WO2015151883A1 - Procédé pour la détection d'une substance cible - Google Patents

Procédé pour la détection d'une substance cible Download PDF

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Publication number
WO2015151883A1
WO2015151883A1 PCT/JP2015/058576 JP2015058576W WO2015151883A1 WO 2015151883 A1 WO2015151883 A1 WO 2015151883A1 JP 2015058576 W JP2015058576 W JP 2015058576W WO 2015151883 A1 WO2015151883 A1 WO 2015151883A1
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Prior art keywords
capture
target substance
polynucleotide
detection
solid phase
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PCT/JP2015/058576
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English (en)
Japanese (ja)
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孝介 丹羽
廣田　寿一
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日本碍子株式会社
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Priority to JP2015549888A priority Critical patent/JP6076500B2/ja
Publication of WO2015151883A1 publication Critical patent/WO2015151883A1/fr

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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • the present specification relates to a method for detecting a target substance.
  • This application is a related application of Japanese Patent Application No. 2014-73412, which is a Japanese patent application filed on March 31, 2014, and claims priority based on this Japanese application. All the contents that were made are used.
  • a method in which a target substance other than a nucleic acid, such as an antigen in a test solution, is captured using an antibody immobilized on a solid phase carrier or the like as a capture element and detected by a so-called sandwich method.
  • a target substance other than a nucleic acid such as an antigen in a test solution
  • an antibody array in which a plurality of antibodies are immobilized on a sheet-like solid phase carrier, or an immunochromatography (lateral flow assay) in which one or more antibodies are immobilized on a strip-like porous solid phase carrier ) Is well known (Patent Document 2).
  • an antibody which is a protein
  • a solid support in advance. Since proteins are generally hydrophobic, they tend to be physically adsorbed on a hydrophobic solid support. Therefore, from the viewpoint of immobilizing the antibody, a solid phase carrier made of a material such as hydrophobic nitrocellulose or nylon is preferable.
  • the target substance is a protein
  • the non-specific adsorption of the protein to the solid phase carrier must be suppressed, so that the solid phase carrier surface is blocked with BSA, gelatin, serum, skim milk, casein, etc.
  • Setting conditions for blocking is not easy because it requires optimization of conditions depending on the type of target substance.
  • This specification solves such conventional problems and provides a more practical method for detecting a target substance.
  • This specification also provides a detection device, a detection kit, and the like suitable for such a detection method.
  • the present inventors examined various problems in detecting a target substance by immobilizing a protein on a solid phase carrier and utilizing a specific interaction between the protein and the target substance.
  • various interactions related to the detection of the target substance on the solid phase carrier can be performed by interposing a polynucleotide-based interaction.
  • a method for detecting a target substance other than a nucleic acid Detecting the target substance on a solid phase carrier on which a first capture having a polynucleotide is immobilized via at least a part of the polynucleotide; With A method of detecting the target substance by utilizing at least a specific interaction exhibited by the first capture.
  • the specific interaction includes the following (a) to (b): (A) a specific interaction acting between a polynucleotide and the target substance (b) including any one of a specific interaction acting between a protein and the target substance Method.
  • the second capture includes a nucleic acid ligand.
  • the second capture captures a specific interaction with at least a part of the polynucleotide of the first capture, whereby the target substance is placed on the solid phase carrier.
  • the method according to any one of (4) to (8) which is a detection step.
  • the detection method according to any one of (1) to (9), wherein the target substance is a protein.
  • the detection method according to any one of (1) to (10), wherein the target substance is detected using an antibody or an antigen that specifically binds to the target substance.
  • the solid phase carrier is made of a hydrophilic material.
  • a detection device for detecting a target substance A solid support; A first capture having a polynucleotide retained on the solid support; A second capture that exhibits a specific interaction with the polynucleotide; A device comprising: (16) The device according to (15), wherein the second capture includes a tag that exerts a specific interaction with at least a part of the polynucleotide.
  • a detection kit for detecting a target substance A solid support; A first capture having a polynucleotide retained on the solid support, the first capture retained via at least a portion of the polynucleotide; A device comprising: With A kit capable of detecting the target substance via a second capture that exhibits a specific interaction with at least a part of the polynucleotide. (20) The kit according to (19), comprising the second capture as a reagent separate from the device. (21) The kit according to (19) or (20), further comprising a labeling element that specifically binds to the target substance.
  • the present disclosure relates to a target substance detection method, a detection device, a detection kit, and the like.
  • 1A and 1B illustrate an overview of the present disclosure.
  • a target substance other than a nucleic acid can be detected on a solid-phase carrier that is a first capture having a polynucleotide and is immobilized via at least a part thereof.
  • the target substance can be detected using at least the specific interaction exhibited by the first capture.
  • the first capture having a polynucleotide is immobilized on the solid phase carrier via at least a part of the polynucleotide, and the target substance is captured on the solid phase carrier via the first capture.
  • the above problems relating to the protein immobilization ability and adsorption ability of the solid phase carrier can be avoided.
  • at least one of the problems related to protein immobilization cost, immobilization workability, detection sensitivity and accuracy due to decrease in capture ability due to omission, inactivation, excessive immobilization, and blocking are solved. .
  • the detection ability is not lowered as in the conventional case, and the detection ability can be increased.
  • Specific interactions for detecting a target substance include, as shown in FIGS. 1A and 1B, (A) Specific interaction that acts between the polynucleotide and the target substance (b) Any specific interaction that acts between the polypeptide and the target substance can be included. Further, In addition to the interaction (a) or (b) (C) Specific interactions acting between polynucleotides are appropriately combined.
  • the amount of protein as a capture element can be reduced, so that the cost can be reduced and a plurality of, preferably a large number of second captures can be used to detect a large number of target substances. It is suitable for.
  • the target substance refers to a substance other than nucleic acid that does not contain nucleic acid, and is not particularly limited as long as it is other than nucleic acid.
  • examples include proteins (including peptides), polysaccharides, oligosaccharides, monosaccharides, organic compounds, lipids, other organic materials, and inorganic materials.
  • the specific interaction is an interaction other than a covalent bond such as a van der Waals force, such as a hydrogen bond, an electrostatic bond, an ionic bond, a dipole-dipole interaction, a hydrophobic bond, or the like.
  • a covalent bond such as a van der Waals force, such as a hydrogen bond, an electrostatic bond, an ionic bond, a dipole-dipole interaction, a hydrophobic bond, or the like.
  • a covalent bond such as a van der Waals force, such as a hydrogen bond, an electrostatic bond, an ionic bond, a dipole-dipole interaction, a hydrophobic bond, or the like.
  • the method for detecting a target substance of the present disclosure can comprise a step of detecting the target substance on a solid phase carrier on which a first capture having a polynucleotide is immobilized.
  • the target substance can be detected by utilizing a specific interaction based on at least the first capture polynucleotide.
  • the target substance is detected using at least the first capture.
  • the first capture will be described, and various detection modes using the first capture will be described.
  • the first capture has a polynucleotide.
  • the first capture may comprise only other polynucleotides, or may include other regions such as an immobilization region that may include a functional group for immobilization on a solid phase carrier to be described later. Also good.
  • the immobilization area and the like will be described later. Further, a polypeptide chain or the like may be provided.
  • the polynucleotide in the first capture can be a site for specific interaction with the target substance or the second capture described below. Furthermore, it can be set as the fixed area
  • Polynucleotide means a polymer of nucleotides such as deoxyribonucleotides and ribonucleotides or their equivalents as nucleotides.
  • a polynucleotide generally has a backbone in which ribose or deoxyribose is linked by a phosphate ester bond.
  • the equivalent may be a PNA having a backbone in which N- (2-aminoethyl) glycine is bonded by an amide bond and capable of holding a base that can be held by DNA or the like, or an alkylene with a phosphate ester bond.
  • It may be an artificial nucleic acid that has a backbone bonded in the above manner and can similarly hold a base, or L-form DNA may be used.
  • the backbone in a polynucleotide is typically mainly composed of a phosphate ester skeleton of deoxyribose.
  • a polynucleotide has D-form ribose or deoxyribose in the backbone of the main chain.
  • ribose or deoxyribose in the main chain skeleton for example in the case of PNA, a main chain structure capable of exhibiting the same hybridization ability as that of the main chain of such D-form sugar chain is provided.
  • the base that can be held by the polynucleotide can hold an artificial base that is appropriately modified as necessary, in addition to natural bases such as adenine, thymine, cytosine, guanine, and uracil.
  • natural bases such as adenine, thymine, cytosine, guanine, and uracil.
  • Those skilled in the art can appropriately select such artificial bases from various known modified bases whose functions are modified with respect to fluorescence, stability, base pairing, and the like.
  • the polynucleotide may be single-stranded or double-stranded. Alternatively, it may be partially double-stranded with a single-stranded region at one end or at both ends.
  • a partial double-stranded nucleic acid can be obtained, for example, by using a primer having a linking site capable of suppressing or stopping the polymerase reaction, as disclosed in International Publication No. 2013/038534.
  • a primer having a linking site capable of suppressing or stopping the polymerase reaction as disclosed in International Publication No. 2013/038534.
  • a partial double-stranded nucleic acid having a single-stranded region at both ends can be obtained by using a primer containing such a linking site on both ends, and only one end can be obtained by using such a primer. Only a partial double-stranded nucleic acid having a single-stranded region can be obtained.
  • Polynucleotides are not particularly limited in the number of polymerizations, but can be, for example, about several to 300. Typically, the number is about 10 to 200.
  • the first capture may include a polynucleotide region that exhibits the specific interaction of (c), that is, a specific interaction with another polynucleotide. Such a first capture is used for detection of a target substance using the specific interaction of (a) and (c), and detection of a target substance using the specific interaction of (b) and (c). Used.
  • the polynucleotide can be provided as a hybridization region capable of hybridization based on hydrogen bonding between base pairs.
  • Such a polynucleotide preferably has at least a single-stranded region.
  • the hybridization region may be a base sequence that causes desired hybridization by base pairing.
  • the functional base sequence is not particularly limited, and may be a naturally derived base sequence or an artificial base sequence.
  • the natural base sequence is preferably an artificial sequence in consideration of cross hybridization.
  • Examples of the artificial sequence include probe sequences (SEQ ID NOs: 1 to 100) capable of specific hybridization.
  • the first capture can also include a polynucleotide that exhibits a specific interaction of (a), that is, a specific interaction with a target substance.
  • a polynucleotide that exhibits a specific interaction of (a), that is, a specific interaction with a target substance.
  • Such first capture is used for a target substance detection mode that uses only the specific interaction of (a), and a target substance that uses the specific interaction of (a) and (c).
  • a polynucleotide can be provided with a nucleic acid ligand region such as an aptamer that exhibits a specific interaction with various target substances other than nucleic acids in nature.
  • the action of nucleic acid ligands includes binding to a target substance, catalytically changing the target substance, suppressing the action of the target substance, promoting the reaction between the target substance and other molecules, etc. Is included.
  • the nucleic acid ligand is not confirmed to be bound to the target substance, and may be a nucleic acid ligand candidate.
  • a nucleic acid ligand candidate By using such a nucleic acid ligand candidate as the first capture polynucleotide, a nucleic acid ligand for a specific target substance can be screened by this detection method.
  • the form of the nucleic acid ligand candidate is not particularly limited. It can take a form similar to a natural or artificially designed nucleic acid ligand candidate.
  • the first capture can include a polypeptide that exhibits the specific interaction (b), that is, the specific interaction with the target substance.
  • Such first capture is used in a detection mode of a target substance that utilizes the specific interaction of (a) and (b), or a detection mode of a target substance that uses only the specific interaction of (b).
  • a polypeptide has a polypeptide chain in a relationship such as an antigen-antibody reaction, a ligand-receptor, an enzyme and a substrate, which has a specific interaction with various target substances other than nucleic acids in nature.
  • Such polypeptides can typically be antibodies or antigens, ligands or receptors, enzymes or substrates, and the like. The production of a capture comprising such a polypeptide with a polynucleotide will be described in connection with a second capture later.
  • the first capture can include an immobilization region for immobilization on a solid support, for example.
  • the immobilization region include a polynucleotide chain added for immobilization, in which a reactive group capable of covalent bonding is introduced into a part of the functional group. Introduction of functional groups for such immobilization is well known to those skilled in the art.
  • the first capture can include a polypyrimidine region as an immobilization region. That is, a polynucleotide as an immobilization region can be provided.
  • the polypyrimidine region consists of a base sequence in which two or more pyrimidine bases are continuous. When two or more pyrimidine bases are continuous, the ability to bind to a solid phase carrier is exhibited by irradiation with electromagnetic waves such as ultraviolet rays. The expression of such binding ability is not necessarily theoretically clear, but may be related to the formation of pyrimidine dimers. Examples of pyrimidine bases include thymine, cytosine, uracil, and 5-methylcytosine. Thymine is preferred.
  • two or more pyrimidine bases may be continuous, and the continuous pyrimidine bases may be the same or different.
  • the polypyrimidine region preferably comprises 4 or more, more preferably 5 or more, and still more preferably 10 or more continuous pyrimidine bases. This is because the number of consecutive pyrimidine bases greatly contributes to the immobilization ability.
  • an upper limit is not specifically limited, It is preferable that it is 30 or less, More preferably, it is 20 or less.
  • the first capture may comprise two or more polypyrimidine regions.
  • these regions may be continuous, or one or more other nucleotides may be interposed.
  • Two or more polypyrimidine regions may each be composed of the same pyrimidine base with one or more other nucleotides interposed therebetween.
  • Solid phase carrier and detection device Such a first capture is immobilized on a solid phase carrier via at least a part of the polynucleotide to constitute a target substance detection device.
  • the solid phase carrier is not particularly limited as long as the polynucleotide can be immobilized and the solid phase can be maintained in hybridization in the presence of a liquid.
  • the material of the solid phase carrier is not particularly limited, and examples thereof include polymer materials such as various plastics, ceramics including glass, and metals.
  • the solid phase carrier is easily fixed with the polynucleotide.
  • a material suitable for immobilization of a polynucleotide for example, in the case of a polynucleotide having a polypyrimidine region as an immobilization region, based on nitrocellulose, a cellulose-based polymer such as cellulose acetate, hydrophilic polyvinylidene fluoride (PVDF), polyethersulfone, hydrophilic nylon, fibers composed of amylose, composite fibers containing amylose (such as amylose-cellulose), and the like.
  • PVDF hydrophilic polyvinylidene fluoride
  • polyethersulfone polyethersulfone
  • hydrophilic nylon fibers composed of amylose, composite fibers containing amylose (such as amylose-cellulose), and the like.
  • the target substance is not adsorbed nonspecifically on the solid phase carrier.
  • nitrocellulose is used as a reference, and it is difficult to adsorb protein nonspecifically, that is, as a material having low background noise, hydrophilic PVDF, polyethersulfone and hydrophilic PTFE, amylose And composite fibers containing amylose (such as amylose-cellulose).
  • the degree of nonspecific adsorption of the target substance can be evaluated by applying the target substance to a solid phase carrier of various materials on which the polynucleotide is not immobilized / immobilized and confirming the detection amount.
  • the solid phase carrier is made of a hydrophilic material. It is preferable. From the above results, preferable solid phase carrier materials include hydrophilic PVDF, polyethersulfone, materials containing them, and materials exhibiting surface characteristics equivalent to these.
  • the solid phase carrier may be dense or porous.
  • a porous solid phase carrier is suitable as a carrier for chromatography (for development type hybridization) based on hybridization (affinity) between polynucleotides.
  • a conventionally known carrier capable of moving a liquid by a capillary phenomenon can be adopted.
  • a cellulose-based material such as filter paper can be preferably used.
  • the solid phase carrier does not necessarily need to be composed of a single solid phase material. As long as the hybridization medium can be moved by capillary action as a whole, it may be connected by a plurality of solid phase carriers.
  • the shape of the solid phase carrier is not particularly limited, and various forms can be adopted. For example, in addition to particles such as a spherical shape and an indefinite shape, stick-like bodies, and rod-like bodies, plate-like shapes such as a sheet shape and a film shape.
  • the shape of the solid phase carrier can be appropriately set according to the form of hybridization (immersion type / deployment type).
  • the solid phase carrier is preferably not subjected to blocking treatment with BSA or the like, but this is not necessarily the case.
  • the solid phase carrier in particular, an antibody (which may be preliminarily provided with a labeling substance or the like) that specifically binds to a target substance described later is held in advance on the solid phase carrier subjected to development-type hybridization. It may be.
  • the first capture is immobilized on a solid support in an appropriately patterned form.
  • the polynucleotide is immobilized in various forms. A known covalent bond is exemplified. Further, for example, when a polypyrimidine region is provided as an immobilization region, it is immobilized by irradiating an electromagnetic wave to a polynucleotide supplied on a solid phase carrier. This immobilization is thought to be due to the interaction between the dimer formed by electromagnetic wave irradiation on the polypyrimidine region and the solid support surface. Therefore, it can be said that the polynucleotide is immobilized on the solid phase carrier via the polypyrimidine region.
  • the pattern of immobilization of the first capture on the solid phase carrier is not particularly limited and can take various forms. Depending on the form of the solid phase carrier, when immobilizing on a solid phase carrier such as a sheet, strip or rod, the position of the position where the polynucleotide is immobilized, such as an array or stripe. It is preferable that the information is fixed in a pattern suitable for acquisition.
  • Target substance detection mode In the detection step of the present detection method, it is sufficient that the target substance is detected using at least a specific interaction based on the polynucleotide. Specific interaction with a polynucleotide is generally achieved by a hybridization process.
  • the hybridization of the polynucleotide is performed by immersing the solid phase carrier in the hybridization medium, and the development solution as the hybridization medium is supplied to a part of the solid phase carrier and the development solution is supplied to the solid phase carrier. It is known to develop hybridization, which is a form of chromatography by moving the protein by a capillary phenomenon. Either form can be applied to the present detection step.
  • a detection mode a mode in which a target substance is detected only through a first capture immobilized on a solid phase carrier, a first capture, and a second capture that has a specific interaction with the polynucleotide. And a mode of detecting a target substance via Hereinafter, these detection modes will be described.
  • the first capture includes a polynucleotide that interacts with a target substance other than a nucleic acid.
  • a target substance other than a nucleic acid.
  • problems relating to protein immobilization can be avoided, and a specific interaction between the polynucleotide and the target substance can be avoided.
  • the target substance can be detected with high sensitivity based on the action.
  • the aspect of the first capture polynucleotide is determined according to the target substance.
  • the polynucleotide may be a nucleic acid ligand such as an aptamer that binds to a specific protein, a protein binding region known to bind to a specific protein, a polynucleotide comprising a base sequence recognized by an antibody, or the like. Can do.
  • the first capture includes a polypeptide that interacts with a target substance other than a nucleic acid.
  • a target substance other than a nucleic acid.
  • the target substance can be detected with high sensitivity based on the specific interaction between the target substances.
  • the aspect of the first capture polypeptide is determined depending on the target substance.
  • the polypeptide can be a receptor, antibody, antigen, or the like that binds to a specific protein.
  • detection of the target substance using the first capture will be described.
  • the detection step may use immersion type hybridization or may use development type hybridization.
  • FIG. 1B One embodiment using the second capture is the embodiment shown in FIG. 1B.
  • the upper aspect is an aspect using (a) a specific interaction acting between the polynucleotide and the target substance and (c) a specific interaction acting between the polynucleotide
  • the lower aspect is (B) A specific interaction that acts between the polypeptide and the target substance and (c) a specific interaction that acts between the polynucleotides.
  • a first capture and a second capture are provided, the second capture comprising a first polynucleotide region that has a specific interaction with the polynucleotide of the first capture.
  • a second polynucleotide region that exhibits a specific interaction with the target substance can be provided.
  • the target substance since the target substance is not detected using the directly immobilized protein on the solid phase carrier, problems related to protein immobilization can be avoided, and based on two kinds of specific interactions.
  • the target substance can be detected with high sensitivity.
  • the first capture includes an immobilization region for immobilization on the solid phase carrier, and also the second capture and the specific interaction of (c), that is, other polycrystals.
  • Polynucleotide regions that interact specifically with nucleotides can be provided.
  • the first polynucleotide region included in the second capture may be any one that can specifically hybridize with at least a part of the polynucleotide region of the first capture, and the same aspect as the tag described above is applied. be able to.
  • hybridization ability and a predetermined three-dimensional structure can be constructed by applying a predetermined base sequence according to the type of the target substance.
  • a nucleic acid ligand such as an aptamer that binds to a specific protein, a protein binding region known to bind to a specific protein, or the like can be applied.
  • the second capture of this aspect generally comprises a polynucleotide form as a whole.
  • the second capture is preferably single-stranded for exerting a specific interaction with the first polynucleotide, etc., but as described above, the single-stranded region protruding only at one end May be a partial double-stranded nucleic acid, or a partial double-stranded nucleic acid having a single-stranded region at both ends.
  • the lower aspect of FIG. 1B includes a first capture and a second capture, similar to the upper aspect of FIG. 1B, and the second capture has a specific interaction with the polynucleotide region of the first capture.
  • a polypeptide having a tag and a specific interaction with the target substance can be provided. Also according to this aspect, the same effect as before can be obtained.
  • the tag included in the second capture may be any tag that can specifically hybridize with at least a part of the polynucleotide of the first capture. Therefore, the tag may be a polynucleotide or a functionally equivalent tag.
  • the polymer capable of hybridizing with a polynucleotide to form a complementary strand various types of skeletal forms and base forms of polynucleotides can be applied as in the first capture described above.
  • the tag provided in the second capture is preferably single-stranded to ensure the ability to hybridize with the first capture, but the tag itself is single-stranded and the tag is placed only on one end. It may be a partial double-stranded nucleic acid provided as a protruding single-stranded region.
  • the polypeptide provided in the second capture may be any polypeptide that exhibits a specific interaction with the target substance.
  • the specific interaction with the target substance here refers to, for example, an antibody or antigen in an antigen-antibody reaction, a receptor in a ligand-receptor, an enzyme in a substrate-enzyme reaction, an RNA aptamer, a DNA aptamer or the like and a target substance And the like as a target substance.
  • an antibody it is preferable to use an antibody from the viewpoint of being specifically applicable to many target substances.
  • Such a protein such as an antibody can be appropriately produced or obtained by those skilled in the art once the target substance is identified.
  • a well-known technique can be adopted for the complexing of the polynucleotide tag and the polypeptide in the second capture.
  • a bifunctional compound into an amino group or the like in a polypeptide
  • the tag and the protein can be linked by reacting with a predetermined functional group such as a group.
  • one terminal carboxyl polypeptide can be linked to an amino group or the like in the polypeptide.
  • a known peptide-oligonucleotide conjugate (POC) method can be applied.
  • a protein functional group for example, an amino group, a carboxyl group, or a sulfhydryl group
  • a linker for example, an amino group or a sulfhydryl group
  • an amino group or a sulfhydryl group is previously introduced into the 3′- or 5′-terminal hydroxyl group of the polynucleotide.
  • an amino group may be introduced at the 5 ′ end of an oligonucleotide using commercially available AMINOLINK-2 (Applied Biosystems) or 5′-Amino-Modifiers (Glen Research) [BAConnollyn & P. Rider: Nucleic Acids Res., 13,4485 (1985), BSSproat et al .: Nucleic Acids Res., 15, 4837 (1987)].
  • a sulfhydryl group can be introduced at the 5 ′ end of an oligonucleotide using commercially available 5′-Thiol-Modifiers (manufactured by Glen Research) [BAConnolly & P.Rider: Nucleic Acids Res., 13 4485 (1985), BSSproat et al .: Nucleic® Acids® Res., 15, 4837 (1987)].
  • an amino group or a sulfhydryl group can also be introduced at the 3 ′ end of the oligonucleotide.
  • the 3′-amino oligonucleotide derivative was reacted with N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP; manufactured by Pharmacia), and then reduced with dithiothreitol to give a sulfhydryl group at the 3 ′ end.
  • SPDP N-succinimidyl-3- (2-pyridyldithio) propionate
  • the protein can be bound using the amino group or sulfhydryl group introduced into the polynucleotide.
  • Various methods can be used to bind an amino group or sulfhydryl group introduced at the end of a polynucleotide to a protein [AHBlair & TIGhose: J. Immunol. Methods, 59, 129 (1983), Kitakawa Tsuneo: Organic Synthesis Chem., 42, 283 (1984)].
  • a maleimide group-containing active ester such as N- ( ⁇ -maleimidobutyryloxy) succinimide or N- ( ⁇ -maleimidocaproyloxy) succinimide
  • crosslinking to synthesize a second capture Also good.
  • the first capture, the second capture, and the target substance need only be able to form a complex for detection based on their specific interactions. There is no question of the order. Also, as before, any form of hybridization can be applied.
  • the second capture and the target substance may be supplied to the first capture on the solid phase carrier.
  • a complex for detection in which the three are complexed can be obtained on the solid phase carrier.
  • the second capture and the target substance may be combined in advance prior to the detection step.
  • the second capture Prior to the detection step, the second capture is hybridized in advance to the first capture on the solid phase carrier, and then the complex for detection is detected by the interaction between the second capture and the target substance. You may form a body. May be.
  • the second capture may be supplied to the first capture on the solid phase carrier, and a complex may be formed along with these hybridizations.
  • the second capture is fixed in advance to a part of the solid phase carrier, and the target substance and the first capture are specifically interacted with the movement of the hybridization medium. You may be comprised so that there may exist an effect
  • such a complex can be detected using a known labeling element as appropriate according to the type of the target substance.
  • the labeling element only needs to be able to impart a labeling substance or a labeling substance binding substance capable of binding to the labeling substance to the complex.
  • a person skilled in the art can appropriately set the timing for applying the labeling element to the complex.
  • the labeling element may be applied to the target substance in advance, or the labeling element may be applied to the target substance in the detection process.
  • the labeling substance is not particularly limited, but typically, a labeling substance using fluorescence, radioactivity, enzyme (for example, peroxidase, alkaline phosphatase, etc.), phosphorescence, chemiluminescence, coloring or the like can be mentioned.
  • the labeling substance is preferably a luminescent substance or a coloring substance that presents luminescence or coloring that can be detected visually (with the naked eye).
  • This kind of labeling substance typically includes various dyes, various pigments, luminol, isoluminol, acridinium compounds, olefins, enol ethers, enamines, aryl vinyl ethers, dioxene, aryl imidazoles, lucigenin, luciferin and eclion.
  • a chemiluminescent substance is mentioned.
  • particles such as latex particles labeled with such a labeling substance are also included.
  • colloids or sols including gold colloids or sols or silver colloids or sols can be mentioned.
  • a metal particle, an inorganic particle, etc. are mentioned.
  • the labeling substance binding substance for example, protein-protein interaction, low molecular weight compound-protein interaction and the like can be used.
  • antibodies and antigens in an antigen-antibody reaction biotin in an avidin (streptavidin) -biotin system, digoxigenin in an anti-digoxigenin (DIG) -digoxigenin (DIG) system, or a hapten represented by FITC in an anti-FITC-FITC system Etc.
  • the labeling substance finally used for detection is modified to include, for example, an antigen, an antibody, streptavidin, anti-FITC and the like as a site for binding to the labeling substance binding substance.
  • the detection step it is preferable to detect using an antibody that specifically binds the target substance.
  • an antibody that specifically binds the target substance This is because antibodies can be applied to various target substances and are easily available.
  • the antibody itself can be provided with a labeling substance or a labeling substance binding substance. Bound antibodies to which a labeling element is bound are well known to those skilled in the art.
  • a target substance such as a protein other than a nucleic acid can be detected on a solid phase carrier by utilizing a specific interaction between polynucleotides.
  • a target substance such as a protein other than a nucleic acid
  • various problems related to protein immobilization can be avoided and the target substance can be detected efficiently with high detection sensitivity.
  • the detection device disclosed in the present specification includes a solid phase carrier, a first capture held on the solid phase carrier, and a second capture that has a specific interaction with the polynucleotide of the first capture. be able to. According to this detection device, various problems related to protein immobilization for protein-protein interaction can be avoided, and a detection device excellent in sensitivity and the like can be provided. In addition, since the second capture is provided in advance, it is not necessary to supply the second capture separately in the detection process.
  • the detection device includes a hydrophilic solid phase carrier.
  • the detection device includes a porous solid phase carrier.
  • This detection device has a first capture immobilized on a solid support.
  • the immobilization form of the first capture to the solid phase carrier is not particularly limited, and may be any of various conventionally known immobilization forms.
  • the first capture has a polypyrimidine region, it is preferably immobilized on the solid phase carrier via the region.
  • the detection device further includes a second capture.
  • the second capture may be provided based on a specific interaction between the polynucleotides relative to the first capture.
  • Such a detection device can be manufactured, for example, by performing the following steps.
  • the first capture is immobilized on the solid phase carrier, and then the second capture is supplied to the first capture to form a complex.
  • a known method can be applied to immobilize the first capture. If the first capture has a polypyrimidine region, the first capture is supplied to the solid support in the form of a solution, and a droplet containing the first capture on the solid support is required. Can be immobilized on a solid support by irradiation with electromagnetic waves.
  • Electromagnetic wave irradiation conditions for immobilization is not particularly limited, it is preferable that wavelength is ultraviolet 220 nm ⁇ 380 nm, the amount of irradiation is preferably 10 ⁇ 5000mJ / cm 2, more preferably 100 ⁇ 2000mJ / cm 2 .
  • the second capture is supplied as, for example, a solution to the first capture immobilization region on the solid phase carrier so as to exert a specific interaction to form a complex in the immobilization region.
  • the first capture and the second capture are combined in advance, and this is supplied as a solution to the immobilization region on the solid phase carrier to immobilize the first capture in the same manner as described above. May be.
  • the detection device may hold the second capture on the solid phase carrier independently of the first capture.
  • the region to which the development solution is supplied or the movement destination of the development solution until the first capture immobilization region is reached the region to which the development solution is supplied or the movement destination of the development solution until the first capture immobilization region is reached.
  • the second capture can be held at any location and can be moved with the developing solution. By doing so, the complex of the target substance and the second capture can be supplied to the first capture. In addition, the second capture can be easily fixed.
  • the solid phase carrier of the detection device may further hold a labeling element such as an antibody with a labeling substance or a labeling substance-binding substance, if necessary.
  • a labeling element such as an antibody with a labeling substance or a labeling substance-binding substance, if necessary.
  • Such a labeling element is movably held together with the developing solution at a site where the developing solution is supplied or a destination of the developing solution and reaching the first capture immobilization region. It may be left.
  • the detection kit disclosed in the present specification includes a detection device including at least a first capture on a solid phase carrier, and can be configured to detect a target substance via the second capture. According to this detection kit, a target substance can be detected efficiently with good detection sensitivity and without using an excessive amount of antibody or the like.
  • the kit of the present disclosure may include the second capture in the various aspects already described on the solid phase carrier, or the first capture as a reagent used together with the detection device separately from the solid phase carrier. May be.
  • this detection kit may include a labeling element including a labeling substance or a labeling substance binding substance on a solid phase carrier or as a reagent independent of the solid phase carrier.
  • immobilization was performed by irradiating with UV light of about 300 mJ / cm 2 at a wavelength including a component of 280 nm using a UV irradiation device (XL-1500 UV Crosslinker) manufactured by Spectroline.
  • a UV irradiation device XL-1500 UV Crosslinker manufactured by Spectroline.
  • Hydrophobic PVDF has a particularly non-uniform spot state, which is assumed to be repelled due to low wettability between the spot solution and the material surface, and the spot state is more non-uniform than other materials. It was.
  • Hybri Solution (0.5% Tween20-1% BSA-PBS) 200.0 ⁇ l Synthetic DNA (1 ⁇ M) 4.0 ⁇ l 204.0 ⁇ l total
  • the DNA array was transferred to a tube containing a washing solution (0.1% Tween20-1 mM EDTA-TBS) and washed in a 37 ° C. heat block (37 ° C. ⁇ 1 min, 37 ° C. ⁇ 10 min, 37 ° C. ⁇ 1 min). .
  • the DNA array was transferred to a tube containing an aqueous solution in which biotin-HRP and streptavidin were mixed and reacted at room temperature for 20 minutes.
  • the DNA array was transferred to a tube containing a washing solution (0.1% Tween20-1 mM EDTA-TBS) and washed (room temperature ⁇ 1 min, room temperature ⁇ 10 min, room temperature ⁇ 1 min).
  • FIG. 2 shows images and numerical values after the color development reaction of the DNA microarray.
  • the material types with the same or better sample detection performance than nitrocellulose generally used in immunochromatography are hydrophilic PVDF, cellulose acetate / nitrocellulose, polyethersulfone, hydrophilic Nylon was mentioned.
  • hydrophilic PVDF, polyethersulfone, and hydrophilic PTFE had lower noise than nitrocellulose.
  • the cause of background noise is considered to be caused by non-specific adsorption of protein-derived components (HRP, streptavidin, etc.) in the reagents used in the evaluation process to the membrane material.
  • HRP protein-derived components
  • streptavidin etc.
  • Hydrophilic PVDF and polyethersulfone are known as materials with low non-specific adsorption of protein, and this time we confirmed that a capture DNA probe equivalent to or higher than nitrocellulose can be immobilized. A material with less nonspecific adsorption and capable of detecting a DNA sample could be obtained.
  • Nitrocellulose manufactured by Advantech Toyo
  • Hydrophilic PVDF Merck Millipore
  • immobilization was performed by irradiating with UV light of about 300 mJ / cm 2 at a wavelength including a component of 280 nm using a UV irradiation device (XL-1500 UV Crosslinker) manufactured by Spectroline.
  • a UV irradiation device XL-1500 UV Crosslinker manufactured by Spectroline.
  • the latex stock solution used is a polystyrene latex bead containing a blue colorant coated with avidin (streptavidin) and prepared with a chromatographic developing solution to an arbitrary concentration. did. Further, Phosphate buffered saline was used as the chromatographic developing solution.
  • FIG. 3 shows images and numerical values after development.
  • hydrophilic PVDF is higher in sample detection performance and lower in background noise than nitrocellulose.
  • the cause of background noise is considered to be due to nonspecific adsorption of protein-derived components (streptavidin coated with beads in latex solution) in the reagent used in the evaluation process to nitrocellulose.
  • single-stranded DNA complementary to the single-stranded DNA sequence fixed to Pos1 of the DNA array used in Example 1 etc. was introduced into proteins using various cross-linking agents.
  • a second capture in this disclosure was made. The procedure is as follows, and the outline of the reaction is also shown.
  • Example 3 Protein detection by first capture and second capture on selected membrane: immersion hybridization system
  • the second capture produced in Example 3 was evaluated according to the procedure described below using a nitrocellulose and hydrophilic PVDF material DNA array.
  • Example 1 made of various materials is immersed in a sampling tube containing a single-stranded DNA-binding Rabbit IgG (second capture) solution prepared so as to be 10 ⁇ g / ml. In order to hybridize the first capture and the second capture, a reaction was performed at 25 ° C. for 1 hour.
  • the DNA array that had been subjected to (1) was immersed in a sampling tube containing 3% BSA / PBST and blocked at room temperature for 1 hour. (3) The blocking solution was removed and washing with PBST was performed.
  • the hydrophilic PVDF can detect the protein sample more easily than the nitrocellulose material (it can be visually determined without performing the digitization process). From the above results, it was possible to reliably suppress the generation of noise. Conventionally, even if blocking is performed, noise generation cannot always be suppressed.
  • hydrophilic PVDF makes it easy to detect a protein sample (it can be visually judged without performing a quantification process).
  • Protein detection evaluation was performed using hydrophilic PVDF material (Merck Millipore, pore size 0.4 ⁇ m) as a membrane, and aptamer as a first capture.
  • the Pos1 sequence is an aptamer sequence specific to Ochratoxin
  • the Pos2 sequence is an aptamer sequence specific to Thrombin
  • each of them has polyT (20) added to the 5 ′ side. .
  • biotinylation with D-biotin and succinimidyl ester was performed using Albumin (Human Serum Albumins: Sigma-Aldrich) to obtain a biotin-Albumin solution.
  • Albumin Human Serum Albumins: Sigma-Aldrich
  • Sample composition developing solution 20.0 ⁇ l Latex solution 2.0 ⁇ l Biotin-*** solution 5.0 ⁇ l TE buffer 13.0 ⁇ l Total 42.0 ⁇ l *** ⁇ Thrombin or Albumin
  • the latex stock solution used is a polystyrene latex bead containing a blue colorant coated with avidin (streptavidin) and prepared with a chromatographic developing solution to an arbitrary concentration. did. Further, Phosphate buffered saline was used as the chromatographic developing solution.
  • Protein detection evaluation was performed using hydrophilic PVDF material (Merck Millipore, pore size 0.4 ⁇ m) as a membrane and aptamer as a second capture.
  • the Pos3 sequence is a tag79 sequence
  • the Pos4 sequence is a polyT (20) added to the 5 ′ side of the tag02 sequence.
  • Tag79 and tag02 correspond to D1_79 and D1_02 in Supplementary Table 1 of Analytical Biochemistry_364_1_2007_78-85.
  • Sample composition developing solution 20.0 ⁇ l Latex solution 2.0 ⁇ l Biotin-*** solution 5.0 ⁇ l Complementary oligo DNA solution (1 ⁇ M, each) 4.2 ⁇ l TE buffer 10.8 ⁇ l total 42.0 ⁇ l ***: Thrombin or Albumin
  • each sequence in the table consists of a continuous synthesis of an aptamer sequence specific to Ochratoxin on the 3 ′ side of the complementary sequence of tag79, and an aptamer specific to Thrombin on the 3 ′ side of the complementary sequence of tag02.
  • Each of the sequences synthesized by connecting the sequences is shown.
  • the latex solution used is the same as in Example 6.
  • the Thrombin detection probe site (Pos4) is colored (Ochratoxin detection probe: no coloration of Pos3), and when the Albumin sample is reacted, Pos3,4 Both were confirmed not to react.

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Abstract

Le problème à résoudre dans le cadre de cette invention consiste fournir un procédé plus pratique pour la détection d'une substance cible. La solution proposée consiste en un procédé pour détecter une substance cible autre qu'un acide nucléique. La présente invention comprend une étape de détection de la substance cible sur un support en phase solide possédant une première capture, qui possède un polynucléotide, immobilisée par l'intermédiaire d'au moins une partie du polynucléotide, la substance cible étant conçue pour être détectée à l'aide d'au moins l'interaction spécifique présentée par la première capture.
PCT/JP2015/058576 2014-03-31 2015-03-20 Procédé pour la détection d'une substance cible WO2015151883A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019069372A1 (fr) * 2017-10-03 2019-04-11 株式会社ニコン Procédé de mesure de cible de détection, support d'ancrage de sonde de capture, kit de détection et dispositif de fluide

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10253632A (ja) * 1997-03-10 1998-09-25 Nissui Pharm Co Ltd 分析方法、キット及び装置
JP2001281246A (ja) * 2000-01-26 2001-10-10 Nisshinbo Ind Inc 固定化核酸及び核酸の検出法
JP2003294751A (ja) * 2002-02-01 2003-10-15 Nisshinbo Ind Inc 生体分子の担体への固定法
JP2006523462A (ja) * 2003-04-18 2006-10-19 ベックマン　コールター　インコーポレイテッド 多重結合アッセイ用オリゴヌクレオチド対
JP2008232914A (ja) * 2007-03-22 2008-10-02 Canon Inc 標的物質検出素子、標的物質検出方法、標的物質検出素子の製造方法
JP2009529658A (ja) * 2006-02-21 2009-08-20 トラスティーズ・オブ・タフツ・カレッジ 標的分析物検出および溶液中の標的分析物濃度の決定のための方法およびアレイ

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10253632A (ja) * 1997-03-10 1998-09-25 Nissui Pharm Co Ltd 分析方法、キット及び装置
JP2001281246A (ja) * 2000-01-26 2001-10-10 Nisshinbo Ind Inc 固定化核酸及び核酸の検出法
JP2003294751A (ja) * 2002-02-01 2003-10-15 Nisshinbo Ind Inc 生体分子の担体への固定法
JP2006523462A (ja) * 2003-04-18 2006-10-19 ベックマン　コールター　インコーポレイテッド 多重結合アッセイ用オリゴヌクレオチド対
JP2009529658A (ja) * 2006-02-21 2009-08-20 トラスティーズ・オブ・タフツ・カレッジ 標的分析物検出および溶液中の標的分析物濃度の決定のための方法およびアレイ
JP2008232914A (ja) * 2007-03-22 2008-10-02 Canon Inc 標的物質検出素子、標的物質検出方法、標的物質検出素子の製造方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019069372A1 (fr) * 2017-10-03 2019-04-11 株式会社ニコン Procédé de mesure de cible de détection, support d'ancrage de sonde de capture, kit de détection et dispositif de fluide

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