JP6076500B2 - Target substance detection method - Google Patents

Target substance detection method Download PDF

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JP6076500B2
JP6076500B2 JP2015549888A JP2015549888A JP6076500B2 JP 6076500 B2 JP6076500 B2 JP 6076500B2 JP 2015549888 A JP2015549888 A JP 2015549888A JP 2015549888 A JP2015549888 A JP 2015549888A JP 6076500 B2 JP6076500 B2 JP 6076500B2
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capture
target substance
polynucleotide
solid phase
protein
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JPWO2015151883A1 (en
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孝介 丹羽
孝介 丹羽
廣田 寿一
寿一 廣田
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日本碍子株式会社
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Priority to PCT/JP2015/058576 priority patent/WO2015151883A1/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306Solid-phase reaction mechanisms

Description

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, and is described in this Japanese application. All the contents that were made are used.

  A method is known in which an antibody in which a target substance other than a nucleic acid such as an antigen in a test solution is immobilized on a solid phase carrier or the like is captured as a capture element and detected by a so-called sandwich method. For example, an antibody array (Patent Document 1) 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).

  In any of these techniques, an antibody that is a protein is immobilized on a solid phase carrier 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.

Japanese Patent Laid-Open No. 2003-294751 JP 2001-281246 A

  However, when 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. There is a need to. Setting conditions for blocking is not easy because it requires optimization of conditions depending on the type of target substance.

  Further, when the antibody is immobilized only by physical adsorption, there is a problem that the antibody is difficult to be retained and easily dropped off. On the other hand, in order to firmly immobilize the protein on the solid phase carrier, it is possible to use an interaction such as biotin-avidin or to utilize a covalent bond between the solid phase carrier surface and the antibody. However, immobilization of antibodies by biotin-avidin binding is often not realistic from the viewpoint of workability and cost. Moreover, in order to form a covalent bond, generally heat processing is required, but this heat processing will reduce the capture function of an antibody. Furthermore, since the antibody is a protein, it becomes inactive over time after immobilization on the solid phase carrier, and the capture function decreases.

  From the above, in such a detection method, it is important to maintain the sensitivity of specific detection based on protein-protein interaction or the like and to eliminate false positives and false negatives. At present, the actual situation is that an excessive amount of antibody is applied in order to ensure the necessary detection sensitivity and accuracy over a certain period.

  The present 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 investigated various problems in detecting a target substance by immobilizing the protein on a solid phase carrier and utilizing a specific interaction between the protein and the target substance. As a result, in the detection of the target substance, in addition to or instead of the 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. I found that I could solve a problem. That is, it was found that various problems can be solved by using a polynucleotide as a capture for capturing a target substance other than a nucleic acid on a solid phase carrier. According to the present specification, the following means are provided.

(1) 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.
(2) 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.
(3) Furthermore, in addition to the interaction (a) or (b), the following (c):
(C) The method according to (2), wherein the target substance is detected using a specific interaction acting between polynucleotides.
(4) The method according to any one of (1) to (3), wherein the target substance is detected via a second capture that exhibits a specific interaction with at least a part of the polynucleotide.
(5) The method according to (4), wherein the second capture includes a tag that exhibits a specific interaction with at least a part of the polynucleotide.
(6) The method according to (5), wherein the second capture includes a protein.
(7) The method according to (6), wherein the protein of the second capture is an antibody or an antigen.
(8) The method according to (4) or (5), wherein the second capture includes a nucleic acid ligand.
(9) In the detection step, 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 step of detecting.
(10) The detection method according to any one of (1) to (9), wherein the target substance is a protein.
(11) 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.
(12) The detection method according to any one of (1) to (11), wherein the solid phase carrier is made of a hydrophilic material.
(13) The detection method according to any one of (1) to (12), wherein the solid phase carrier is porous or flat.
(14) The detection method according to any one of (1) to (13), wherein the detection step is performed by chromatography in which the hybridization medium containing at least the target substance is moved in a predetermined direction. .
(15) 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.
(17) The device according to (16), wherein the second capture includes a protein.
(18) The device according to any one of (15) to (17), wherein the solid phase carrier is hydrophilic and porous.
(19) 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.

It is a figure which illustrates the outline | summary of the detection aspect of this indication. It is a figure which illustrates the outline | summary of the other detection aspect of this indication. It is a figure which shows the evaluation result in Example 1. FIG. It is a figure which shows the evaluation result in Example 2. FIG. It is a figure which shows the evaluation result in Example 4. FIG. It is a figure which shows the evaluation result in Example 5. FIG. It is a figure which shows the evaluation result in Example 6. FIG. It is a figure which shows the evaluation result in Example 7. FIG.

  The disclosure of the present specification relates to a method for detecting a target substance, a detection device, a detection kit, and the like. 1A and 1B illustrate an overview of the present disclosure.

  According to 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 immobilized via at least a part thereof. In the detection, the target substance can be detected using at least the specific interaction exhibited by the first capture. By using this method to detect the target substance, the problem of conflicting protein adsorption ability on the solid phase carrier (I want to fix the capture protein but avoid nonspecific protein adsorption) is avoided. be able to.

  That is, 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. Will be. As a result, the above problems relating to the protein immobilization ability and adsorption ability of the solid phase carrier can be avoided. Specifically, 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. .

  On the other hand, the detection of the target substance uses the specific interaction produced by the first capture, so that 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.

  Specific examples include (a) only, (b) only, (a) + (c), (b) + (c), and the like. Conventionally, detection is based only on (b).

  Further, according to the present disclosure, 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.

  In this specification, 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.

  In this specification, 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. In other words, it means actions that are performed specifically to each other.

  Hereinafter, the present disclosure will be described in detail with reference to the drawings as appropriate.

  Hereinafter, representative and non-limiting specific examples of the present disclosure will be described in detail with reference to the drawings as appropriate. This detailed description is intended merely to provide those skilled in the art with details for practicing the preferred embodiments of the present invention and is not intended to limit the scope of the present disclosure. Further, the additional features and inventions disclosed below can be used separately from or together with other features and inventions in order to provide further improved methods for detecting target substances other than nucleic acids.

  Further, the combinations of features and steps disclosed in the following detailed description are not essential in carrying out the present disclosure in the broadest sense, and are particularly only for explaining representative specific examples of the present disclosure. It is described. Moreover, various features of the representative embodiments described above and below, as well as those described in the independent and dependent claims, are described herein in providing additional and useful embodiments of the present disclosure. They do not have to be combined in the specific examples given or in the order listed.

  All features described in this specification and / or claims, apart from the configuration of the features described in the examples and / or claims, are individually disclosed as limitations on the original disclosure and claimed specific matters. And are intended to be disclosed independently of each other. Further, all numerical ranges and group or group descriptions are intended to disclose intermediate configurations thereof as a limitation to the original disclosure and claimed subject matter.

(Target substance detection method)
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. In the detection step, the target substance can be detected by utilizing a specific interaction based on at least the first capture polynucleotide.

  In this detection method, the target substance is detected using at least the first capture. Hereinafter, first, the first capture will be described, and various detection modes using the first capture will be described.

(First capture)
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 | region to a solid-phase carrier.

  A 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 bound by an amide bond and capable of retaining a base that can be retained by DNA or the like, or 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 composed mainly of a phosphate ester skeleton of deoxyribose. In general, a polynucleotide has D-form ribose or deoxyribose in the backbone of the main chain. In the case of not having 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.

  In addition to the natural bases such as adenine, thymine, cytosine, guanine, and uracil, the base that can be held by the polynucleotide can also hold an artificial base that is appropriately modified as necessary. 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. Such 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. Among the pair of primers, 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 of the ends can be obtained by using such a primer. Only a partial double-stranded nucleic acid having a single-stranded region can be obtained.

  The number of polymerizations of the polynucleotide is not particularly limited, but can be, for example, about several to 300. Typically, the number is about 10 to about 200.

  The first capture may comprise a polynucleotide region that exerts a 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. Typically, 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. Therefore, 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. 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). More specifically, such 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. In general, 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. 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.

  In addition, the first capture may 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). More specifically, such 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. Can be provided. 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, for example, an immobilization region for immobilization on a solid phase carrier. Examples of 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 comprise 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.

  In the polypyrimidine region, two or more pyrimidine bases may be continuous, and the continuous pyrimidine bases may be the same or different. In consideration of immobilization ability, cost, etc., it is preferable that two or more identical pyrimidine bases are continuous, and if the same pyrimidine base has a region where two or more consecutive, it is further separately of another type and You may have a continuous region of the same pyrimidine base.

  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. Although 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. When two or more polypyrimidine regions are composed of different pyrimidine bases, 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.

  In consideration of detection of the target substance using the first capture, it is preferable that the solid phase carrier is easily fixed with the polynucleotide. For example, as 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. Whether or not it is suitable for immobilization of a polynucleotide can be evaluated by confirming the detection amount by applying a target substance to a solid phase carrier of various materials and the type of immobilization region and immobilization technique.

  Moreover, it is preferable that the target substance is not adsorbed nonspecifically to the solid phase carrier. For example, when protein is a target substance, 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.

  Since the first capture has a polynucleotide and is immobilized via at least a part of the polynucleotide, when the target substance is a generally hydrophobic material such as a protein, 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.

  Further, the solid support may be dense or porous. In particular, a porous solid phase carrier is suitable as a carrier for chromatography (for development type hybridization) based on hybridization (affinity) between polynucleotides. As the porous carrier, a conventionally known carrier capable of moving a liquid by a capillary phenomenon can be adopted. For example, in addition to the solid phase carrier material already described, a cellulose-based material such as filter paper can be preferably used. Further, 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. When the first capture is fixed in a stick shape, a rod shape, or a flat plate shape, it is suitable for confirming the position information of the fixed position by fixing the first capture at a specific position or arrangement. In addition, the shape of the solid phase carrier can be appropriately set according to the form of hybridization (immersion type / deployment type).

  In the present disclosure, the solid phase carrier is preferably not subjected to blocking treatment with BSA or the like, but this is not necessarily the case.

  In addition, 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.

  As 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.

(Mode in which target substance is detected using first capture)
As an aspect of detecting the target substance only by the first capture, there is an aspect shown in the second row of FIG. 1A. In the uppermost embodiment, (a) a target substance is detected by utilizing a specific interaction between the polynucleotide and the target substance, and in the next stage, (b) a specific interaction between the polypeptide and the target substance. To detect the target substance.

  In the upper aspect, the first capture includes a polynucleotide that interacts with a target substance other than a nucleic acid. According to this embodiment, since the target substance is not detected using the directly immobilized protein on the solid phase carrier, problems relating to protein immobilization can be avoided, and 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. In this aspect, the aspect of the first capture polynucleotide is determined according to the target substance. For example, 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.

  In the lower embodiment, the first capture includes a polypeptide that interacts with a target substance other than a nucleic acid. According to this embodiment, since the target substance is not detected using the directly immobilized protein on the solid phase carrier, the problem related to protein immobilization can be avoided, as well as the uppermost embodiment. The target substance can be detected with high sensitivity based on the specific interaction between the target substances. In this aspect, the aspect of the first capture polypeptide is determined depending on the target substance. For example, the polypeptide can be a receptor, antibody, antigen, or the like that binds to a specific protein.

  Next, detection of a target substance using the first capture will be described. In detection, as shown in FIG. 1A, it is only necessary that the first capture and the target substance form a complex for detection based on their specific interaction. Further, the detection step may use immersion type hybridization or may use development type hybridization.

(Mode of detecting a target substance using the first capture and the second capture)
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, and 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.

  In the upper aspect of FIG. 1B, 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. In addition, a second polynucleotide region that exhibits a specific interaction with the target substance can be provided. According to this embodiment, 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.

  In this embodiment, as described above, 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. For the second polynucleotide region, 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. As the second polynucleotide region, for example, 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 embodiment 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. As 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, and a nucleic acid ligand and the target substance And the like as a target substance. As such a protein, 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 known technique can be employed for the complexing of the polynucleotide tag and the polypeptide in the second capture. For example, while introducing a bifunctional compound into an amino group or the like in a polypeptide, the amino at the site for binding to another functional group of the compound and a part of the tag or a protein separately attached to the tag The tag and the protein can be linked by reacting with a predetermined functional group such as a group. Alternatively, one terminal carboxyl polypeptide can be linked to an amino group or the like in the polypeptide.

  As such a linking method, a method in a known peptide-oligonucleotide conjugate (POC) can be applied. For example, the protein functional group (for example, amino group, carboxyl group, sulfhydryl group) can be bound to the 3′- or 5′-terminal hydroxyl group of a polynucleotide or a linker. Typically, an amino group or a sulfhydryl group is previously introduced into the 3′- or 5′-terminal hydroxyl group of the polynucleotide. For example, an amino group may be introduced at the 5 ′ end of the oligonucleotide using commercially available AMINOLINK-2 (Applied Biosystems) or 5′-Amino-Modifiers (Glen Research) [BAConnolly & P. Rider: Nucleic Acids Res., 13, 4485 (1985), BSSproat et al .: Nucleic Acids Res., 15, 4837 (1987)]. Similarly, 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)]. On the other hand, an amino group or a sulfhydryl group can also be introduced at the 3 ′ end of the oligonucleotide. For example, using a commercially available 3′-amine-ON CPG carrier (Clontech), a (3′-amino-2-hydroxyl-propyl) phosphate group [(3-amino-2-hydroxyl-propyl) is attached to the 3 ′ end. ) phosphate] [B. Oberhauser & E. Wagner: Nucleic Acids Res., 20,533 (1992)]. Next, 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. Can be introduced.

  Subsequently, the amino group or sulfhydryl group introduced into the polynucleotide can be used to bind the protein. Various methods can be used to bind the amino group or sulfhydryl group introduced at the end of the polynucleotide to the protein [AHBlair & TIGhose: J. Immunol. Methods, 59, 129 (1983), Kitakawa Tsuneo: Organic Synthesis Chem., 42, 283 (1984)]. For example, by reacting with a maleimide group-containing active ester (bifunctional binding reagent), such as N- (γ-maleimidobutyryloxy) succinimide or N- (ε-maleimidocaproyloxy) succinimide, and crosslinking to synthesize a second capture Also good.

  Next, detection of a target substance using the first capture and the second capture will be described. In the detection, as shown in FIG. 1B, 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.

  For example, the second capture and the target substance may be supplied to the first capture on the solid phase carrier. Thereby, a complex for detection in which the three are complexed can be obtained on the solid phase carrier. In this case, the second capture and the target substance may be combined in advance prior to the detection step. 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. Furthermore, in the detection step, 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.

  In the case of using the development type hybridization, 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 | action.

  Various hybridization conditions in the above detection step are well known to those skilled in the art, and those skilled in the art can implement the detection step by appropriately adopting known conditions.

  Furthermore, in the detection step, 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. For example, prior to the detection step, 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 and 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. Moreover, particles such as latex particles labeled with such a labeling substance are also included. Furthermore, colloids or sols including gold colloids or sols or silver colloids or sols can be mentioned. Furthermore, a metal particle, an inorganic particle, etc. are mentioned.

  As the labeling substance-binding substance, for example, protein-protein interaction, low molecular compound-protein interaction and the like can be used. For example, haptens represented by antibodies and antigens in antigen-antibody reaction, biotin in avidin (streptavidin) -biotin system, digoxigenin in anti-digoxigenin (DIG) -digoxigenin (DIG) system, or FITC in anti-FITC-FITC system Etc. In this case, 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.

  In the detection step, detection is preferably performed using an antibody that specifically binds the target substance. This is because antibodies can be applied to various target substances and are easily available. In this case, 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.

  As described above, according to this detection method, 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. Compared to the case of using only the specific interaction between protein and protein, various problems related to protein immobilization can be avoided and the target substance can be detected efficiently with high detection sensitivity.

(Detection device and manufacturing method thereof)
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 various aspects of the solid phase carrier, the first capture, and the second capture in this detection device can be appropriately applied with the previously described aspects. Preferably, the detection device includes a hydrophilic solid phase carrier. In addition, preferably, the detection device includes a porous solid phase carrier.

  In this detection device, the first capture is immobilized on a solid phase carrier. 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. When 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 220Nm~380nm, irradiation dose is preferably 10~5000mJ / cm 2, more preferably 100 to 2000 mJ / 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. To.

  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.

  In the detection device, the second capture may be held on the solid phase carrier independently of the first capture. In particular, when the present detection device is used for development-type hybridization, 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 provided with a labeling substance or a labeling substance binding substance as 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.

(Detection kit for target substance detection)
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.

  Further, the 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.

  Hereinafter, although the example which materialized this indication is described, the following example does not limit this indication.

(1) Production of single-stranded DNA probe-immobilized membrane for evaluation: for immersion type hybridization A spot solution containing a first capture consisting of a base sequence shown in the following table is described in JP-A-2003-75305. Using a GESHOT (registered trademark) spotter using NGK's discharge unit (inkjet method), spotting was carried out on various membrane materials in a predetermined pattern. The membrane sheet material used was as follows. The pore diameter of each material was unified to 0.45 μm.

・ Nitrocellulose (manufactured by Advantech Toyo)
・ Hydrophilic PVDF (Merck Millipore)
・ Hydrophobic PVDF (Merck Millipore)
・ Cellulose acetate / nitrocellulose (Merck Millipore)
・ Polyethersulfone (Merck Millipore)
・ Hydrophilic PTFE (Merck Millipore)
・ Hydrophilic nylon (Merck Millipore)

After the spot, 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 apparatus (XL-1500 UV Crosslinker) manufactured by Spectroline.

  When the uniformity of the image after spotting was observed for each membrane material, it was confirmed that the membrane was spotted almost uniformly except for the hydrophobic PVDF. 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.

  In addition to the above, a spot solution not containing the first capture was spotted by the same method as described above, and an ultraviolet ray irradiated was prepared.

(2) Evaluation of single-stranded DNA probe-immobilized membrane: immersion type hybridization The membrane was evaluated by the following procedure.
(1) A DNA microarray made of various materials was immersed in a sampling tube containing 3% BSA / PBST (0.05% (v / v) Tween-20 / PBS), and blocked at room temperature for 2 hours.
(2) The blocking solution was removed and PBST was washed (twice).
(3) Using an aqueous solution (1 μM) in which the synthetic DNA shown in Table 2 is dissolved (1 μM), a hybrid sample solution having the following composition is prepared, and 200 μl of each hybrid is placed in a sampling tube containing various membranes. Samples were added and hybridized for 30 minutes at room temperature.

Hybri Solution (0.5% Tween20-1% BSA-PBS) 200.0μl
Synthetic DNA (1 μM) 4.0 μl
204.0 μl total

(4) 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). .
(5) 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.
(6) 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).
(7) Using a washed membrane type DNA array, TMB Peroxidase Substarate Kit, 3,3 ′, 5,5′-tetramethylbenzidine, manufactured by Vector Laboratories, was subjected to a color reaction for about 5 minutes at room temperature.
(8) The color-reacted DNA array was captured as a Tif image using a scanner (EP-805AW: EPSON), and the image was digitized using GenePix Pro software. ). FIG. 2 shows images and numerical values after the color development reaction of the DNA microarray.

  As shown in FIG. 2, 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. On the other hand, in terms of background noise, it was confirmed that 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. Compared to nitrocellulose, PVDF and polyethersulfone can be used as membrane materials that can take advantage of both sample detection capability and background noise. 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.

  From the above results, it is possible to utilize the advantages of both sample detection ability and background noise if materials other than those examined this time are materials with low nonspecific adsorption of proteins and the capture can be fixed to the material. It was confirmed that it was possible to obtain a membrane material.

(1) Production of single-stranded DNA probe-immobilized membrane for evaluation: For development-type hybridization A spot solution containing a first capture consisting of a base sequence shown in the following table is described in JP-A-2003-75305. NGSHOT (registered trademark) spotter using a discharge unit (inkjet method), spotted in a predetermined pattern on various membrane materials prepared for development-type hybridization. The membrane sheet material used was as follows. The pore diameter of each material was unified to 0.45 μm.

・ Nitrocellulose (manufactured by Advantech Toyo)
・ Hydrophilic PVDF (Merck Millipore)

After the spot, 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 apparatus (XL-1500 UV Crosslinker) manufactured by Spectroline.

  When the uniformity of the image after spotting was observed for each of the various membrane materials, it was confirmed that the spotting was almost uniform except for the hydrophobic PVDF as in Example 1. In addition to the above, a spot solution containing no capture DNA probe was spotted by the same method as described above, and an ultraviolet ray was irradiated.

(2) Evaluation of single-stranded DNA probe-immobilized membrane: development-type hybridization Using an aqueous solution (200 nM) in which the synthetic DNA shown in Table 2 was dissolved, a hybridized sample solution having the following composition was prepared.
Sample composition developing solution 20.0 μl
Latex solution 2.0 μl
Sample (200nM) 4.2 μl
TE buffer 15.8 μl
Subtotal 42.0 μl

  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.

(Hybridization and color reaction)
42 μl of each of the above prepared samples was added to a 0.2 ml tube and developed chromatography was inserted to start the reaction. The sample solution was all sucked up in about 20 minutes, and the reaction was completed. After completion of the reaction, the chromatography, which is a development hybridization carrier, was air-dried, and the reaction site was visually confirmed and an image was taken. Furthermore, the developed membrane was captured as a Tif image with a scanner (EP-805AW: EPSON), and the image was digitized using GenePix Pro software (the location of Pos1 capture DNA fixation in each image was digitized). . FIG. 3 shows images and numerical values after development.

  As shown in FIG. 3, in the sample detection by development type chromatography, it was confirmed that 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.

  From the above results, it is possible to utilize the advantages of both sample detection ability and background noise if materials other than those examined this time are materials with low nonspecific adsorption of proteins and the capture can be fixed to the material. It was confirmed that it was possible to obtain a membrane material.

  In this example, 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.

(1) Sulfo-AC 5 -SPDP (N- {6- [3- (2-Pyridyldithio) propionamido] hexanoyloxy} sulfosuccinimide, sodium salt / Dojindo Laboratories, Inc.), one of the Hetero-bifunctional Reagents A 50 mM DMSO solution was prepared.
(2) Rabbit IgG was dissolved in PBS-EDTA to prepare 5 mg / ml.
(3) To 1 ml of the solution of (2), 15 μl of the Sulfo-AC 5 -SPDP solution prepared in (1) was added and reacted at 25 ° C. for 1 hour.
(4) After completion of the reaction, purification was performed using Desalting Columns (Zeba Spin Desalting Columns).
(5) Separately, in order to measure the amount of Rabbit IgG bound to Sulfo-AC 5 -SPDP, react with DTT solution and measure the amount of released Pyridine 2-thione to bind Sulfo-AC 5 -SPDP. The amount of Rabbit IgG was calculated (converted to mole).
(6) As a result of (5), add 3 equivalents of thiol-modified single-stranded DNA solution (Table 4) in molar equivalent to the amount of Rabbit IgG bound to Sulfo-AC 5 -SPDP and mix gently. Then, the reaction was carried out at 25 ° C. for 16 hours.
(7) After the reaction was completed, unreacted thiol-modified single-stranded DNA was removed by ultrafiltration.
(8) Separately, Sulfo-AC 5 -SPDP was confirmed to be cross-linked between Rabbit IgG and SH-modified single-stranded DNA by measuring the amount of Pyridine 2-thione released by the reaction of (6) did.

(Protein detection by first capture and second capture on selected membrane: immersion hybridization system)
Of the membrane materials used in the evaluation in Example 1, the second capture produced in Example 3 was evaluated according to the procedure described below using a nitrocellulose and hydrophilic PVDF material DNA array.

(1) The DNA array of 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 μg / ml. In order to hybridize the first capture and the second capture, a reaction was performed at 25 ° C. for 1 hour.
(2) 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.
(4) Using biotin-labeled anti-IgG antibody (for example, Goat anti Rabbit IgG) diluted 1000 times with PBST as an antibody (target substance), (3) Add it to the tube and react at 37 ° C for 30 minutes It was.
(5) The antibody solution was removed and washed with PBST.
(6) 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.
(7) Hereinafter, evaluation was performed by operating in the same manner as in (6) to (8) of Example 1. The results are shown in FIG.

  As shown in FIG. 4, it was confirmed that 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.

(Protein detection by the first capture and the second capture on the selected membrane: deployable hybridization system)
Using the nitrocellulose and hydrophilic PVDF material-based DNA array produced in Example 2, detection evaluation of the protein produced in Example 3 was performed according to the procedure described below.

(1) Add a biotin-labeled anti-IgG antibody PBST solution (diluted 1000 times) to a sampling tube containing a single-stranded DNA-bound Rabbit IgG solution prepared to 10 μg / ml, and react at 37 ° C for 30 minutes. It was.
(2) During the execution of (1), development type chromatography was inserted into a sampling tube containing 30 μl of 3% BSA / PBST solution, and the solution was sucked up to perform chromatography blocking.
(3) After the completion of (1) and (2), a hybrid sample solution having the following composition was prepared, and the reaction was started in the same manner as in Example 2. Note that the same developing solution and latex storage solution as those in Example 2 were used.
(4) Then, it operated similarly to Example 2 and evaluated. The results are shown in FIG.

Sample composition developing solution 20.0 μl
Latex solution 2.0 μl
Sample (200nM) 10.0 μl
3% BSA / PBST 10.0 μl
Total 42.0 μl

  As shown in FIG. 5, as in Example 4, it was confirmed that hydrophilic PVDF facilitates detection of a protein sample (it can be visually determined without performing numerical processing).

(Protein detection by first capture on selected membrane)
Protein detection evaluation was performed using hydrophilic PVDF material (Merck Millipore, pore size 0.4 μm) as a membrane, and aptamer as a first capture.

(1) Preparation of single-stranded DNA probe for evaluation (first capture) immobilization membrane: for development-type hybridization A spot solution containing a first capture consisting of a base sequence shown in the following table is disclosed in JP-A-2003-75305. The membrane material prepared for unfolded hybridization was spotted in a predetermined pattern using a GENISHOT (registered trademark) spotter using a discharge unit (inkjet method) described in Japanese Patent Publication No. Gazette.

  In the table, the Pos1 sequence is an aptamer sequence specific to Ochratoxin, the Pos2 sequence is an aptamer sequence specific to Thrombin, and each of them has polyT (20) added to the 5 ′ side. .

After the spot, 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 apparatus (XL-1500 UV Crosslinker) manufactured by Spectroline. As a result of observing the uniformity of the image after spotting, it was confirmed that the spot was uniformly spotted as in the previous example.

(2) Sample detection using a single-stranded DNA probe-immobilized membrane: development-type hybridization First, using D-biotin, succinimidyl ester (Life Technologies), biotinylation of Thrombin (human-derived α-Thrombin: Cosmo Bio) Went. Each solution of D-biotin, succinimidyl ester, and Thrombin was added to a coupling buffer solution (sodium carbonate 0.1M, pH 9.4) and reacted at 4 ° C. for 2 hours. Thereafter, Glycine was added to block unreacted D-biotin and succinimidyl ester to obtain a biotin-Thrombin solution.

  Instead of Thrombin, biotinylation with D-biotin and succinimidyl ester was performed using Albumin (Human Serum Albumins: Sigma-Aldrich) to obtain a biotin-Albumin solution.

Subsequently, a sample solution having the following composition was prepared.
Sample composition developing solution 20.0 μl
Latex solution 2.0 μl
Biotin-*** solution 5.0 μl
TE buffer 13.0 μl
Total 42.0 μl
*** → 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.

(Hybridization and color reaction)
42 μl of each of the above prepared samples was added to a 0.2 ml tube and developed chromatography was inserted to start the reaction. The sample solution was all sucked up in about 20 minutes, and the reaction was completed. After completion of the reaction, the chromatography, which is a development hybridization carrier, was air-dried, and the reaction site was visually confirmed and an image was taken. The developed image is shown in FIG.

  As shown in FIG. 6, when the Thrombin sample was reacted, the site (Pos2) where the Thrombin-specific aptamer was immobilized was colored (Ochratoxin: no coloration of Pos1), and when the Albumin sample was reacted, Pos1, Both were confirmed not to react.

  From the above results, it was confirmed that it was possible to detect only the target protein.

(Protein detection by the first capture and the second capture on the selected membrane)
Protein detection evaluation was performed using hydrophilic PVDF material (Merck Millipore, pore size 0.4 μm) as a membrane and aptamer as a second capture.

(1) Production of single-stranded DNA probe-immobilized membrane for evaluation: For development-type hybridization A spot solution containing a first capture consisting of a base sequence shown in the following table is described in JP-A-2003-75305. The membrane material prepared for development-type hybridization was spotted in a predetermined pattern using a GENISHOT (registered trademark) spotter using NGK Corporation using a discharge unit (inkjet method).

The Pos3 sequence is a tag79 sequence, and 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.

After the spot, 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 apparatus (XL-1500 UV Crosslinker) manufactured by Spectroline. As a result of observing the uniformity of the image after spotting, it was confirmed that the spot was uniformly spotted as in the previous example.

(2) Sample detection with a single-stranded DNA probe-fixed membrane: development-type hybridization In the same manner as in Example 6, a biotin-Thrombin solution and a biotin-albumin solution were obtained.

Subsequently, a sample solution having the following composition was prepared.
Sample composition developing solution 20.0 μl
Latex solution 2.0 μl
Biotin-*** solution 5.0 μl
Complementary oligo DNA solution (1μM, each) 4.2 μl
TE buffer 10.8 μl
total 42.0 μl
***: Thrombin or Albumin

  As the complementary oligo DNA, the one shown in the table below prepared in advance at an equal concentration and mixed in an equal amount was used. 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.

(Hybridization and color reaction)
42 μl of each of the prepared samples was added to a 0.2 ml tube, and the reaction was completed in the same manner as in Example 6. The reaction site was visually confirmed and an image was taken. The developed image is shown in FIG.

  As shown in FIG. 7, when the Thrombin sample is reacted, 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.

  From the above results, it was found that only the target protein can be detected using the first capture and the second capture.

SEQ ID NOs: 1-113: Probe

Claims (23)

  1. A detection device for detecting a protein as a target substance,
    A solid support;
    A first capture having a polynucleotide retained on the solid support;
    With
    The first capture is immobilized on the solid support via at least a portion of the polynucleotide;
    The solid phase carrier is a hydrophilic material other than nitrocellulose, and is superior in the immobilization ability of the first capture than the nitrocellulose and inferior in the nonspecific adsorption ability of the protein than the nitrocellulose. Selected
    It said first capture is immobilized on the solid phase support via a polyethylene thymine region as said at least part,
    The device, wherein the solid phase carrier contains any material selected from the group consisting of hydrophilic polyvinylidene fluoride, polyethersulfone and amylose .
  2. The device of claim 1 , wherein the device is for visual detection.
  3. It said solid support is a porous, according to claim 1 or 2 device.
  4. Wherein exhibit the specific interaction between the polynucleotide comprises a second capture to capture the target substance, according to any one of claims 1 to 3 devices.
  5. 5. The device of claim 4 , wherein the second capture comprises a tag that has a specific interaction with at least a portion of the polynucleotide.
  6. The device of claim 4 or 5 , wherein the second capture comprises a protein.
  7. The device of claim 6 , wherein the protein of the second capture is an antibody or an antigen.
  8. The device of claim 6 or 7 , wherein the second capture comprises a nucleic acid ligand.
  9. The device according to claim 1, wherein the solid phase carrier is porous or flat.
  10. The detecting device is a device for detecting the protein by chromatography, according to any one of claims 1-9 device.
  11. The device according to any one of claims 1 to 9 , wherein the detection device is a device for detecting a protein by immersion hybridization.
  12. A method for producing a detection device for detecting a protein as a target substance, comprising:
    A first capture having a polynucleotide with respect to a solid phase carrier, the first capture having a polypyrimidine region as at least a part of the polynucleotide is supplied, and the first capture is performed by electromagnetic wave irradiation. Immobilizing the solid phase carrier;
    With
    The solid phase carrier is a hydrophilic material other than nitrocellulose, and is superior in the immobilization ability of the first capture than the nitrocellulose and inferior in the nonspecific adsorption ability of the protein than the nitrocellulose. Selected method.
  13. A protein detection method comprising:
    A step of detecting the target substance on the solid phase carrier using the detection device according to any one of claims 1 to 12 ,
    With
    A method of detecting the target substance by utilizing at least a specific interaction exhibited by the first capture.
  14. The specific interaction includes the following (a) to (b):
    (A) a polynucleotide comprising any one of the specific interaction acting between the specific interaction that interacts with (b) a protein with the target substance between the target substance, according to claim 13 Method.
  15. Furthermore, in addition to the interaction (a) or (b), the following (c):
    (C) The method according to claim 14 , wherein the target substance is detected using a specific interaction acting between polynucleotides.
  16. The detection device comprises a second capture that has a specific interaction with at least a portion of the polynucleotide;
    The method according to any one of claims 13 to 15 , wherein the target substance is detected via the second capture.
  17. In the detection step, the target substance is detected on the solid phase carrier by causing the second capture to have a specific interaction with at least a part of the polynucleotide of the first capture. The method of claim 16 , wherein
  18. The detection method according to claim 13 , wherein the target substance is detected using an antibody or an antigen that specifically binds to the target substance.
  19. The detection method according to any one of claims 13 to 18 , wherein the detection step is performed by chromatography in which a hybridization medium containing at least the target substance is moved in a predetermined direction.
  20. The detection method according to claim 13 , wherein the detection step is performed by immersion type hybridization between a hybridization medium containing at least the target substance and the solid phase carrier.
  21. A detection kit for detecting a protein as a target substance,
    A solid support;
    A first capture having a polynucleotide retained on the solid support;
    With
    The first capture is immobilized on the solid support via at least a portion of the polynucleotide;
    The solid phase carrier is a hydrophilic material other than nitrocellulose, and is superior in the immobilization ability of the first capture than the nitrocellulose and inferior in the nonspecific adsorption ability of the protein than the nitrocellulose. Selected
    It said first capture is immobilized on the solid phase support via a polyethylene thymine region as said at least part, kit,
  22. Further, a second capture that captures the target substance by performing a specific interaction with the polynucleotide is provided on the solid phase carrier, or the second capture is used as a reagent separate from the device. The kit according to claim 21 , comprising:
  23. The kit according to claim 21 or 22 , further comprising a labeling element that specifically binds to the target substance.
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JP2001281246A (en) * 2000-01-26 2001-10-10 Nisshinbo Ind Inc Method for detecting immobilization nucleic acid and nucleic acid
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JP2009529658A (en) * 2006-02-21 2009-08-20 トラスティーズ・オブ・タフツ・カレッジTrustees Of Tufts College The methods and arrays for determination of target analyte concentration of a target analyte detection and in solution

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JP2001281246A (en) * 2000-01-26 2001-10-10 Nisshinbo Ind Inc Method for detecting immobilization nucleic acid and nucleic acid
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