide a nucleic acid molecule that is easy to manufacture and handle and has a binding property to a rodent-derived IgG antibody having a binding property equal to or higher than that of an antibody.
- the nucleic acid molecule of the present invention has a specific binding property to a rodent-derived IgG antibody, and has a dissociation constant of 1 ⁇ M or less.
- the unit M of concentration is equal to 1 mol / dm 3 or 1 mol / L.
- the binding agent for rodent-derived IgG antibody of the present invention is characterized by containing the nucleic acid molecule of the present invention.
- the detection reagent for the rodent-derived IgG antibody of the present invention is characterized by containing a binding agent for the rodent-derived IgG antibody of the present invention.
- the rodent-derived IgG antibody detection kit of the present invention is characterized by including the detection reagent for the rodent-derived IgG antibody of the present invention.
- the nucleic acid molecule of the present invention can bind with high specificity to a rodent-derived IgG antibody.
- the nucleic acid molecule of the present invention is easier to manufacture and handle than antibodies.
- FIG. 1 is a schematic diagram showing predicted secondary structures of various aptamers.
- FIG. 2 is a schematic diagram showing predicted secondary structures of various aptamers.
- FIG. 3 is a schematic diagram showing predicted secondary structures of various aptamers.
- FIG. 4 is a photograph showing the results of Northwestern blotting.
- FIG. 5 is a graph showing the analysis result by BIACORE.
- FIG. 6 is a graph showing the analysis result by BIACORE.
- FIG. 7 is a graph showing the analysis result by BIACORE.
- FIG. 8 is a graph showing the analysis result by BIACORE.
- FIG. 9 is a graph showing an analysis result by BIACORE.
- FIG. 10 is a graph showing the analysis result by BIACORE.
- FIG. 10 is a graph showing the analysis result by BIACORE.
- FIG. 11 is an electrophoretogram showing the analysis results of aptamer stability.
- FIG. 12 is a graph showing the analysis results of aptamer stability.
- FIG. 13 is an electrophoresis photograph showing the analysis result of the specificity of binding between aptamer and mouse-derived IgG antibody.
- the nucleic acid molecule of the present invention has a specific binding property to a rodent-derived IgG antibody, and has a dissociation constant of 1 ⁇ M or less.
- the dissociation constant is preferably 200 nM or less, more preferably 100 nM or less, and still more preferably 10 nM or less.
- a rodent refers to a mammal reticulata (rodent). Examples of rodents include mice and rats.
- the nucleic acid molecule of the present invention is preferably a nucleic acid molecule that specifically binds to mouse-derived IgG with the dissociation constant.
- the nucleic acid molecule of the present invention is preferably a nucleic acid molecule that specifically binds to rat-derived IgG with the dissociation constant.
- the nucleic acid molecule of the present invention may be, for example, single-stranded or double-stranded.
- the single strand include single-stranded RNA and single-stranded DNA
- examples of the double-stranded nucleic acid include double-stranded RNA and double-stranded DNA.
- the nucleic acid molecule of the present invention may be made into a single strand by denaturation or the like before use, for example.
- the nucleic acid molecule of the present invention may be, for example, an RNA molecule or a DNA molecule, but is preferably an RNA molecule.
- the nucleic acid molecule of the present invention may be DNA or RNA, for example, as described above, but the nucleotide residues constituting the nucleic acid molecule include deoxyribonucleotides that are DNA structural units and RNA structural units. As well as ribonucleotides.
- the nucleic acid molecule of the present invention is not limited by the number of strands such as ssDNA, ssRNA, dsDNA, dsRNA, or whether the nucleic acid is modified.
- the base in the nucleotide may be, for example, natural bases (non-artificial bases) such as adenine (a), cytosine (c), guanine (g), thymine (t), and uracil (u). Further, it may be an artificial base such as a modified base or a modified base having the same function as the natural base (a, c, g, t or u). Examples of the artificial base having the same function as described above include an artificial base capable of binding to cytosine (c) instead of guanine (g), and an artificial base capable of binding to guanine (g) instead of cytosine (c).
- an artificial base capable of binding to thymine (t) or uracil (u) an artificial base capable of binding to adenine (a) instead of thymine (t), and uracil (u)
- an artificial base capable of binding to adenine (a) examples of the modified base include methylated base, 2'-fluorouracil, 2'-aminouracil, 2'-O-methyluracil, 2-thiouracil and the like.
- nucleic acid molecule of the present invention may include, for example, peptide nucleic acids such as PNA, LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylenebridged Nucleic Acids) and the like.
- the nucleic acid molecule of the present invention is, for example, a single-stranded nucleic acid molecule and may be represented by the following general formula (I).
- the N 1 , N 2 , N 3 , N 4 , N 5 , N 6 , N 7 and N 8 represent nucleotide residues
- the n 1 , n 2 , n 3 , n 4 , n 5 , n 6 , n 7 and n 8 represent the number of the nucleotide residues N 1 , N 2 , N 3 , N 4 , N 5 , N 6 , N 7 and N 8 , respectively.
- 1 , N 3 , N 5 and N 7 can each form a loop structure
- the N 2 and N 8 can be hydrogen bonded to each other to form a stem structure
- the N 4 and N 6 can To form a stem structure by hydrogen bonding.
- loop structure can be formed includes, for example, that a loop structure is actually formed and that a loop structure can be formed depending on conditions even if the loop structure is not formed.
- the fact that “a loop structure can be formed” includes, for example, both experimentally confirmed and predicted by simulation of a computer or the like.
- a stem structure can be formed includes, for example, that a stem structure is actually formed and that a stem structure can be formed depending on conditions even if the stem structure is not formed.
- being able to form a stem structure includes, for example, both experimentally confirmed and predicted by simulation of a computer or the like.
- the number of nucleotide residues in the entire single-stranded nucleic acid is not particularly limited, but is, for example, in the range of 7 to 150 residues, and preferably in the range of 10 to 100 residues. More preferably, it is in the range of 15 to 60 residues.
- the nucleotide residue number n 1 of said N 1 is not particularly limited, for example, in the range of 0 to 40 residues, preferably in the range of 1 to 25 residues, more preferably, 4 to 10 residues The range of the group.
- the number of N 2 nucleotide residues n 2 is not particularly limited, but is, for example, in the range of 1 to 40 residues, preferably in the range of 2 to 20 residues, and more preferably in the range of 2 to 8 residues.
- the nucleotide residue number n 3 of said N 3 is not particularly limited, for example, in the range of 1 to 50 residues, preferably in the range of 1 to 30 residues, more preferably 1 to 15 residues The range of the group.
- the nucleotide residue number n 4 of the N 4 is not particularly limited, for example, in the range of 1 to 40 residues, preferably in the range of 1 to 20 residues, more preferably 1 to 10 residues The range of the group.
- the number of N 5 nucleotide residues n 5 is not particularly limited, but is, for example, in the range of 1 to 50 residues, preferably in the range of 1 to 30 residues, and more preferably 1 to 15 residues.
- the nucleotide residue number n 6 of the N 6 is not particularly limited, for example, in the range of 1 to 40 residues, preferably in the range of 1 to 20 residues, more preferably 1 to 10 residues The range of the group.
- the nucleotide residue number n 7 of the N 7 is not particularly limited, for example, in the range of 1 to 50 residues, preferably in the range of 1 to 30 residues, more preferably 1 to 10 residues The range of the group.
- the number of N 8 nucleotide residues n 8 is not particularly limited, but is, for example, in the range of 1 to 40 residues, preferably in the range of 2 to 20 residues, and more preferably in the range of 2 to 8 residues.
- the number of nucleotide residues n 1 , n 2 , n 3 , n 4 , n 5 , n 6 , n 7 and n 8 is not particularly limited, but preferably satisfies the following equations and inequalities.
- n 2 n 8
- n 4 n 6 , (n 3 + n 7 )> n 5
- the nucleic acid molecule represented by the formula (I) may be a DNA molecule or an RNA molecule, but is preferably an RNA molecule.
- the nucleic acid molecule of the present invention is an RNA molecule, it is preferably resistant to RNase.
- the method of making the RNase resistant is not particularly limited. For example, a method of modifying a part of the nucleotide residue of the RNA nucleic acid molecule of the present invention by methylation, etc. Or the method of LNA etc. is mention
- a part of the nucleotide residues is a modified nucleotide residue.
- the modified nucleotide residue is preferably a methylated nucleotide residue, for example.
- the modification site in the nucleotide residue is preferably a ribose site, for example.
- the base is a pyrimidine base, for example, the 2'-position and the 4'-position are preferably modified, and when the base is a purine base, for example, the 2'-position and the 4'-position are preferably modified.
- a method of binding tens of kDa PEG (polyethylene glycol) or deoxythymidine to the 5 'end or 3' end is also included.
- the number of nucleotide residues in the entire single-stranded nucleic acid is not particularly limited, but is, for example, in the range of 7 to 150 residues, and preferably in the range of 10 to 100 residues. More preferably, it is in the range of 15 to 60 residues.
- the site of the modified nucleotide residue is not particularly limited, but is preferably the end of a region where a stem structure can be formed and the end of a region where a loop structure can be formed.
- a part of the nucleotide residue is, for example, at least one of LNA and DNA.
- the nucleic acid molecule represented by the formula (I) is an RNA molecule as described above, it is preferable that a part or all of the nucleotide residue is, for example, at least one of LNA and DNA.
- the RNA molecule may include both LNA and DNA.
- the number of LNA nucleotide monomers is not particularly limited, but is, for example, in the range of 1 to 150 residues, preferably in the range of 1 to 100 residues, and more preferably in the range of 1 to 150 residues. It is in the range of 10 residues.
- the length of the LNA included in the formula (I) is not particularly limited, but is, for example, in the range of 1 to 10 residues, preferably 2 residues.
- the number of deoxyribonucleotide monomers constituting the DNA is not particularly limited, but is, for example, in the range of 1 to 150 residues, preferably in the range of 1 to 100 residues, and more preferably. Is in the range of 1-30 residues.
- the length of the DNA contained in (I) is not particularly limited, but is, for example, in the range of 1 to 30 residues, preferably in the range of 3 to 10 residues, and more preferably 4 A range of residues.
- the nucleic acid molecule represented by the formula (I) is an RNA molecule and contains DNA
- the DNA is contained in a site where the stem structure can be formed in the formula (I).
- the nucleic acid molecule represented by the formula (I) is an RNA molecule and contains LNA
- it is preferable that the LNA is also contained in the site where the stem structure can be formed in the formula (I).
- the formula (I) it is preferable that all or part of N 2 and N 7 contain LNA.
- a label compound is preferably bonded to at least one of the 5 'end and the 3' end, and more preferably, the label compound is bonded to the 5 'end.
- the label compound is not particularly limited.
- the fluorophore is not particularly limited, and examples thereof include fluorescein and its derivatives, rhodamine and its derivatives, dansyl chloride and its derivatives, umbelliferone and the like.
- the enzyme is not particularly limited, and examples thereof include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, and amylase.
- the radioisotope is not particularly limited. For example, iodine ( 131 I, 125 I, 123 I, 121 I), phosphorus ( 32 P), sulfur ( 35 S), metals (for example, 68 Ga, 67 Ga, 68 Ge, 54 Mn, 99 Mo, 99 Tc, 133 Xe, etc.), tritium and the like.
- luminescent substances such as NADH-, FMNH2-, acridinium ester, and luminol
- bioluminescent substances such as luciferase and luciferin
- biofluorescent proteins such as GFP.
- the nucleic acid molecule of the present invention preferably comprises, for example, at least one sequence selected from the group consisting of the following sequences.
- “dN” such as dA, dG, dC and dT represents a deoxyribonucleotide residue
- “ N ” such as G and C represents an LNA nucleotide residue
- “mN” such as mC and mU. Indicates a methylated ribonucleotide residue.
- the mouse IgG antibody is preferably at least one of a subtype 1 IgG antibody, a subtype 2a IgG antibody, and a subtype 3 IgG antibody.
- the subtypes of mouse-derived IgG antibodies are not particularly limited.
- subtypes 1, subtypes 2a and subtypes 3 and IgG antibodies having different subtypes optionally having these are mixed. Is mentioned.
- the nucleic acid molecule of the present invention is formed by specifically binding a nucleic acid molecule and a target substance using a nucleic acid molecule such as a so-called RNA pool and an IgG antibody derived from a rodent such as a mouse as a target substance.
- the nucleic acid molecule / target substance complex can be produced by a method of selecting only the nucleic acid molecule involved in the formation of the complex. As such a method, for example, after obtaining a nucleic acid molecule / target substance complex using a method called SELEX method (Systematic Evolution of Ligand by EXPonential enrichment), or using a carrier such as agarose gel or polyacrylamide gel. Examples thereof include a method for recovering only the nucleic acid molecules involved in the formation of this complex.
- SELEX method Systematic Evolution of Ligand by EXPonential enrichment
- the nucleic acid molecule of the present invention is obtained by recovering an RNA pool-target substance complex obtained by reacting an RNA pool and a target substance according to the SELEX method or a method according to this method. Only RNA pools involved in complex formation can be recovered and manufactured.
- RNA pool is a region in which about 20 to 120 bases selected from the group consisting of A, G, C and U, and substitutions of this base are linked (this region is hereinafter referred to as a “random region”). .)) Refers to a mixture of genes collectively referring to gene sequences having. Therefore, the RNA pool preferably includes 4 20 to 4 120 (10 12 to 10 72 ) types of genes, and preferably includes 4 30 to 4 60 (10 18 to 10 36 ) types of genes.
- the base substitute include appropriately substituted bases such as halogens such as fluorine, chlorine, bromine and iodine, and alkyl groups such as methyl, ethyl and propyl.
- the RNA pool is not limited in other structures as long as it has a random region. However, when the nucleic acid molecule of the present invention is produced according to the SELEX method, the 5 ′ end and / or the 3 ′ end of the random region are described later. It preferably has a primer region used in PCR and the like, and a DNA-dependent RNA polymerase recognition region.
- the random region includes a DNA-dependent RNA polymerase recognition region (hereinafter referred to as “RNA polymerase recognition region”) such as a T7 promoter from the 5 ′ end side, and a DNA-dependent DNA polymerase primer region ( Hereinafter, this region is referred to as “5 ′ terminal primer region”), a random region is connected to the 3 ′ end of this 5 ′ terminal primer region, and further to the 3 ′ end of this random region. It may have a structure in which a primer region of a DNA-dependent DNA polymerase (hereinafter, this region is referred to as “3 ′ terminal primer region”) is linked.
- the RNA pool may have a known region that assists in binding to the target substance.
- the RNA pool may have a part of the random region having the same sequence in each RNA pool.
- the random region is obtained by performing gene amplification based on the PCR method using an initial pool in which U in the random region of the RNA pool is replaced with T as a template, and a DNA-dependent RNA polymerase such as T7 polymerase. It may be prepared by reacting. In addition, a gene complementary to the initial pool is synthesized, and a primer composed of an RNA polymerase recognition region and a sequence complementary to the 5 ′ terminal primer region is annealed to the gene complementary to this primer in the initial pool. It may be prepared based on the PCR method.
- the RNA pool synthesized in this manner and the target rodent-derived IgG antibody are bound via intermolecular forces such as hydrogen bonding.
- the binding method include a method in which the RNA pool and the target substance are incubated for a certain period of time in a buffer solution that maintains a function such as binding of the target substance. In this way, an RNA pool-target substance complex is formed in the buffer.
- the buffer contains RNA pools and target substances that were not involved in the formation of the complex.
- a nucleic acid molecule that binds to the target substance is used.
- a method of removing an RNA pool that was not involved in the formation of a complex present in the buffer solution may be used. Examples of this method include a method that utilizes a difference in the adsorptivity to a specific component of the target substance and the RNA pool, and a method that utilizes a difference in molecular weight between the complex and the RNA pool.
- the above-mentioned RNA pool-target substance complex can be prepared using a membrane having an adsorptivity to the target substance such as nitrocellulose.
- the RNA pool-target substance complex is adsorbed on the membrane, and the RNA pool involved in the formation of the complex from the RNA pool-target substance complex remaining on the membrane is, for example, A method of recovering the RNA pool after releasing the binding between the RNA pool and the target substance in this complex can be mentioned.
- RNA pool-target substance complex As a method using the difference in molecular weight between the RNA pool-target substance complex and the RNA pool, a pore such as an agarose gel that can pass through the RNA pool but cannot pass through the RNA pool-target substance complex is used. There is a method in which the RNA pool-target substance complex and the RNA pool are electrically separated using a carrier having, and the RNA pool involved in the formation of the complex is recovered from this complex.
- gene amplification is performed using the RNA pool involved in the formation of the complex recovered from the RNA pool-target substance complex thus obtained.
- Examples of the gene amplification method include a method using a 5'-end primer region, a 3'-end primer region, and an RNA polymerase recognition region contained in the RNA pool.
- RNA dependence such as avian myeloblastosis virus-derived reverse transcriptase (AMV Reverse Transscriptase) using a gene fragment complementary to the 3 'terminal primer region of the RNA pool involved in complex formation as a primer
- AMV Reverse Transscriptase avian myeloblastosis virus-derived reverse transcriptase
- a PCR reaction using a DNA-dependent DNA polymerase is carried out using the 5 ′ terminal primer region and the 3 ′ terminal primer region contained in this cDNA.
- the RNA pool may be amplified by performing an in vitro transcription reaction using a DNA-dependent RNA polymerase using the RNA polymerase recognition region contained in the obtained gene product.
- RNA pool-target substance complex A nucleic acid molecule that specifically binds to an IgG antibody derived from mouse, rat or the like as a target substance, that is, a nucleic acid molecule having binding property to an IgG antibody derived from rodent can be obtained.
- the nucleic acid molecule of the present invention can be obtained as described above, but a part of the obtained nucleic acid molecule is modified with reference to the result of using the secondary structure prediction method based on the base sequence. It may be.
- the secondary structure prediction is particularly limited as long as it is a method for searching for secondary structure candidates of nucleic acid molecules and predicting secondary structures that are energetically stable among the searched secondary structure candidates. Absent. For example, two nucleic acid molecules obtained by dividing the base sequence of a nucleic acid molecule into a stem region constituting a Watson-Crick base pair and a single-stranded region such as a loop structure composed of bases other than this stem region. Secondary structure prediction based on minimizing the energy function of the secondary structure candidate may be used.
- the secondary structure prediction based on minimizing the energy function of this secondary structure candidate will be described.
- a candidate for a base constituting a Watson-Crick type base pair and a single-stranded region candidate other than this base pair candidate are searched. Except for combinations that cannot be theoretically taken, such as the bases that make up the base pair candidates and the bases that make up the single-stranded region candidates overlap among all the combinations of searched base pair candidates and single-stranded region candidates Identify secondary structure candidates.
- the energy function of the secondary structure candidate is calculated, and the secondary structure that minimizes the calculated energy function is searched.
- the free energy in this secondary structure candidate is A method of using an energy function of a secondary structure candidate may be used.
- the secondary structure having the smallest energy function is set as the secondary structure of the base sequence of the target nucleic acid molecule.
- the nucleic acid molecule of the present invention refers to the result of the secondary structure thus obtained, by substitution or deletion of a base constituting a characteristic site of the secondary structure, or of the secondary structure. It may be modified by insertion of a base into a characteristic part. For example, a part of the base constituting the stem region and / or the single-stranded region of the secondary structure may be substituted with the nucleic acid molecule prepared as described above as the parent molecule. Further, a part of the base constituting the stem region and / or the single-stranded region of the secondary structure may be deleted. In addition, a stem length and / or a single-stranded region may be shortened / extended by inserting one or more bases into the stem region and / or single-stranded region of the secondary structure.
- the nucleic acid molecule of the present invention preferably has a stem region having a stem length of 3 or more at the end of the nucleic acid molecule in the secondary structure of the nucleic acid molecule obtained by using this secondary structure prediction.
- the base adjacent to the terminal base on the single-stranded region side of the stem region having a stem length of 3 or more is preferably a base other than adenine.
- the stem region having a stem length of 3 or more is preferably composed of only guanine residues and cytosine residues. These further improve the binding to rodent-derived IgG antibodies.
- the nucleic acid molecule of the present invention is characterized by having binding properties to IgG antibodies derived from rodents such as mice. Therefore, the use of the nucleic acid molecule of the present invention is not particularly limited as long as it uses the binding property to an IgG antibody derived from a rodent such as a mouse, and the SDS-PAGE (SDS polyacrylamide electrophoresis) method.
- SDS-PAGE SDS polyacrylamide electrophoresis
- an ELISA method enzyme-linked immunosorbent assay
- a complex containing this object for detection of electrophoretic objects performed according to the above, for the detection of an ELISA method (enzyme-linked immunosorbent assay) or a complex containing this object, according to the Northwestern method It may be used for detection of a migration target carried out in the above, or an IgG antibody derived from a rodent such as a mouse or purification using this antibody.
- the nucleic acid molecule of the present invention when used in the SDS-PAGE method, it may be used for detecting the migrated protein, and rodents such as mice used for detecting the migrated protein. It may be used to detect IgG antibodies derived from the species.
- the nucleic acid molecule of the present invention when used in the ELISA method, it may be used for detecting IgG derived from rodents such as mice to be measured, and for detecting the measurement target. It may be used to detect IgG antibodies derived from rodents such as mice.
- the nucleic acid molecule of the present invention when used in the Northwestern method, it may be used to detect an IgG antibody derived from a rodent such as a mouse to be electrophoresed. It may be used to detect IgG antibodies derived from rodents such as mice, which are used for detection of proteins.
- the nucleic acid molecule of the present invention when used for purification of IgG derived from rodents such as mice, it can be used in a form suitable for purifying IgG antibodies derived from rodents such as mice to be purified.
- the nucleic acid molecule of the present invention may be used by binding to beads made of agarose or synthetic resin.
- beads made of agarose or synthetic resin As a form couple
- nucleic acid molecule of the present invention examples include a reagent containing the obtained nucleic acid molecule, a kit having this reagent, and the like utilizing the binding property to an IgG antibody derived from a rodent such as a mouse.
- a detection kit for detecting binding to an IgG antibody derived from a rodent such as a mouse using the above-described reagent containing the nucleic acid molecule of the present invention may be used.
- the measurement object of such a kit may be a liquid system such as a solution or a suspension, or may be a solid material such as a cultured cell or tissue section.
- a substance necessary for detecting the binding between the target substance and the nucleic acid molecule may be appropriately selected, and this is used for the detection kit. What is necessary is just to select suitably with a nucleic acid molecule.
- the detection kit of the present invention may be performed in accordance with the SDS-PAGE method (SDS polyacrylamide electrophoresis), and in this case, the reagent contained in the detection kit is the subject of electrophoresis. It may be a nucleic acid molecule that binds to a rodent IgG derived from a mouse or the like used for protein detection, or a nucleic acid molecule that binds to a rodent IgG derived from a mouse or the like to be subjected to electrophoresis. May be.
- SDS-PAGE method SDS polyacrylamide electrophoresis
- the detection kit of the present invention may be performed according to the ELISA method.
- the reagent contained in the detection kit is binding to IgG derived from rodents such as mice to be measured.
- the detection kit of the present invention may be performed according to the Northwestern method.
- a reagent contained in the detection kit a mouse or the like used for detection of a protein to be electrophoresed is used. It may be a nucleic acid molecule having a binding property to a rodent-derived IgG, or a nucleic acid molecule having a binding property to a rodent-derived IgG such as a mouse to be subjected to electrophoresis.
- the detection method of the present invention comprises: An immunoprecipitation step in which a detection target in a sample is an antigen, and the detection target and an antibody are reacted to perform immunoprecipitation; A separation step in which the immunoprecipitate is denatured and separated from other components of the sample; A first reaction step of reacting an antibody with the antigen of the immunoprecipitate separated in the separation step; A second reaction step of reacting a binding agent that specifically binds to the antibody in the antibody reaction step, The binder of the present invention is used as the binder in the second reaction step.
- the nucleic acid molecule (especially RNA molecule) of the present invention specifically binds to rodent-derived IgG such as mouse and has low specificity to denatured rodent-derived IgG such as mouse. Therefore, when the nucleic acid molecule of the present invention (in particular, RNA molecule) is used, for example, when detecting by Western blotting using mouse IgG after SDS-PAGE, it rarely binds to an antibody modified with SDS. It is possible to detect a detection object clearly while suppressing noise. Therefore, in the detection method of the present invention, it is preferable that the separation step is SDS-PAGE, and the first reaction step and the second reaction step are Western blot methods.
- mice IgG aptamers for mouse IgG antibodies used in the following Examples.
- Modification indicates modification of each sequence. Specifically, “5′-biotin” means that biotin is bound to the 5 ′ end, “dA 5mer” means 5mer polydeoxynucleotide residues, “stem LNA-2bp” and “stem LNA-4bp”.
- stem DNA-2 bp or 4 bp LNA in the region capable of forming a stem structure stem DNA-2 bp "and” stem DNA-4 bp "have 2 bp or 4 bp in the region capable of forming a stem structure
- + DT has a deoxythymidine residue at the 3 ′ end
- “methylation 21C”, “methylation 31C” and “methylation 10U” are the 21st C, 31st C, or 10th U, respectively. Indicates that it is methylated.
- MIG-m041 is an aptamer having a 4 bp DNA in a region where the 10th uracil residue is methylated and a stem structure can be formed.
- MIG-m042 is an aptamer having a 2-bp LNA in a region where the 9th uracil residue is methylated and can form a stem structure in MIG-m034.
- FIG. 4 is a photograph showing the results of Northwestern blotting. 4, (a) shows the result of MIG-m034, (b) shows the result of MIG-m041, and (c) shows the result of MIG-m042, and in each figure, lane M is biotinylated. Molecular weight markers, lane 1 is the result of using 200 ng FLAG-BAP, lane 2 is 100 ng FLAG-BAP, and lane 3 is the result using 50 ng FLAG-BAP. As shown in FIG. 4, even when any aptamer was used, a signal specific to the antigen could be detected. Among them, MIG-m041 and MIG-m042 were able to confirm signals sufficiently even though the aptamer concentration was a low concentration of 10 nM. [Example 2]
- the binding between the mouse IgG aptamer and various subtypes of mouse-derived IgG antibodies was analyzed.
- the aptamer the above-mentioned aptamers of MIG-m034, MIG-m041, and MIG-m042 were used.
- the analysis of the binding was performed using BIACORE 3000 (manufactured by GE Healthcare) according to the instructions for use. Further, from the signal intensity (RU: Resonance Unit) measured by the BIACORE, analysis was performed with a 1: 1 Langmuir binding model using BIAevaluation software, and the dissociation constant between each aptamer and mouse IgG antibody was obtained.
- the measurement conditions and method of the BIACORE were as described in the following document.
- FIGS. 5 to 7 are graphs showing changes in signal intensity over time.
- FIG. 5 shows the binding between the subtype 1 mouse IgG antibody and the aptamer
- FIG. 6 shows the binding between the subtype 2a mouse IgG and the aptamer
- FIG. 7 shows the binding between the subtype 3 mouse IgG and the aptamer.
- MIG-m034, MIG-m041 and MIG-m042 from the top in each figure.
- the vertical axis represents the signal intensity (RU) measured by the BIACORE
- the horizontal axis represents the analysis time (second).
- Table 2 shows the dissociation constants of each mouse IgG aptamer with each subtype mouse IgG antibody.
- FIGS. 8 to 10 are graphs showing changes in signal intensity over time, and are the results using subtype 1, subtype 2a and subtype 3 mouse IgG, respectively.
- the dissociation constants between the aptamers and the subtypes of mouse IgG determined from the BIACORE measurement results are shown in the following table. From the results in the following table, it was confirmed that these aptamers are very versatile as secondary antibody reagents and have excellent binding power.
- Example 2 since it was considered from Example 1 that the base pair in the stem region and U in the vicinity thereof do not participate in the binding of mouse IgG, a clone having a systematic modified base was prepared and the Kd value was determined. Since the binding force to the mouse antibody subtype is almost the same for any clone, the stem region may not be involved in the binding for any subtype, and the aptamer binding to each subtype The style was considered the same.
- the aptamer was added to the FBS solution, and this sample was kept at room temperature (20 ° C.) for 30 minutes, 1 hour, 2 hours, and 4 hours.
- An equal amount of 2 ⁇ Urea sample solution to which RNase inhibitor (RNAsecure; manufactured by Ambion) was added was added to the sample after holding and treated at 60 ° C. for 10 minutes to stop the nuclease reaction.
- the sampled RNA was added to a 15% 7M urea polyacrylamide gel (1 ⁇ TBE, 140 mm ⁇ 70 mm ⁇ 1 mm gel plate) at 10 to 20 ng per lane and subjected to electrophoresis at a constant voltage of 200 V for 80 to 90 minutes.
- the gel was stained with SYBR Gold (manufactured by Invitrogen) and photographed on a UV transilluminator.
- the full length aptamer band was used with ImageJ 1.37v (Abramoff, MD, Magellahaes, PJ, Ram, SJ “Image Processing with ImageJ” Biophotonics International, 11, 7, 36-42, 2004.). Densitometric analysis was performed and digitized to evaluate the stability of each aptamer. In the sample, the final concentration of the aptamer was 10 ⁇ M, and the final concentration of the FBS was 1%. MIG-m034, MIG-m041 and MIG-m042 were used as the aptamers.
- FIG. 11 is an electrophoretogram of the aptamer sample after being held for a certain time.
- m034, m041 and m042 each indicate the type of aptamer.
- M is an RNA marker
- 1 to 5 are results of retention times of 0, 0.5, 1.0, 2.0, and 4.0 hours, respectively. The retention time was 0 hour when the aptamer was added to the FBS solution.
- FIG. 12 is a graph showing changes in band intensity over time in electrophoresis.
- the vertical axis represents relative intensity (%), and the horizontal axis represents retention time (hr).
- the half-life of the aptamer in 1/100 diluted FBS was calculated as 1 to 2 hours. From the above results, it was found that the half-life of the aptamer can be extended by modifying a site that is not involved in binding to IgG.
- Example 1 the specificity of binding of the MIG-m041 aptamer to a mouse-derived IgG antibody was evaluated by North Western blotting.
- the experimental conditions were anti-FLAG mouse IgG (0.5 ⁇ g), anti-FLAG mouse IgG (0.5 ⁇ g) and FLAG-BAP protein (100 ng) as antigen, and FLAG-BAP protein (100 ng) as aptamer.
- the same procedure as in Example 1 was performed except that 20 nM MIG-m041 was used. [Comparative Example 1]
- Example 5 Western blotting was performed on the above antigen. Blotting was performed in the same manner as in Example 5 except that a biotinylated anti-mouse IgG goat antibody (AP124B manufactured by CHEMICON) was used instead of the MIG-m041 aptamer.
- a biotinylated anti-mouse IgG goat antibody API124B manufactured by CHEMICON
- Example 5 and Comparative Example 1 are shown in FIG.
- the figure (a) is a photograph showing the result of the North Western blot method of Example 5, and the figure (b) is a photograph showing the result of the Western blot method of Comparative Example 1.
- lane M is a biotinylated molecular weight marker
- lane 1 is anti-FLAG mouse IgG (0.5 ⁇ g)
- lane 2 is anti-FLAG mouse IgG (0.5 ⁇ g) and FLAG- BAP protein (100 ng)
- lane 3 is the result of using FLAG-BAP protein (100 ng).
- FIG. 4A and 4B lane M is a biotinylated molecular weight marker
- lane 1 is anti-FLAG mouse IgG (0.5 ⁇ g)
- lane 2 is anti-FLAG mouse IgG (0.5 ⁇ g)
- FLAG- BAP protein 100 ng
- lane 3 is the result of using FLAG-BAP protein (100 ng).
- the nucleic acid molecule of the present invention can be applied to all fields in which rodent-derived IgG antibodies are used, and can be applied to a wide range of fields such as clinical tests and biochemical tests.
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Abstract
Description
前記本発明の核酸分子は、前述の通り、げっ歯類由来IgG抗体に対し特異的結合性を有し、解離定数が、1μM以下であることを特徴とする。前記解離定数は、好ましくは200nM以下、より好ましくは100nM以下、さらに好ましくは、10nM以下である。本発明において、げっ歯類とは、哺乳網ネズミ目(げっ歯目)のことをいう。げっ歯類としては、例えば、マウス、ラット等があげられる。本発明の核酸分子は、マウス由来IgGに前記解離定数で特異的に結合する核酸分子が好ましい。同様に、本発明の核酸分子は、ラット由来IgGに前記解離定数で特異的に結合する核酸分子が好ましい。
前記N1のヌクレオチド残基数n1は、特に制限されないが、例えば、0~40残基の範囲であり、好ましくは、1~25残基の範囲であり、より好ましくは、4~10残基の範囲である。
前記N2のヌクレオチド残基数n2は、特に制限されないが、例えば、1~40残基の範囲であり、好ましくは、2~20残基の範囲であり、より好ましくは、2~8残基の範囲である。
前記N3のヌクレオチド残基数n3は、特に制限されないが、例えば、1~50残基の範囲であり、好ましくは、1~30残基の範囲であり、より好ましくは、1~15残基の範囲である。
前記N4のヌクレオチド残基数n4は、特に制限されないが、例えば、1~40残基の範囲であり、好ましくは、1~20残基の範囲であり、より好ましくは、1~10残基の範囲である。
前記N5のヌクレオチド残基数n5は、特に制限されないが、例えば、1~50残基の範囲であり、好ましくは、1~30残基の範囲であり、より好ましくは、1~15残基の範囲である。
前記N6のヌクレオチド残基数n6は、特に制限されないが、例えば、1~40残基の範囲であり、好ましくは、1~20残基の範囲であり、より好ましくは、1~10残基の範囲である。
前記N7のヌクレオチド残基数n7は、特に制限されないが、例えば、1~50残基の範囲であり、好ましくは、1~30残基の範囲であり、より好ましくは、1~10残基の範囲である。
前記N8のヌクレオチド残基数n8は、特に制限されないが、例えば、1~40残基の範囲であり、好ましくは、2~20残基の範囲であり、より好ましくは、2~8残基の範囲である。
以上のヌクレオチド残基数n1、n2、n3、n4、n5、n6、n7およびn8は、特に制限されないが、以下の等式および不等式を満たすことが好ましい。
n2=n8、n4=n6、(n3+n7)>n5
前記式(I)において、LNAヌクレオチドモノマーの数は、特に制限されないが、例えば、1~150残基の範囲であり、好ましくは、1~100残基の範囲であり、より好ましくは、1~10残基の範囲である。
また、前記式(I)に含まれるLNAの長さは、特に制限されないが、例えば、1~10残基の範囲であり、好ましくは2残基である。前記式(I)において、DNAを構成するデオキシリボヌクレオチドモノマーの数は、特に制限されないが、例えば、1~150残基の範囲であり、好ましくは、1~100残基の範囲であり、より好ましくは、1~30残基の範囲である。また、前記(I)に含まれるDNAの長さは、特に制限されないが、例えば、1~30残基の範囲であり、好ましくは、3~10残基の範囲であり、より好ましくは、4残基の範囲である。
配列番号1
dAdAdAdAdA-CGCUGAAGAGAAGACGGAAGGAGACGAAGCG-dT
配列番号2
dAdAdAdAdA-GCUGAAGAGAAGACGGAAGGAGACGAAGC-dT
配列番号3
dAdAdAdAdA-dGdCGCUGAAGAGAAGACGGAAGGAGACGAAGCdGdC
配列番号4
dAdAdAdAdA-dGdCdGdCUGAAGAGAAGACGGAAGGAGACGAAdGdCdGdC
配列番号5
dAdAdAdAdA-GCGCmUGAAGAGAAGACGGAAGGAGACGAAGCGC
配列番号6
dAdAdAdAdA-dGdCdGdCmUGAAGAGAAGACGGAAGGAGACGAAdGdCdGdC
配列番号7
dAdAdAdAdA-CGCmUGAAGAGAAGACGGAAGGAGACGAAGCG-dT
本発明の核酸分子は、SELEX法に従って、またこの方法に準じた方法で、RNAプールと標的物質とを反応させて得られるRNAプール-標的物質複合体を回収した後、この複合体から、この複合体の形成に関与したRNAプールのみを回収して、製造することが可能である。
本発明の核酸分子は、上記の通りに得ることが出来るが、その塩基配列に基づいた二次構造予測の手法を用いた結果を参照して、得た核酸分子の一部を改変されたものであってもよい。この二次構造予測としては、核酸分子の二次構造の候補を探索し、この探索された二次構造候補のうち、エネルギー的に安定な二次構造を予測する方法であれば、特に制約はない。例えば、核酸分子の塩基配列を、ワトソン・クリック型等の塩基対を構成するステム領域と、このステム領域以外の塩基で構成されるループ構造等の一本鎖領域とに分割して得た二次構造候補のエネルギー関数を最小化することに基づく二次構造予測であってもよい。
本発明の核酸分子は、上記の通り、マウス等のげっ歯類由来のIgG抗体に結合性を有することを特徴とするものである。従って、本発明の核酸分子の用途としては、マウス等のげっ歯類由来のIgG抗体への結合性を利用する用途であれば特に制約はなく、SDS-PAGE(SDSポリアクリルアミド電気泳動法)法に準じて行われる泳動対象物の検出用として、ELISA法(酵素免疫測定法;Enzyme-Linked ImmunoSorbent Assay)の測定の対象物若しくはこの対象物を含む複合体の検出用として、ノースウエスタン法に準じて行われる泳動対象物の検出用として、又はマウス等のげっ歯類由来のIgG抗体若しくはこの抗体を利用した精製用として用いられてもよい。
本発明の核酸分子の応用例としては、得た核酸分子を含有する試薬、この試薬を有するキット等、マウス等のげっ歯類由来のIgG抗体への結合性を利用したものが挙げられる。例えば、上記の本発明の核酸分子を含有する試薬を用いて、マウス等のげっ歯類由来のIgG抗体への結合を検出する検出キットとしてもよい。このようなキットの測定対象物としては、溶液、懸濁液のような液体系であってもよく、培養細胞や組織切片等、固形物であってもよい。
試料中の検出対象物を抗原とし、前記検出対象物と抗体とを反応させて免疫沈降させる免疫沈降工程と、
前記免疫沈降物を変性させて前記試料の他の成分から分離する分離工程と、
前記分離工程で分離した免疫沈降物の前記抗原に抗体を反応させる第1反応工程と、
前記抗体反応工程の抗体に対し特異的に結合する結合剤を反応させる第2反応工程とを有し、
前記第2反応工程の前記結合剤として、前記本発明の結合剤を用いることを特徴とする。
[実施例2]
(文献)
Yoshihito Yoshida*, Nobuya Sakai*, Hiromi Masuda,
Makio Furuichi, Fumiko Nishikawa, Satoshi Nishikawa,
Hiroshi Mizuno and Iwao Waga:
“Rabbit antibody detection with RNA aptamers”
Analytical Biochemistry, Vol. 375, pp. 217-222, (2008)
[実施例3]
[実施例5]
[比較例1]
Claims (16)
- げっ歯類由来IgG抗体に対し特異的結合性を有し、解離定数が1μM以下であることを特徴とする核酸分子。
- 前記式(I)において、
前記一本鎖核酸全体のヌクレオチド残基の数は、7~150残基の範囲であり、
前記N1のヌクレオチド残基数n1は、0~40残基の範囲であり、
前記N2のヌクレオチド残基数n2は、1~40残基の範囲であり、
前記N3のヌクレオチド残基数n3は、1~50残基の範囲であり、
前記N4のヌクレオチド残基数n4は、1~40残基の範囲であり、
前記N5のヌクレオチド残基数n5は、1~50残基の範囲であり、
前記N6のヌクレオチド残基数n6は、1~40残基の範囲であり、
前記N7のヌクレオチド残基数n7は、1~50残基の範囲であり、
前記N8のヌクレオチド残基数n8は、1~40残基の範囲である、
ことを特徴とする請求項2記載の核酸分子。 - 前記式(I)において、前記ヌクレオチド残基の一部がメチル化ヌクレオチド残基であることを特徴とする請求項2または3記載の核酸分子。
- RNA分子であることを特徴とする請求項1から4のいずれか一項に記載の核酸分子。
- 前記式(I)において、前記ヌクレオチド残基の一部がLNAおよびDNAの少なくとも一方であることを特徴とする請求項5記載の核酸分子。
- 前記式(I)において、5’末端にラベル化合物が結合していることを特徴とする請求項2から6のいずれか一項に記載の核酸分子。
- 前記ラベル化合物が、ビオチンであることを特徴とする請求項7記載の核酸分子。
- 下記に示す配列からなる群から選択された少なくとも一つの配列からなることを特徴とする請求項1から8のいずれか一項に記載の核酸分子。
配列番号1
dAdAdAdAdA-CGCUGAAGAGAAGACGGAAGGAGACGAAGCG-dT
配列番号2
dAdAdAdAdA-GCUGAAGAGAAGACGGAAGGAGACGAAGC-dT
配列番号3
dAdAdAdAdA-dGdCGCUGAAGAGAAGACGGAAGGAGACGAAGCdGdC
配列番号4
dAdAdAdAdA-dGdCdGdCUGAAGAGAAGACGGAAGGAGACGAAdGdCdGdC
配列番号5
dAdAdAdAdA-GCGCmUGAAGAGAAGACGGAAGGAGACGAAGCGC
配列番号6
dAdAdAdAdA-dGdCdGdCmUGAAGAGAAGACGGAAGGAGACGAAdGdCdGdC
配列番号7
dAdAdAdAdA-CGCmUGAAGAGAAGACGGAAGGAGACGAAGCG-dT - 前記げっ歯類由来IgGが、マウス由来IgGであることを特徴とする請求項1から9のいずれか一項に記載の核酸分子。
- 前記マウス由来IgG抗体が、サブタイプ1のIgG抗体、サブタイプ2aのIgG抗体およびサブタイプ3のIgG抗体のうち少なくとも一つであることを特徴とする請求項10記載の核酸分子。
- 請求項1から11のいずれか一項に記載の核酸分子を含むことを特徴とするげっ歯類由来IgG抗体に対する結合剤。
- 請求項12記載の結合剤を含むことを特徴とするげっ歯類IgG由来抗体の検出試薬。
- 請求項13記載の検出試薬を含むことを特徴とするげっ歯類由来IgG抗体の検出キット。
- 試料中の検出対象物を抗原とし、前記検出対象物と抗体とを反応させて免疫沈降させる免疫沈降工程と、
前記免疫沈降物を変性させて前記試料の他の成分から分離する分離工程と、
前記分離工程で分離した免疫沈降物の前記抗原に抗体を反応させる第1反応工程と、
前記抗体反応工程の抗体に対し特異的に結合する結合剤を反応させる第2反応工程とを有し、
前記第2反応工程の前記結合剤として、請求項12記載の結合剤を用いることを特徴とする検出方法。 - 前記分離工程が、SDS-PAGEであり、前記第1反応工程および前記第2反応工程がウエスタンブロット法であることを特徴とする請求項15記載の検出方法。
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US13/511,618 US8852954B2 (en) | 2009-08-21 | 2009-08-21 | Nucleic acid molecule having binding affinity to rodent-derived IgG antibody, binder, detection reagent, and detection kit |
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WO2011021308A1 (ja) * | 2009-08-21 | 2011-02-24 | Necソフト株式会社 | げっ歯類由来IgG抗体に結合性を有する核酸分子、結合剤、検出試薬および検出キット |
WO2015001856A1 (ja) * | 2013-07-01 | 2015-01-08 | Necソリューションイノベータ株式会社 | 属性推定システム |
EP3491135A1 (en) * | 2016-07-28 | 2019-06-05 | Laboratoire Français du Fractionnement et des Biotechnologies | Method for obtaining aptamers |
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JP2009046494A (ja) | 1996-11-19 | 2009-03-05 | Roche Diagnostics Gmbh | モノクローナル抗体又はポリクローナル抗体の安定な凍結乾燥された医薬製剤 |
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WO2011021308A1 (ja) * | 2009-08-21 | 2011-02-24 | Necソフト株式会社 | げっ歯類由来IgG抗体に結合性を有する核酸分子、結合剤、検出試薬および検出キット |
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JP2009046494A (ja) | 1996-11-19 | 2009-03-05 | Roche Diagnostics Gmbh | モノクローナル抗体又はポリクローナル抗体の安定な凍結乾燥された医薬製剤 |
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JP2018027024A (ja) * | 2016-08-15 | 2018-02-22 | 国立大学法人東京農工大学 | アプタマー及び抗体検出方法 |
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US8852954B2 (en) | 2014-10-07 |
US9557339B2 (en) | 2017-01-31 |
JP5692811B2 (ja) | 2015-04-01 |
AU2009351455B2 (en) | 2014-07-03 |
JPWO2011021308A1 (ja) | 2013-01-17 |
US20140370618A1 (en) | 2014-12-18 |
EP2489734A4 (en) | 2013-07-31 |
US20130022967A1 (en) | 2013-01-24 |
EP2489734A1 (en) | 2012-08-22 |
AU2009351455A1 (en) | 2012-08-23 |
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