JPWO2010125932A1 - Fusion protein-containing assembly, method for producing the same, and assay method using the assembly - Google Patents

Abstract

Provided are an assay method that is highly sensitive and highly accurate and dramatically improved in rapidity, a fusion protein-containing assembly used in the assay method, and a method for producing the assembly. The fusion protein-containing assembly of the present invention comprises a target substance, a target virus or a target cell, a protein (A) that specifically associates with one or more sites α, and a target substance, target virus, or target cell having the sites α. It is characterized by containing at least a fusion protein containing a total of two or more proteins (B) that specifically associate with one or more sites β and a fluorescent dye or enzyme.

Description

  The present invention relates to an assembly mainly composed of proteins, a production method thereof, and an assay method using the assembly. More specifically, the present invention relates to a fusion protein-containing assembly comprising two types of proteins specifically associated with an analyte and a fluorescent dye or enzyme, a method for producing the same, and surface plasmon excitation enhanced fluorescence spectroscopy (SPFS; Surface). The present invention relates to an assay method using the aggregate based on the principle of Plasmon-field enhanced Fluorescence Spectroscopy).

  With surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), irradiation is performed by generating a dense wave (surface plasmon) on the surface of the metal thin film under the condition that the irradiated laser light is attenuated by total reflection (ATR) on the gold thin film surface. The amount of photons in the laser light is increased to several tens to several hundreds times (electric field enhancement effect of surface plasmons), and by this, the fluorescent dye in the vicinity of the gold thin film is efficiently excited. It is a method that can detect an analyte.

  As an example of a biosensor or biochip based on the principle of SPFS, Patent Document 1 discloses that a ligand (primary antibody) immobilization film using carboxymethyldextran is arranged on the surface of a metal substrate, and surface plasmon is used. A method of detecting a fluorescent dye associated with an antigen with an enhanced electric field is shown.

  However, since the method described in Patent Document 1 is a so-called heterogeneous reaction system that uses an antibody immobilized on a substrate, the amount and degree of freedom of immobilization of the antibody are limited, and the efficiency of the antigen-antibody reaction There was a problem of low.

  As a method for improving the reaction efficiency, a so-called homogeneous reaction system using a free antibody is conceivable. However, when combined with an SPFS biosensor, there is a problem of how to recover the antigen-antibody reaction product to the detection portion. It was.

  On the other hand, Patent Document 2 discloses a method in which an antibody against an antigen held by a blood cell or the like is used to agglutinate the blood cell, trap it with a filter, and detect it.

  However, the method described in Patent Document 2 is a method for aggregating relatively large cells such as blood cells in which the target substance holds a plurality of the same epitope, and has no versatility for molecules such as proteins and nucleic acids. Furthermore, after trapping the aggregate with a filter, it is necessary to further react the fluorescent substance and the chemiluminescent substance, and the operation is complicated although the sensitivity is low.

Japanese Patent No. 3294605 JP 2008-256457 A

  The present invention has been made in view of the above problems and situations, and the problem to be solved is an assay method that is highly sensitive, highly accurate, and dramatically improved in rapidity, and a fusion protein-containing assembly used in the assay method. It is to provide a body and a method for producing the assembly.

  As a result of intensive studies to solve the above problems, the present inventors have found that a protein (A) specific to the site α of the analyte and a protein (B) specific to the site β of the analyte having the site α. ) And a fluorescent protein or enzyme-containing assembly comprising a fluorescent dye or enzyme was used in a sandwich immunoassay method using SPFS, and the rapidity and sensitivity were dramatically improved, and the present invention was completed.

  That is, the said subject which concerns on this invention is solved by the following means.

1. Protein (A) that specifically associates with one or more sites α of target substance, target virus or target cell, and specifically associates with one or more sites β of target substance, target virus or target cell having the site α Or at least a fusion protein comprising two or more proteins (B) and a fluorescent dye or enzyme, or
A fusion protein comprising a total of two or more proteins (A) that specifically associate with one or more site α possessed by a target substance, target virus or target cell, and a fluorescent dye or enzyme, and a target material having the site α, A fusion protein-containing assembly comprising at least a total of two or more proteins (B) that specifically associate with one or more sites β of a target virus or target cell and a fusion protein containing a fluorescent dye or an enzyme body.

  2. Biotin is immobilized on each of the proteins (A) and (B), and a fluorescent dye or enzyme is immobilized on at least one of the protein (A), protein (B), biotin, and avidin, and the fusion protein The fusion protein-containing assembly according to item 1, wherein the protein comprises a total of 4 proteins (A) and (B) and 1 avidin.

3. 3. The fusion protein-containing assembly according to item 1 or 2, wherein each of the proteins (A) and (B) is an antibody, a Fab fragment, a Fab ′ fragment or a F (ab ′) ₂ fragment. body.

  4). The proteins (A) and (B) are both antibodies, and the fluorescent dye or enzyme is immobilized only on the antibody, protein A, protein G or protein A / G alone, Alternatively, the antibody and protein A, protein G, or protein A / G are immobilized, and the fusion protein is a total of 2 to 5 of the proteins (A) and (B) and protein A, protein G, or protein A. The fusion protein-containing assembly according to any one of items 1 to 3, wherein / G is included.

  5). 5. The fusion protein-containing assembly according to any one of items 1 to 4, wherein the sites α and β are the same.

  6). 6. The fusion protein-containing assembly according to any one of items 1 to 5, wherein the enzyme is alkaline phosphatase, β-galactosidase, β-glucosidase, or glucose oxidase.

7). A method for producing a fusion protein-containing assembly comprising the following steps (a), (b) and (c):
Step (a): a protein (A) that specifically associates with one or more sites α of the target substance, target virus, or target cell, and one or more sites β of the target substance, target virus, or target cell having the site α Immobilizing a fluorescent dye or enzyme on at least one of a protein consisting of a protein (B) that specifically associates with biotin, biotin and avidin;
Step (b): Biotin by immobilizing biotin to which the fluorescent dye or enzyme may be immobilized on each of the proteins (A) and (B) to which the fluorescent dye or enzyme may be immobilized. Steps for obtaining modified proteins (A) and (B) and step (c): mixing so as to contain one or more biotinylated proteins (A) and (B) to which fluorescent dyes or enzymes may be immobilized, respectively. And
By further mixing the biotinylated protein (A) and (B) with avidin in which a fluorescent dye or an enzyme may be immobilized so as to have a molar ratio of 4: 1 or more, Obtaining a fusion protein-containing assembly.

8). 8. The method for producing a fusion protein-containing assembly according to item 7, wherein each of the proteins (A) and (B) is an antibody, a Fab fragment, a Fab ′ fragment, or a F (ab ′) ₂ fragment. .

  9. Item 9. The method for producing a fusion protein-containing assembly according to Item 7 or 8, comprising at least one of steps (d) and (e) below and (f).

Step (d): antibody (A) that specifically associates with one or more sites α of the target substance, target virus or target cell, and one or more sites β of the target substance, target virus or target cell having the site α Immobilizing a fluorescent dye or an enzyme on each of the antibodies (B) that specifically associate with
Step (e): Protein A, Protein G or Protein A / G is immobilized with a fluorescent dye or enzyme, and Step (f): The fluorescent dye or enzyme may be immobilized with the antibody (A) and ( B) are mixed so that each contains at least one, and the total of the antibodies (A) and (B) may be combined with protein A, protein G or protein A / G (However, when the fluorescent dye or enzyme is not immobilized on the antibodies (A) and (B), the fluorescent dye or enzyme is immobilized on the protein A, protein G, or protein A / G used.) Are further mixed so that the molar ratio is 2: 1 or more to obtain an aggregate.

  10. 10. The method for producing a fusion protein-containing assembly according to any one of items 7 to 9, wherein the sites α and β are the same.

  11. Item 8. The method for producing a fusion protein-containing assembly according to any one of Items 7 to 10, wherein the enzyme is alkaline phosphatase, β-galactosidase, β-glucosidase, or glucose oxidase. .

12 An assay method comprising the following steps (g) to (l);
Step (g): A target substance, target virus or target cell contained in the specimen by bringing the fusion protein-containing assembly according to any one of paragraphs 1 to 6 and the specimen into contact with each other. Forming an aggregate via
Step (h): a step of isolating only the aggregate by passing the reaction product obtained through the step (g) through a filter,
Step (i): a step of reacting the aggregate obtained through the step (h) with a fluorescent substrate or a quencher substrate to produce a fluorescent dye (E) or a quencher, respectively (however, the above step (g) , Only when a fusion protein-containing assembly comprising the enzyme according to any one of claims 1 to 6 is used).
Step (j): Spacer layer comprising a transparent flat substrate, a metal thin film formed on one surface of the substrate, and a dielectric formed on the other surface of the metal thin film not in contact with the substrate Or the surface of the spacer layer or the surface of the fluorescent dye (F) layer of a plasmon excitation sensor having at least a fluorescent dye (F) layer formed on the other surface not in contact with the metal thin film. A step of contacting the aggregate obtained through (h) or the fluorescent dye (E) obtained through the above step (i) or a quencher;
Step (k): The plasmon excitation sensor obtained in step (j) was excited by being irradiated with laser light from the other surface of the substrate on which the thin film was not formed via a prism. A step of measuring the amount of fluorescence emitted from the fluorescent dye, and a step (l): a step of calculating the amount of the target substance, the amount of the target virus or the amount of the target cell from the measurement result obtained in the step (k) .

  13. 13. The assay method according to item 12, wherein the immune reaction field in steps (g) and (h) and the SPFS detection field in steps (j) and (k) are independent of each other.

  14 14. The assay method according to item 12 or 13, wherein the metal thin film is formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum.

  15. 15. The assay method according to 14 above, wherein the metal is gold.

16. Item 16. The assay method according to any one of Items 12 to 15, wherein the dielectric includes silicon dioxide (SiO ₄ ) or titanium dioxide (TiO ₂ ).

  When the assembly of the present invention is used in an assay method, (1) since it is not necessary to immobilize an antibody or the like on the sensor base for SPFS measurement, there is a problem related to the amount of immobilized antibody and the nonspecificity to the sensor base. (2) The reaction efficiency is improved because the immune reaction field is a liquid-liquid system, and (3) the immune reaction field and the SPFS detection field can be completely separated. Immune reaction conditions and detection conditions can be optimized, and it is difficult to be influenced by scattering noise. (4) A homogeneous and extremely efficient antibody-antigen reaction can be realized. Because it is concentrated, the speed and sensitivity are dramatically improved over ordinary SPFS, or (5) an enzyme immobilized on a fusion protein (this enzyme converts a fluorescent substrate into a fluorescent dye (E)) Touch reaction Either, or it catalyzes a reaction to convert the quencher substrates quencher. By), it is possible to amplify the signal.

Schematic diagram of an embodiment of a fusion protein (I) containing a fluorescent dye Schematic diagram of one embodiment of fusion protein (I) containing an enzyme Schematic diagram of one embodiment of a fusion protein (II) containing a fluorescent dye It is a schematic diagram of one aspect | mode of the fusion protein (II) containing an enzyme, Comprising: The fluorescent substrate 13 is hydrolyzed by the enzyme 3b fix | immobilized in this fusion protein (II) 11, and it converts into fluorescent dye (E) 14 Schematically shows the reaction Schematic diagram of one embodiment of a fusion protein (I) -containing assembly containing a fluorescent dye Schematic diagram of one embodiment of a fusion protein (I) -containing assembly containing an enzyme Schematic diagram of the aggregate 21 formed by associating the fusion protein (II) 12 containing a fluorescent dye with each other via the target substance, target virus or target cell 7 Schematic diagram of an aggregate 21 formed by associating fusion proteins (II) 12 containing enzymes via target substances, target viruses or target cells 7 The figure which showed typically the assay method which concerns on the fusion protein containing assembly containing the fluorescent dye of this invention The figure which showed typically the assay method concerning the fusion protein containing assembly containing the enzyme of this invention

The fusion protein-containing assembly of the present invention comprises a protein (A) that specifically associates with one or more sites α possessed by a target substance, target virus, or target cell, and a target substance, target virus, or target cell having the site α. At least a fusion protein comprising a total of two or more proteins (B) that specifically associate with one or more sites β and a fluorescent dye or enzyme, or
A fusion protein comprising a total of two or more proteins (A) that specifically associate with one or more site α possessed by a target substance, target virus or target cell, and a fluorescent dye or enzyme, and a target material having the site α, It is characterized by containing at least a total of two or more proteins (B) that specifically associate with one or more sites β of the target virus or target cell and a fusion protein containing a fluorescent dye or an enzyme. In other words, the fusion protein-containing assembly of the present invention comprises a protein (A) that specifically associates with a target substance, a target virus, or a target cell that has at least one site α, and a target substance, target virus, or An assembly comprising a fusion protein comprising a total of at least one of at least one protein (B) that specifically associates with one or more sites β of a target cell and a fluorescent dye or enzyme, wherein the protein (A) And (B) each having one or more.

  This feature is a technical feature common to the inventions according to claims 1 to 16. In the present invention, as described below, the “enzyme” according to the present invention is an enzyme that catalyzes a reaction for converting a “fluorescent substrate” into a “fluorescent dye”, or a “quencher substrate” is a “quenching agent”. It is characterized by using an enzyme that catalyzes a reaction for conversion to.

As an embodiment of the present invention, from the viewpoint of expression of the effects of the present invention, biotin is immobilized on the protein, and a fluorescent dye or enzyme is immobilized on at least one of the protein, biotin and avidin, and the fusion The protein preferably contains a total of four of the proteins (A) and (B) and one avidin, and in particular, each of the proteins (A) and (B) is an antibody, Fab fragment, Fab ′ fragment or F Preferably it is an (ab ′) ₂ fragment.

  The proteins (A) and (B) are both antibodies, and the fluorescent dye or enzyme is immobilized only on the antibody, or only on protein A, protein G, or protein A / G. Or the antibody and protein A, protein G, or protein A / G, and the fusion protein comprises a total of 2 to 5 of the proteins (A) and (B) and protein A, protein G or protein It is also preferable that A / G is included.

  The sites α and β may be the same.

  The enzyme is preferably alkaline phosphatase, β-galactosidase, β-glucosidase or glucose oxidase.

  The method for producing a fusion protein-containing assembly of the present invention includes the steps (a), (b) and (c).

In the production method, each of the proteins (A) and (B) is preferably an antibody, a Fab fragment, a Fab ′ fragment or a F (ab ′) ₂ fragment.

  In addition, the method for producing a fusion protein-containing assembly of the present invention is characterized by including at least one of the steps (d) and (e) and (f).

  In the production method, the sites α and β may be the same.

  The enzyme is preferably alkaline phosphatase, β-galactosidase, β-glucosidase or glucose oxidase.

  The assay method of the present invention comprises the following steps (g) to (l).

  It is preferable that the immune reaction field in the steps (g) and (h) and the SPFS detection field in the steps (j) and (k) are independent from each other.

  The metal thin film is preferably formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, and the metal is more preferably made of gold.

The dielectric preferably includes silicon dioxide (SiO ₄ ) or titanium dioxide (TiO ₂ ).

  Hereinafter, the present invention, its components, and modes and modes for carrying out the present invention will be described in detail.

<Aggregate>
The fusion protein-containing assembly of the present invention comprises a protein (A) that specifically associates with one or more sites α possessed by a target substance, target virus, or target cell, and a target substance, target virus, or target cell having the site α. At least a fusion protein comprising a total of two or more proteins (B) that specifically associate with one or more sites β and a fluorescent dye or enzyme, or
A fusion protein comprising a total of two or more proteins (A) that specifically associate with one or more site α possessed by a target substance, target virus or target cell, and a fluorescent dye or enzyme, and a target material having the site α, It is characterized by containing at least a total of two or more proteins (B) that specifically associate with one or more sites β of the target virus or target cell and a fusion protein containing a fluorescent dye or an enzyme.

  In other words, the aggregate of the present invention includes “a protein (A) that specifically associates with one or more sites α of target substance, target virus or target cell” and “target substance, target virus or “Assembly” containing “fusion protein” containing at least one of a total of at least one of proteins (B) specifically associated with one or more sites β of target cells and “fluorescent dye” or “enzyme” And having one or more of each of the proteins (A) and (B).

  That is, the aggregate of the present invention includes a plurality of such fusion proteins, and a plurality of fusion proteins are formed through a target substance, a target virus, or a target cell. .) Is different.

  As long as the expression of the effect of the present invention is not inhibited, the aggregate of the present invention contains any useful substance that does not inhibit the specific association between the site of the target substance, the target virus or the target cell, and the protein. You may let them.

  The concentration of the fusion protein contained in the aggregate of the present invention is desirably higher, preferably at least 10 nM or more, more preferably 100 nM or more, and particularly preferably 1 μM or more.

  The protein (A) and the protein (B) contained in the aggregate of the present invention are each at least one, preferably 9: 1 to 1: 9, more preferably 8: 2 to 2: 8, and more preferably It is expressed in a ratio of 7: 3 to 3: 7, particularly preferably 6: 4 to 4: 6, and most preferably 5: 5. The closer the number contained in the aggregate of proteins (A) and (B) is to the ratio of 5: 5 (the closer the number is), the more preferable it is because aggregates can be formed efficiently and stably. In such an embodiment, the total number contained in the aggregate of the protein (A) and the protein (B) is constant.

(Target substance, target virus, target cell)
In the present invention, each of “target substance”, “target virus” and “target cell” (hereinafter collectively referred to simply as “analyte”) represents a site where the protein can specifically associate (bind). If there are two or more independently, it may be a non-living substance that is a substance (a chemical substance that is a simple substance or a compound) or a virus, or it may be a single-cell or multi-cellular organism, and is not particularly limited. Absent.

  Examples of the “target substance” include nucleic acids (DNA that may be single-stranded or double-stranded, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., or nucleosides, nucleotides, and their Modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or their modified molecules, complexes, etc. These molecules may be molecular fragments. More specifically, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signaling substance, a hormone, or the like, and is not particularly limited.

  Examples of the “target virus” include viruses that infect animals such as influenza virus, hepatitis virus, human immunodeficiency virus (HIV), viruses that infect plants, or bacteriophages that infect bacteria. There is no particular limitation.

  “Target cell” refers to a cell derived from, for example, vertebrates and invertebrates including mammals such as humans, plants, algae, fungi, bacteria, and the like. Examples include Mycobacterium tuberculosis containing mycolic acid.

  There are two or more and the completely independent sites that the target substance, target virus, or target cell can specifically associate with (combine with) the protein (that is, one or more sites α and the sites). is one or more sites β that are completely different from α.) or the sites α and β are the same. Note that “same” includes not only completely identical but also partially identical (partially overlapping).

  However, when the sites α and β are the same, the following proteins (A) and (B) cannot be used.

(Proteins (A) and (B))
In the present invention, the “protein (A) specifically associated with one or more sites α possessed by the target substance, target virus or target cell” (hereinafter also simply referred to as “protein (A)”) is as described above. The target substance, target virus, or target cell is not particularly limited as long as it is a protein that specifically associates (bonds) with one or more sites α, for example, various monoclonal antibodies ( Fab fragment, Fab ′ fragment and F (ab ′) ₂ fragment are also included.), MDP1 (Mycobacterial DNA-binding protein 1) and the like. In the present invention, “protein” includes polypeptides and oligopeptides.

  Further, in the present invention, “a protein (B) that specifically associates with one or more sites β of the target substance, target virus, or target cell having the site α” (hereinafter also simply referred to as “protein (B)”). ) Is particularly a protein in which the target substance, target virus or target cell as described above has one or more sites α and one or more sites β and specifically associates (bonds) via the sites β. It is not limited, and specific examples include the same kind as the protein (A).

(Fluorescent dye)
In the present invention, the “fluorescent dye” is a general term for substances that emit fluorescence by irradiating with predetermined excitation light or using the electric field effect, and the “fluorescence” refers to various substances such as phosphorescence. Luminescence is also included.

  The fluorescent dye used in the present invention is not particularly limited as long as it is not quenched due to light absorption by a metal thin film described later, and may be any known fluorescent dye. In general, fluorescent dyes with large Stokes shifts that allow the use of a fluorometer with a filter rather than a monochromator and also increase the efficiency of detection are preferred.

  Examples of such fluorescent dyes include fluorescein family fluorescent dyes (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan Co., Ltd.), and hexachlorofluorescein family fluorescent dyes. (Applied Biosystems Japan), Coumarin family fluorescent dye (Invitrogen), Rhodamine family fluorescent dye (GE Healthcare Biosciences), Cyanine family fluorescent dye, Indocarbocyanine family fluorescent dye, oxazine family fluorescent dye, thiazine family fluorescent dye, squaraine family fluorescent dye, chelated lanthanide family Fluorescent dye of BODIPY (registered trademark) family (manufactured by Invitrogen), naphthalenesulfonic acid family fluorescent dye, pyrene family fluorescent dye, triphenylmethane family fluorescent dye, Alexa Fluor (Registered trademark) dye series (manufactured by Invitrogen Corp.) and the like, and further, U.S. Patent Nos. 6,406,297, 6,221,604, 5,994,063, The fluorescent dyes described in US Pat. Nos. 5,808,044, 5,880,287, 5,556,959, and 5,135,717 can also be used in the present invention.

  Table 1 shows the absorption wavelength (nm) and emission wavelength (nm) of typical fluorescent dyes included in these families.

The fluorescent dye is not limited to the organic fluorescent dye. For example, rare earth complex fluorescent dyes such as Eu and Tb can also be used as fluorescent dyes in the present invention. In general, rare earth complexes have a large wavelength difference between an excitation wavelength (about 310 to 340 nm) and an emission wavelength (about 615 nm for an Eu complex and 545 nm for a Tb complex), and a long fluorescence lifetime of several hundred microseconds or more. is there. An example of a commercially available rare earth complex fluorescent dye is ATBTA-Eu ³⁺ . Furthermore, it is represented by blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (DsRed), or allophycocyanin (APC; LyoFlogen (registered trademark)). Fluorescent fine particles such as latex protein and latex that can be used as the fluorescent dye in the present invention.

  In the present invention, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength in a wavelength region where light absorption by the metal species forming the metal thin film is small when performing fluorescence measurement described later. For example, when gold is used as the metal thin film, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength of 600 nm or more in order to minimize the influence of light absorption by gold. Therefore, in this case, it is particularly desirable to use a fluorescent dye having a maximum fluorescence wavelength in the near-infrared region such as Cy5, Alexa Fluor (registered trademark) 647. The use of a fluorescent dye having the maximum fluorescence wavelength in the near-infrared region can minimize the influence of light absorption by iron derived from blood cell components in the blood. Is also useful. On the other hand, when silver is used as the metal thin film, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength of 400 nm or more.

  These fluorescent dyes may be used alone or in combination of two or more.

(enzyme)
In the present invention, as an “enzyme” according to the present invention, an enzyme (C) that catalyzes a reaction for converting a “fluorescent substrate” to a “fluorescent dye (E)”, or a “quenching agent substrate” as a “quenching agent”. An enzyme (D) that catalyzes a reaction to be converted is used.

  Examples of such an enzyme (C) include alkaline phosphatase (ALP), peroxidase (POD), galactosidase (GAL) and the like.

  In addition, the enzyme (D) can be used as an enzyme reaction (D-1) by activating a “quencher substrate” blocked by a protecting group, activating the quencher by an enzyme reaction, or as an enzyme reaction (D-2). The pH is lowered by a quencher activated by an enzymatic reaction using a specific “quencher substrate”.

  Among the enzymes (D), those used for the enzyme reaction (D-1) include, for example, β-galactosidase (βGAL), β-glucosidase (βGlu), alkaline phosphatase (ALP), etc. As what is used for D-2), glucose oxidase (GOD) etc. are mentioned, for example.

  These enzymes can be used alone or in combination of two or more.

  The “fluorescent substrate”, “fluorescent dye (E)”, “quenching agent substrate” and “quenching agent” will be described later.

[Fusion protein]
The fusion protein constituting the aggregate of the present invention contains at least one of protein (A) and protein (B) in total of 2 or more and a fluorescent dye or enzyme, and includes the following fusion proteins (I) and (II) is mentioned as a preferred embodiment.

(Fusion protein (I))
As illustrated in FIGS. 1 and 2, in the fusion protein (I) 10, the protein (A) 1 and the protein (B) 2 may be both antibodies, and the fluorescent dye 3a or the enzyme 3b, And biotin 4 are immobilized (not shown, but the fluorescent dye or enzyme can be immobilized on biotin and / or avidin in addition to the antibody. For example, the fluorescent dye or enzyme can be immobilized on the antibody, biotin and avidin. 4), a total of four such proteins (A) 1 and (B) 2 and one avidin 5 are included.

  In addition, the antibody which fusion protein (I) 10 shown in FIG.1 and FIG.2 has two proteins (A) 1 and two proteins (B) 2, However, this invention is limited to this aspect. In addition, an embodiment having only four proteins (A) is also included.

  In the present invention, avidin includes streptavidin and neutravidin.

(Fusion protein (II))
As illustrated in FIGS. 3 and 4, in the fusion protein (II) 11, the protein (A) 1 and the protein (B) 2 are both antibodies, and the fluorescent dye 3a or the enzyme 3b is an Fc fragment of the antibody. (The fluorescent dye or enzyme can be immobilized on protein A, protein G or protein A / G in addition to the Fc fragment of the antibody. For example, the fluorescent dye or enzyme Fc fragments of (2) and protein A, protein G or protein A / G may be immobilized simultaneously.), Protein (A) 1 and protein (B) 2 in total and protein A6 (not shown) However, protein G or protein A / G may be used in addition to protein A. When protein G or protein A / G is used, an association ) Which may be a protein (A) and protein (B) is the sum is 2-5.) And is intended to include.

  In addition, although the antibody which fusion protein (II) 11 shown in FIG.3 and FIG.4 has is one protein (A) 1 and protein (B) 2, respectively, this invention is not limited to this aspect, An embodiment comprising only protein (A) 1 is also included.

  In the present invention, protein A and protein G may be natural or recombinant.

  Among these fusion proteins, since the aggregates described later can be formed efficiently and stably, the fusion protein having a larger total number of proteins (A) and proteins (B) (that is, a higher concentration) is preferable.

<Method for producing assembly>
The method for producing an aggregate of the present invention has any one of the following production methods (i) to (iv), and the production method (i) comprises the following steps (a), (b) and (c). Manufacturing method (ii) includes the following steps (d), (e) and (f); manufacturing method (iii) includes the following steps (d) and (f); manufacturing method (iv) Includes the following steps (e) and (f).

  Step (a): a protein (A) that specifically associates with one or more sites α of the target substance, target virus, or target cell, and one or more sites β of the target substance, target virus, or target cell having the site α A step of immobilizing a fluorescent dye or enzyme on at least one of a protein (B), biotin and avidin, which specifically associates with the protein.

  Step (b): Biotin by immobilizing biotin on which fluorescent dye or enzyme may be immobilized on each of the proteins (A) and (B) on which fluorescent dye or enzyme may be immobilized. A step of obtaining modified proteins (A) and (B).

  Step (c): The biotinylated proteins (A) and (B) are mixed so as to contain one or more biotinylated proteins (A) and (B) to which fluorescent dyes or enzymes may be immobilized. A step of obtaining an aggregate by further mixing avidin, which may be immobilized on a fluorescent dye or an enzyme, with a molar ratio of 4: 1 or more with respect to the total of the above.

  Step (d): antibody (A) that specifically associates with one or more sites α of the target substance, target virus or target cell, and one or more sites β of the target substance, target virus or target cell having the site α A step of immobilizing a fluorescent dye or an enzyme on each of the antibodies (B) that specifically associate with the antibody.

  Step (e): A step of immobilizing a fluorescent dye or enzyme on protein A, protein G or protein A / G.

  Step (f): Mix the antibodies (A) and (B) to which one or more fluorescent dyes or enzymes may be immobilized so as to contain one or more, respectively, and the total of the antibodies (A) and (B) Then, the protein A, protein G or protein A / G, to which the fluorescent dye or enzyme may be immobilized, is further mixed so that the molar ratio thereof is 2: 1 or more, whereby a fusion protein-containing assembly is obtained. Obtaining step.

[Production method (i)]
The production method (i) includes the above steps (a), (b) and (c), wherein at least one of protein ((A), (B)), biotin and avidin is a fluorescent dye or This is a method for producing a fusion protein (I) -containing assembly on which an enzyme is immobilized.

  Each of the proteins (A) and (B) is preferably any of an antibody (immunoglobulin), a Fab fragment, a Fab ′ fragment and a F (ab ′) fragment.

(Process (a))
Step (a) includes “a protein (A) that specifically associates with one or more sites α of target substance, target virus or target cell” (protein (A)), and “target substance having the site α. A “fluorescent dye” or “enzyme” for at least one of a protein consisting of a protein (B) ”(protein (B)) that specifically associates with one or more sites β of a target virus or target cell, protein (B) Is a step of immobilizing.

  The proteins (A) and (B), and “fluorescent dye” and “enzyme” are the same as those described in the item “Aggregate” above. The same applies to these terms described in the steps (b), (d) and (e).

  Examples of the method for immobilizing an enzyme on protein ((A) and (B)), biotin and / or avidin include an amine coupling method, a thiol coupling method, an aldehyde coupling method, and the like. The present invention is not limited to these methods.

When the fluorescent dye or enzyme is immobilized on the protein (A) or (B), when the protein (A) or (B) is an antibody, the fluorescent dye or enzyme is preferably a constant region (C _H 1 and C _{H L} ), more preferably immobilized on an Fc fragment, and when the protein (A), (B) is a Fab fragment, Fab ′ fragment or F (ab ′) ₂ fragment, the fluorescent dye or enzyme is a constant region (C _H 1 and C _L ) are preferably immobilized.

  In this way, protein (A), protein (B), biotin and avidin to which a fluorescent dye or enzyme is immobilized are referred to as “fluorescent dye or enzyme-labeled protein (A)”, “fluorescent dye or enzyme-labeled protein ( B) "," fluorescent dye or enzyme-labeled biotin "and" fluorescent dye or enzyme-labeled avidin ".

(Process (b))
The step (b) is a “protein (A) that specifically associates with one or more sites α of the target substance, target virus, or target cell” to which the “fluorescent dye” or “enzyme” may be immobilized. And “fluorescent dye” or “enzyme” is immobilized on each of the “protein (B) specifically associated with one or more sites β of the target substance, target virus or target cell having the site α” This is a step of obtaining “biotinylated protein (A)” and “biotinylated protein (B)” by immobilizing good biotin. The “fluorescent dye” or enzyme may be simultaneously immobilized on the proteins (A), (B) and biotin.

  That is, the step (b) means (1) a step of immobilizing biotin on a fluorescent dye or enzyme-labeled protein (A), (B); (2) a fluorescent dye or enzyme label on the protein (A), (B) A step of immobilizing biotin; (3) a step of immobilizing biotin on the proteins (A) and (B); (4) a fluorescent dye or enzyme-labeled antibody (A), and (B) with a fluorescent dye or enzyme-labeled biotin. One of the steps of immobilization.

  Examples of a method for immobilizing biotin on a protein include an amine coupling method, a thiol coupling method, and an aldehyde coupling method. When an enzyme is immobilized on either a protein or biotin. However, the present invention is not limited to these methods.

When proteins (A) and (B) are antibodies, biotin is preferably immobilized on constant regions (C _H 1 and C _L ), more preferably Fc fragments, and proteins (A) and (B) are In the case of a Fab fragment, F (ab ′) fragment or F (ab ′) ₂ fragment, biotin is preferably immobilized on the constant regions (C _H 1 and C _L ).

  The protein (A) and the protein (B) immobilized with biotin in this manner are also referred to as “biotinylated protein (A)” and “biotinylated protein (B)”, respectively.

(Process (c))
In step (c), the biotinylated proteins (A) and (B) are mixed so as to contain one or more biotinylated proteins (A) and (B) to which fluorescent dyes or enzymes may be immobilized. This is a step of obtaining an aggregate by further mixing avidin in which a fluorescent dye or an enzyme may be immobilized with respect to the sum of B) so that the molar ratio thereof is 4: 1 or more.

  That is, in the step (c), at least one biotinylated protein (A) and (B) to which the fluorescent dye or enzyme obtained by the above step (b) may be immobilized, preferably 9: 1 to 1: 9, more preferably 8: 2 to 2: 8, still more preferably 7: 3 to 3: 7, particularly preferably 6: 4 to 4: 6, most preferably 5: 5. Avidin to which a fluorescent dye or an enzyme may be immobilized is added to the total of the biotinylated proteins (A) and (B) in a molar ratio of 4: 1 or more, preferably 4 1 is a step of obtaining a fusion protein (I) -containing assembly as shown in FIG. 1 and FIG.

  When the ratio of the numbers of the biotinylated proteins (A) and (B) and the molar ratio of the biotinylated proteins (A), (B) and the avidin are within the above ranges, the aggregate can be stably stabilized. It is preferable because it can be formed.

[Production method (ii)]
The production method (ii) includes the steps (d), (e) and (f) described above, and comprises an antibody (preferably an antibody constant region (C _H 1 and C _L ), more preferably an antibody Fc). Fragment) and protein A, protein G, or protein A / G is a method for producing a fusion protein (II) -containing assembly in which a fluorescent dye or an enzyme is immobilized.

(Process (d))
Step (d) includes “an antibody (A) that specifically associates with one or more sites α possessed by the target substance, target virus or target cell” and “target substance, target virus or target cell having the site α”. In this step, a “fluorescent dye” or an “enzyme” is immobilized on each of the “antibody (B) specifically associated with one or more sites β”.

  The method for immobilizing the fluorescent dye or the enzyme on each of the antibodies (A) and (B) is the same as the method for immobilizing the protein described in the step (a).

  The antibodies (A) and (B) immobilized with a fluorescent dye or enzyme are also referred to as “) fluorescent dye or enzyme-labeled antibody (A)” and “fluorescent dye or enzyme-labeled antibody (B)”, respectively.

(Process (e))
The step (e) is a step of immobilizing “fluorescent dye” or “enzyme” on “protein A, protein G or protein A / G”.

  “Protein A, Protein G or Protein A / G” is as described above.

  Examples of the method for immobilizing a fluorescent dye or enzyme on protein A, protein G, or protein A / G include an amine coupling method, a thiol coupling method, an aldehyde coupling method, and the like. It is not limited to these methods.

  Protein A, protein G, and protein A / G each having a fluorescent dye or enzyme immobilized thereon are referred to as “fluorescent dye or enzyme-labeled protein A”, “fluorescent dye or enzyme-labeled protein G” and “fluorescent dye or Also referred to as “enzyme-labeled protein A / G”.

(Process (f))
In step (f), the antibodies (A) and (B) to which fluorescent dyes or enzymes may be immobilized are mixed so as to contain at least one antibody, and the total of the antibodies (A) and (B) In contrast, protein A, protein G, or protein A / G, to which a fluorescent dye or enzyme may be immobilized, is further mixed so that the molar ratio thereof is 2: 1 or more to obtain an aggregate. It is a process.

  That is, in the step (f), at least one of the antibodies (A) and (B), preferably 9: 1 to 1: 9, more preferably 8: 2 to 2: 8, still more preferably 7 : 3 to 3: 7, particularly preferably 6: 4 to 4: 6, most preferably 5: 5, and the fluorescent dye is mixed with respect to the sum of the antibodies (A) and (B). Alternatively, Protein A, Protein G, or Protein A / G, to which the enzyme is immobilized, may be further mixed so that the molar ratio thereof is 2: 1 or more, preferably 2: 1. This is a step of obtaining a fusion protein (II) -containing assembly as shown in FIG.

  Ratio of the number of antibodies (A) and (B) to which fluorescent dyes or enzymes may be immobilized, and proteins to which the antibodies (A) and (B) and fluorescent dyes or enzymes may be immobilized It is preferable that the molar ratio of A, protein G, or protein A / G is within the above range because aggregates can be formed efficiently and stably.

[Production method (iii)]
The production method (iii) includes the steps (d) and (f) described above, and is applied only to an antibody (preferably an antibody constant region (C _H 1 and C _L ), more preferably an antibody Fc fragment). This is a method for producing a fusion protein (II) -containing assembly on which a fluorescent dye or an enzyme is immobilized.

[Production method (iv)]
Production method (iv) includes the above steps (e) and (f), and a fusion protein (II) in which a fluorescent dye or an enzyme is immobilized only on protein A, protein G or protein A / G It is a method of manufacturing a containing aggregate.

<Assay method>
The assay method of the present invention includes the following steps (g) to (l).

  Step (g): A step of bringing the aggregate of the present invention into contact with a specimen to form an aggregate via a target substance, target virus or target cell contained in the specimen.

  Step (h): A step of isolating only the aggregate by passing the reaction product obtained through the step (g) through a filter.

  Step (i): a step of reacting the aggregate obtained through the above step (h) with a fluorescent substrate or a quencher substrate to produce a fluorescent dye (E) or a quencher, respectively (however, the above step (g) Only when a fusion protein-containing assembly containing an enzyme is used.)

  Step (j): Spacer layer comprising a transparent flat substrate, a metal thin film formed on one surface of the substrate, and a dielectric formed on the other surface of the metal thin film not in contact with the substrate Or the surface of the spacer layer or the surface of the fluorescent dye (F) layer of a plasmon excitation sensor having at least a fluorescent dye (F) layer formed on the other surface not in contact with the metal thin film. A step of contacting the aggregate obtained through (h) or the fluorescent dye (E) or quencher obtained through the above step (i).

Step (k): The plasmon excitation sensor obtained in the step (j) was excited by being irradiated with laser light from the other surface of the substrate on which the thin film was not formed via a prism. A step of measuring the amount of fluorescence emitted from the fluorescent dye. as well as,
Step (l): A step of calculating the target substance amount, the target virus amount, or the target cell amount from the measurement result obtained in the step (k).

  In the assay method of the present invention, the immune reaction field in the above steps (g) and (h) and the SPFS detection field in the above steps (j) and (k) can be independent of each other. It is preferable because it can be optimized.

[Step (g)]
In the step (g), the “aggregate” of the present invention and the “sample” are “contacted” to form an “aggregate” via the target substance, target virus or target cell contained in the sample. It is a process to do.

  The “aggregate” of the present invention is as described above, and refers to, for example, a plurality of fusion proteins (I) 10 such as the aggregate 20 shown in FIGS. 5 and 6.

(Sample)
Examples of the “specimen” include blood (serum / plasma), urine, nasal fluid, saliva, feces, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.), etc., and appropriately diluted in a desired solvent, buffer solution, etc. May be used. Of these samples, blood, serum, plasma, urine, nasal fluid and saliva are preferred.

(contact)
As a condition for bringing the aggregate into contact with the specimen, the temperature is usually 4 to 50 ° C., preferably 10 to 40 ° C., and the time is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.

(Aggregate)
In the present invention, the “aggregate” is formed by, for example, associating (binding) the fusion protein (II) 12 with the target substance, target virus or target cell 7 as shown in FIGS. As shown in FIGS. 7 and 8, the aggregate 21 can be produced by bringing the fusion protein (I) 10-containing assembly 20 and a specimen into contact with each other in an immune reaction field.

[Step (h)]
The step (h) is a step of isolating only the aggregate by passing the “reactant” obtained through the step (g) through a “filter”.

(Reactant)
The “reactant” is obtained through the above step (g). As shown in FIG. 9 and FIG. 10, among the target substance, the target virus, or the target cell 7, for example, the specimen is the target cell. In the case of containing blood, the “reactant” refers to the aggregate 21, the unreacted fusion protein (II) 12, and the specimen from which the target cells have been removed (this is because the target cells contained in the specimen are fused proteins) (II) was associated with 12 to form an aggregate 21, so that only the target cells were specifically removed from the specimen.

(filter)
The filter used in the present invention does not trap the fusion protein, target substance, target virus, or target cell of the present invention alone, but is not particularly limited as long as it can trap aggregates. Thus, those that trap aggregates of two or more fusion proteins are preferred. As a commercially available product, for example, “Anopore, pore size: 0.02 (μm)” manufactured by Whatman Corporation is suitable.

[Step (i)]
In the step (i), when a fusion protein-containing assembly containing an enzyme is used in the step (g), the aggregate obtained through the step (h) is added to a “fluorescent substrate” or a “quenching agent substrate”. Are reacted to produce “fluorescent dye (E)” or “quenching agent”, respectively.

  Further, in the step (i), after the fluorescent dye (E) or the quencher is formed, the fluorescent dye (E) alone or the quencher is mixed with the filter from the mixture of the aggregate and the fluorescent dye (E) or the quencher. It is preferred to isolate only

(Fluorescent substrate)
The “fluorescent substrate” used in the present invention does not emit fluorescence per se, but is hydrolyzed and converted into a fluorescent dye (E) by the enzyme (C) immobilized on the fusion protein. If there is, the present invention is not particularly limited. 1-8 etc. can be used.

  Among these fluorescent substrates, when the metal thin film included in the “plasmon excitation sensor” described below is formed of a metal including gold, No. in Table 2 from the viewpoint of gold transmittance and excitation wavelength. No. 7 1,3-diciloro-9,9-dimethyl-acid-2-one-7-yl phosphate (DDAO phosphate) (Molecular Probes) is preferred.

  The autofluorescence wavelength of the fluorescent substrate is such that the greater the difference between the autofluorescence wavelength and the fluorescence wavelength of the fluorescent dye (E), the easier it is to avoid the influence of the background signal, and high-accuracy measurement is possible. is not.

(Fluorescent dye (E))
In the present invention, the “fluorescent dye (E)” is a general term for substances that emit fluorescence by irradiating with predetermined excitation light or using the electric field effect, and the “fluorescence” is phosphorescence or the like. Various types of light emission are also included.

  The fluorescent dye (E) used in the present invention is obtained by hydrolyzing the aforementioned fluorescent substrate with the enzyme (C) immobilized on the fusion protein, and quenching caused by light absorption by the metal thin film described later. As long as not receiving, there is no restriction | limiting in particular in the kind, Any of well-known fluorescent dye may be sufficient. In general, fluorescent dyes with large Stokes shifts that allow the use of a fluorometer with a filter rather than a monochromator and also increase the efficiency of detection are preferred.

  Table 2 shows examples of fluorescent substrates used in the present invention.

(Quencher substrate / Quencher)
Β-galactosidase (βGAL) that can be used in the “enzyme reaction (D-1)” desorbs βGal from TG-βGal, which is a “quenching agent substrate”, and removes TokyoGreen (TG), which is a “quenching agent”. Catalyze the reaction that forms.

  In addition, β-glucosidase (βGlu) catalyzes a reaction of desorbing βGlu from TG-βGlu, which is a “quenching agent substrate”, and generating TG, which is a “quenching agent”.

  Note that free TG is a fluorescent dye (F) because it causes a fluorescent dye (F) having an excitation wavelength of 490 nm and a fluorescent wavelength of 475 to 495 nm and Fluorescence Resonance Energy Transfer (FRET). Fluorescence such as terbium (Tb) chelate fluorescence (fluorescence wavelength: 495 nm) or enhanced cyan fluorescence protein (ECFP) (fluorescence wavelength: 475 nm) can be quenched.

  In the present invention, TG is 2-Me TG represented by the following formula,

Also included is 2-Me-4-OMe TG represented by the following formula.

  Alkaline phosphatase (ALP) catalyzes a reaction that hydrolyzes the “Quencher Substrate” AttoPhos® substrate, and the “Quencher” BBT (2 ′-[2-benzthiazoyl] -6′-hydroxylyl). -Benzthiazole). Here, BBT (excitation wavelength: 482 nm), which is also a fluorescent dye (F), can cause terbium (Tb) chelate or ECFP and FRET to quench each other as in the case of TG described above.

  Glucose oxidase (GOD) that can be used in the above-mentioned “enzyme reaction (D-2)” generates gluconolactone and hydrogen peroxide, which are “quenching agents”, by an enzyme reaction using glucose as a “quenching agent substrate”. To do. The generated hydrogen peroxide lowers the pH of the aqueous solution in which hydrogen peroxide is dissolved, and 2-Me-4-OMe TG, 2-OMe-5-Me TG, or 2- Fluorescence intensity such as OMe TG can be reduced (that is, quenched).

  In the present invention, preferred combinations of a quencher substrate, an enzyme, a quencher and a fluorescent dye (F) include those shown in Table 3 below.

  The concentration of such a quencher substrate during feeding is preferably 0.001 to 10,000 μg / mL, more preferably 1 to 1,000 μg / mL.

  The temperature, time, and flow rate at which the liquid is circulated are the same as in step (g).

[Step (j)]
The step (j) includes a “transparent flat substrate”, a “metal thin film” formed on one surface of the substrate, and another surface of the metal thin film that is not in contact with the substrate. The spacer layer surface or the fluorescence of a plasmon excitation sensor having at least a “spacer layer made of a dielectric” or a “fluorescent dye (F) layer” formed on the other surface not in contact with the metal thin film In this step, the surface of the dye (F) layer is brought into contact with the aggregate obtained through the step (h) or the fluorescent dye (E) or the quencher obtained through the step (i).

More specifically, the step (j) includes a “transparent flat substrate”, a “metal thin film” formed on one surface of the substrate, and the other of the metal thin film that is not in contact with the substrate. A plasmon excitation sensor having at least a “spacer layer made of a dielectric” formed on the surface thereof, obtained on the surface of the spacer layer by an aggregate obtained through the step (h) or obtained through the step (i). The step of “contacting” the fluorescent dye (E), or the step (j) includes “transparent flat substrate”, “metal thin film” formed on one surface of the substrate, and the metal thin film. The quencher obtained through the above step (i) is applied to the surface of the fluorescent dye (F) layer of the plasmon excitation sensor having at least a “fluorescent dye (F) layer” formed on the other surface that is not This is the process of “contacting”.

  In the latter step (j), the plasmon excitation sensor includes a “spacer layer made of a dielectric” included in the former step (j) between the “metal thin film” and the “fluorescent dye (F) layer”. You may have.

(Transparent flat substrate)
The “transparent planar substrate” used in the present invention may be made of quartz or glass, or may be made of plastic such as polycarbonate (PC) or cycloolefin polymer (COP), and has a refractive index [nd]. Preferably, it is 1.40 to 2.20, and the thickness (length × width) is not particularly limited as long as the thickness is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm.

  Glass transparent flat substrates are commercially available products such as “BK7” (refractive index [nd] 1.52) and “LaSFN9” (refractive index [nd] 1.85) manufactured by Shot Japan Co., Ltd. "K-PSFn3" (refractive index [nd] 1.84), "K-LaSFn17" (refractive index [nd] 1.88) and "K-LaSFn22" (refractive index [nd]) manufactured by Sumita Optical Glass Co., Ltd. 1.90) and “S-LAL10” (refractive index [nd] 1.72) manufactured by OHARA INC. Are preferred from the viewpoints of optical properties and detergency.

  The transparent flat substrate is preferably cleaned with acid and / or plasma before forming metal protrusions on the surface.

  As the cleaning treatment with an acid, it is preferable to immerse in 0.001 to 1N hydrochloric acid for 1 to 3 hours.

  Examples of the plasma cleaning treatment include a method of immersing in a plasma dry cleaner (“PDC200” manufactured by Yamato Scientific Co., Ltd.) for 0.1 to 30 minutes.

(Metal thin film)
The “metal thin film” formed on one surface of the transparent flat substrate is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably gold. It is desirable to consist of these, and the alloy of these metals may be sufficient. Such metal species are preferable because they are stable against oxidation and increase in electric field due to surface plasmons increases.

  Only when a glass flat substrate is used as the transparent flat substrate, it is preferable to form a thin film of chromium, nickel chromium alloy or titanium in advance because glass and a metal thin film can be bonded more firmly.

  Examples of the method for forming a metal thin film on a transparent flat substrate include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Since it is easy to adjust the thin film formation conditions, it is preferable to form a chromium thin film and / or a metal thin film by sputtering or vapor deposition.

  The thickness of the metal thin film is preferably gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 to 500 nm, and alloys thereof: 5 to 500 nm. The thickness of the thin film is preferably 1 to 20 nm.

  From the viewpoint of the electric field enhancement effect, gold: 20-70 nm, silver: 20-70 nm, aluminum: 10-50 nm, copper: 20-70 nm, platinum: 20-70 nm, and alloys thereof: 10-70 nm are more preferable, and chromium The thickness of the thin film is more preferably 1 to 3 nm.

  When the thickness of the metal thin film is within the above range, surface plasmons are easily generated, which is preferable. Moreover, if it is a metal thin film which has such thickness, a magnitude | size (length x width) will not be specifically limited.

(Spacer layer made of dielectric)
As the dielectric used for forming the “spacer layer made of a dielectric”, various optically transparent inorganic substances, natural or synthetic polymers can be used. Among them, it is preferable to contain silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) because it is excellent in chemical stability, production stability and optical transparency.

  The thickness of the spacer layer made of a dielectric is usually 10 nm to 1 mm, and preferably 30 nm or less, more preferably 10 to 20 nm from the viewpoint of resonance angle stability. On the other hand, it is preferably 200 nm to 1 mm from the viewpoint of electric field enhancement, and more preferably 400 nm to 1,600 nm from the stability of the effect of electric field enhancement.

  Examples of the method for forming the spacer layer made of a dielectric include a sputtering method, an electron beam evaporation method, a thermal evaporation method, a formation method by a chemical reaction using a material such as polysilazane, or an application by a spin coater.

(Fluorescent dye (F) layer)
The “fluorescent dye (F) layer” is a layer in which the fluorescent dye (F) is immobilized on the other surface of the metal thin film that is not in contact with the transparent flat substrate. It can also be formed by coating a composition containing (F) and a polymer on the metal thin film. (2) SAM (self-assembled monolayer) composed of alkanethiol or the like (Self-Assembled Monolayer) It can also be formed by bonding the fluorescent dye (F) onto the metal thin film via

  In the case of (1), the fluorescent dye (F) and the polymer may or may not be chemically bonded, and a SAM made of alkanethiol having a polymerizable group is bonded to the metal thin film. The composition containing the fluorescent dye (F) and the polymer can also be formed by adding and copolymerizing another polymerizable monomer, the fluorescent dye (F) and a polymerization initiator.

  In the case of (2), the fluorescent dye (F) is converted into a metal thin film by binding an alkanethiol having an amino group or a carboxyl group and a fluorescent dye (F) having a group that reacts with these groups and is covalently bonded. Can be immobilized. When a spacer layer made of a dielectric is provided between the fluorescent dye (F) layer and the metal thin film, the spacer layer and the fluorescent dye (F) can be bonded via a conventionally known silane coupling agent. it can.

  Thus, in the case of (1) which forms a layer comprising the fluorescent dye (F) and the polymer, the amount of the fluorescent dye (F) that can be immobilized is large, and the strength of the resulting layer is high, which is preferable. .

  Similarly to the fluorescent dye (E), the “fluorescent dye (F)” is a general term for substances that emit fluorescence by irradiating predetermined excitation light or exciting using a field effect in the present invention, The “fluorescence” includes various light emission such as phosphorescence.

  Examples of the “fluorescent dye (F)” used in the present invention include terbium (Tb) chelate (fluorescence wavelength: 490 nm), enhanced cyan fluorescent protein (ECFP) (fluorescence wavelength: 475 nm), 2-Me-4-OMe. TG, 2-OMe-5-Me TG, 2-OMe TG and the like can be mentioned.

  Many of these fluorescent dyes (F) have high water solubility. In order to fix these fluorescent dyes (F) as a fluorescent dye (F) layer in a polymer by intermolecular interaction, fluorescent dyes (F) By reacting the carboxyl group of F) with the amino group or alcohol of the hydrophobic aromatic ring to form a water-insoluble structure, or by reacting the hydrophobic polymer with the active ester of the fluorescent dye (F) Must be chemically bonded. When the polymer and the fluorescent dye (F) do not have a chemical bond, it is preferable to modify the fluorescent dye (F) so as to have a structure close to a solubility parameter (SP) of the polymer.

  These fluorescent dyes (F) may be used alone or in combination of two or more.

(contact)
The aggregate obtained through the step (h) or the fluorescent dye (E) obtained through the step (i) on the surface of the spacer layer made of a dielectric or the fluorescent dye (F) layer of the plasmon excitation sensor. Or as a method of making a quencher "contact", methods, such as dripping of the isolated fluorescent dye (E) or a solution containing a quencher, spraying, application | coating, are mentioned, for example. Moreover, the method of forming the flow path mentioned later in a plasmon excitation sensor, and making the solution containing fluorescent dye (E) or a quencher contact the surface of a plasmon excitation sensor is also mentioned.

  A “flow channel” is a rectangular tube or cylinder (tube) that can efficiently deliver a small amount of a chemical solution and can change or circulate the solution feeding speed in order to promote the reaction. It is preferable that the vicinity of the place where the plasmon excitation sensor is installed has a rectangular tube structure, and the vicinity of the place where the drug solution is delivered preferably has a cylindrical (tube) shape.

  The material is a homopolymer or copolymer containing methyl methacrylate, styrene or the like as a raw material in the plasmon excitation sensor part; it is made of polyethylene, polyolefin, etc., and silicon rubber, Teflon (registered trademark), polyethylene, polypropylene is used in the drug solution delivery part. Etc. are used.

  In the plasmon excitation sensor unit, from the viewpoint of increasing the contact efficiency with the aggregate and shortening the diffusion distance, the vertical and horizontal cross sections of the channel of the plasmon excitation sensor unit are preferably independently about 100 nm to 1 mm.

  As a method of fixing the plasmon excitation sensor to the channel, in a small lot (laboratory level), first, the channel height is set to 0. 0 on the surface on which the spacer layer made of the dielectric of the plasmon excitation sensor is formed. A polydimethylsiloxane (PDMS) sheet having a thickness of 5 mm is pressure-bonded so as to surround a portion where the metal thin film of the plasmon excitation sensor is formed, and then the polydimethylsiloxane (PDMS) sheet and the plasmon excitation sensor are bonded to each other. A method of fixing with a closing tool such as a screw is preferred.

  In industrially manufactured large lots (factory level), as a method of fixing the plasmon excitation sensor to the flow path, a gold substrate is formed on an integrally molded plastic product or a separately manufactured gold substrate is fixed to the gold surface. After the dielectric layer or fluorescent dye (F) layer is formed and the ligand is immobilized, it can be produced by covering with a plastic integrally molded product corresponding to the top plate of the flow path. If necessary, the prism can be integrated into the flow path.

  The “liquid feeding” is preferably the same as the solvent or buffer in which the specimen is diluted, and examples thereof include phosphate buffered saline (PBS) and Tris buffered saline (TBS), but are not particularly limited. It is not something.

  The temperature and time for circulating the liquid supply vary depending on the type of specimen and are not particularly limited, but are usually 20 to 40 ° C. × 1 to 60 minutes, preferably 37 ° C. × 5 to 15 minutes.

  The initial concentration of the aggregate during the feeding may be 100 μg / mL to 0.001 pg / mL.

  The total amount of liquid feeding, that is, the volume of the flow path is usually 0.001 to 20 mL, preferably 0.1 to 1 mL.

  The flow rate of liquid feeding is usually 1 to 2,000 μL / min, preferably 5 to 500 μL / min.

[Step (k)]
In step (k), the plasmon excitation sensor obtained in step (j) is irradiated with laser light from the other surface of the substrate on which the thin film is not formed via a prism. In this step, the amount of fluorescence emitted from the fluorescent dye is measured.

(Optical system)
The light source used in the assay method of the present invention is not particularly limited as long as it can cause plasmon excitation in a metal thin film. However, in terms of unity of wavelength distribution and intensity of light energy, laser light is used. Is preferably used as the light source. It is desirable to adjust the energy and photon amount immediately before the laser light enters the prism through the optical filter.

  Irradiation with laser light generates surface plasmons on the surface of the metal thin film under the total reflection attenuation condition (ATR). Due to the electric field enhancement effect of the surface plasmon, the fluorescent dye is excited by photons increased to several tens to several hundred times the amount of photons irradiated. In addition, although the photon increase amount by the electric field enhancement effect depends on the refractive index of the transparent flat substrate, the metal species of the metal thin film, and the film thickness thereof, it is usually an increase of about 10 to 20 times for gold.

  In the fluorescent dye, electrons in the molecule are excited by light absorption, move to the first electron excited state in a short time, and when returning from this state (level) to the ground state, the wavelength of the wavelength corresponding to the energy difference Fluoresce.

  As the “laser light”, for example, an LD having a wavelength of 200 to 900 nm, an LD having a wavelength of 0.001 to 1,000 mW, a wavelength of 230 to 800 nm (a resonance wavelength is determined by the metal species used for the metal thin film), and a semiconductor having a wavelength of 0.01 to 100 mW. A laser etc. are mentioned.

  The “prism” is intended to allow the laser light through various filters to efficiently enter the plasmon excitation sensor, and preferably has the same refractive index as that of the transparent flat substrate 1. In the present invention, various prisms for which total reflection conditions can be set can be selected as appropriate, and therefore, there is no particular limitation on the angle and shape. For example, a 60-degree dispersion prism may be used. Examples of such commercially available prisms include those similar to the above-mentioned commercially available “glass-made transparent flat substrate”.

  Examples of the “optical filter” include a neutral density (ND) filter and a diaphragm lens. The “darkening (ND) filter” (or neutral density filter) is intended to adjust the amount of incident laser light. In particular, when a detector with a narrow dynamic range is used, it is preferable to use it for carrying out a highly accurate measurement.

  The “polarizing filter” is used to make the laser light P-polarized light that efficiently generates surface plasmons.

  “Cut filters” are external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component in various places), plasmon scattering light (excitation light originated from plasmon A filter that removes optical noise such as scattered light generated by the influence of structures or deposits on the surface of the excitation sensor, and autofluorescence of the fluorescent dye (E) or (F), for example, an interference filter, Examples include color filters.

  The “collecting lens” is intended to efficiently collect the fluorescent signal on the detector, and may be an arbitrary condensing system. As a simple condensing system, a commercially available objective lens (for example, manufactured by Nikon Corporation or Olympus Corporation) used in a microscope or the like may be used. The magnification of the objective lens is preferably 10 to 100 times.

  As the “SPFS detection unit”, a photomultiplier (a photomultiplier manufactured by Hamamatsu Photonics Co., Ltd.) is preferable from the viewpoint of ultrahigh sensitivity. Also, although the sensitivity is lower than these, a CCD image sensor capable of multipoint measurement is also suitable because it can be viewed as an image and noise light can be easily removed.

[Step (l)]
The step (l) is a step of calculating the target substance amount, the target virus amount or the target cell amount from the measurement result obtained in the step (k).

  More specifically, in step (l), a calibration curve is created by performing measurement with an analyte having a known concentration, and the amount of analyte in the sample to be measured is measured based on the created calibration curve. It is the process of calculating from.

(Assay signal)
The assay signal can be calculated by the following formula, where the signal measured before step (k) is “blank signal”.
Assay signal = | (assay measurement signal) − (blank signal) |

Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these.
(I) In the case of a fusion protein-containing assembly containing a fluorescent dye [Example 1]
(Execution of production method (i) of fusion protein (I) -containing assembly containing fluorescent dye)
As the step (a), anti-alpha fetoprotein (AFP) monoclonal antibody 1D5 and anti-AFP monoclonal antibody 6D2 (2.5 mg / mL, manufactured by Nippon Medical Laboratory) were added to “Monoclonal Antibody Labeling Kit” (Molecular Probe). Alexa Fluor (registered trademark) 647 was immobilized respectively.

  As the step (b), the fluorescence-labeled antibodies 1D5 and 6D2 obtained in the above step (a) were biotinylated using “EZ-Link Maleimide-PEO Solid Phase Biotinylation Kit” (manufactured by PIERCE).

  As step (c), the fluorescently labeled biotinylated antibodies 1D5 and 6D2 obtained in step (b) above and avidin are mixed so that the molar ratio of 1D5: 6D2: avidin is 2: 2: 1. As a result, a fusion protein (I) -containing assembly was produced.

[Example 2]
(Method for producing fusion protein (II) -containing assembly (iv))
As a step (d), Alexa Fluor (registered trademark) 647 was immobilized on each of anti-AFP monoclonal antibody 1D5 and anti-AFP monoclonal antibody 6D2 using “Monoclonal Antibody Labeling Kit” (Molecular Probe).

  As the step (f), the fluorescence-labeled antibody 1D5, the fluorescence-labeled antibody 6D2, and the protein A / G obtained in the step (d) are mixed at a molar ratio of 1D5: 6D2: protein A / G of 1: 1: By mixing so as to be 1, a fusion protein (I) -containing assembly was produced.

[Production Example 1]
(Preparation of Alexa Fluor (registered trademark) 647-labeled secondary antibody)
Alexa Fluor (registered trademark) 647 was immobilized on the anti-AFP monoclonal antibody 6D2 using “Monoclonal Antibody Labeling Kit” (Molecular Probe).

[Example 3]
(Production of plasmon excitation sensor)
A glass transparent flat substrate (“S-LAL 10” manufactured by OHARA INC.) Having a refractive index [nd] of 1.72 and a thickness of 1 mm is plasma-cleaned, and a chromium thin film is formed on one surface of the substrate by sputtering. After that, a gold thin film was further formed on the surface by a sputtering method. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.

A spacer layer made of silicon dioxide (SiO ₂ ) as a dielectric was formed by sputtering on one side of the gold thin film that was not in contact with the chromium thin film. The spacer layer had a thickness of 15 nm.

(Implementation of assay method)
As a step (g), a sample containing 100 μL of the fusion protein (I) -containing assembly (1 mg / mL) produced in Example 1 and 800 μL of AFP (prepared as a 1 ng / mL TBS solution) as a target antigen For 25 minutes.

  As the step (h), the reaction product obtained through the above step (g) is added as a filter to “Vector Spin Micro (pore size: 0.02 μm)” manufactured by Whatman Co., Ltd. until the solution on the filter disappears. Aggregates were isolated by centrifugation. Thereafter, as a washing step, 1,000 μL of TBS containing 0.05% by mass of Tween 20 was added to the filter, and the aggregate was washed again by centrifuging until the solution on the filter disappeared.

  As the step (i), the aggregate trapped on the filter through the above step (h) is resuspended in 50 μL of PBS, and the resulting solution is the plasma obtained in the above (production of plasma excitation sensor). It contacted by sending liquid to the surface of the excitation sensor.

  As the step (j), the prism (SIGMA KOKI Co., Ltd. product) is applied to the plasmon excitation sensor obtained in the step (i) from the other surface of the glass transparent flat substrate on which the gold thin film is not formed. ) Was irradiated with laser light (633 nm, 10 μW), and the signal value when the amount of fluorescence emitted from the excited fluorescent dye was observed from the CCD was measured and used as an “assay measurement signal”.

  The SPFS measurement signal when AFP was 0 ng / mL was defined as “blank signal”.

As the step (k), the assay signal was evaluated by the following formula from the measurement result obtained in the step (j).
Assay signal = | (assay measurement signal) − (blank signal) |
Table 4 shows the obtained results.

[Example 4]
(Production of plasmon excitation sensor)
A plasmon excitation sensor was produced in the same manner as in Example 3.

(Implementation of assay method)
The assay method was performed in the same manner as in Example 3 except that 100 μL of the fusion protein (II) -containing assembly solution (1 mg / mL) obtained in Example 2 was used. Table 4 shows the obtained results.

[Comparative Example 1]
(Production of plasmon excitation sensor)
A glass transparent flat substrate (S-LAL 10 manufactured by OHARA INC.) Having a refractive index [nd] of 1.72 and a thickness of 1 mm is plasma-cleaned, and a chromium thin film is formed on one surface of the substrate by sputtering. A gold thin film was further formed on the surface by sputtering. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.

  The substrate thus obtained is immersed in an ethanol solution containing 1 mM of 10-carboxy-1-decanethiol for 24 hours or more to form a SAM (Self Assembled Monolayer) on one surface of a gold thin film. did. The substrate was removed from the solution, washed with ethanol and isopropanol, and then dried with an air gun.

  A polydimethylsiloxane (PDMS) sheet having a flow path height of 0.5 mm is provided on the surface of the SAM, and the substrate is disposed so that the SAM surface is inside the flow path (however, the silicon rubber spacer is used for liquid feeding). The pressure-sensitive adhesive sheet was pressed from the outside of the flow path, and the flow path sheet and the plasmon excitation sensor were fixed with screws.

(Implementation of assay method)
The obtained plasmon excitation sensor is fixed to the flow path, and ultrapure water is supplied as a liquid for 10 minutes, and then MES (25 ° C., pH 5.0) is supplied for 20 minutes by a peristaltic pump at room temperature (25 ° C.), a flow rate of 500 μL / min. And equilibrated the surface.

  Subsequently, 5 mL of MES (25 ° C., pH 5.0) containing 25 mg / mL of N-hydroxysuccinimide (NHS) and 25 mg / mL of water-soluble carbodiimide (WSC) was supplied and circulated for 20 minutes. After the solution was added, 30 mL of an Acetate solution (25 ° C., pH 6.0) containing 30 ml of anti-α-fetoprotein (AFP) monoclonal antibody (1D5, 2.5 mg / mL, manufactured by Japan Medical Laboratory) was added. By circulating the solution for 1 minute, the primary antibody was immobilized on the SAM. Subsequently, 5 mL of 50 mM Tris (25 ° C., pH 7.4) was circulated for 15 minutes to block unreacted activated ester groups, and the PBS buffered saline containing 1% by mass bovine serum albumin (BSA) was used. Then, non-specific adsorption prevention treatment was performed by circulating the solution for 30 minutes.

  Instead of PBS, 0.8 mL of a PBS solution containing 1 ng / mL of AFP was added and circulated for 25 minutes.

  Washing was carried out by circulating TBS containing 0.05% by mass of Tween 20 for 5 minutes as a solution.

  2.5 mL of the secondary antibody (PBS solution prepared to be 1,000 ng / mL) labeled with Alexa Fluor (registered trademark) 647 obtained in Production Example 1 was added and circulated for 20 minutes.

  Then, it was cleaned by circulating TBS containing 0.05% by mass of Tween 20 for 10 minutes.

  The signal value observed from the CCD was measured and used as an assay measurement signal. The SPFS measurement signal when AFP was 0 ng / mL was used as a blank signal. The assay was evaluated by calculating the same assay signal as in Example 1.

  Table 4 shows the measurement results of the assay method of the present invention.

From Table 4, the assay method of the present invention is extremely high in comparison with the conventional heterogeneous SPFS measurement of Comparative Example 1 by applying the homogeneous assay system using the assembly of the present invention to SPFS measurement. It was found that sensitive measurement is possible.
(II) In the case of a fusion protein-containing assembly containing an enzyme [Example 1]
(Execution of production method (i) of assembly containing enzyme-containing fusion protein (I))
As the step (a), anti-α-fetoprotein (AFP) monoclonal antibody 1D5 and anti-AFP monoclonal antibody 6D2 (2.5 mg / mL, manufactured by Japan Medical Laboratory) were added to “Alkaline Phosphatase Labeling Kit” ((stock) Enzyme-labeled antibodies 1D5 and 6D2 were produced by immobilizing alkaline phosphatase using Dojindo Laboratories).

  As the step (b), the enzyme-labeled antibodies 1D5 and 6D2 obtained in the above step (a) are biotinylated using “EZ-Link Maleimide-PEO Solid Phase Biotinylation Kit” (manufactured by PIERCE). Labeled biotinylated antibodies 1D5 and 6D2 were produced.

  As step (c), the enzyme-labeled biotinylated antibodies 1D5 and 6D2 obtained in step (b) above and avidin are mixed so that the molar ratio of 1D5: 6D2: avidin is 2: 2: 1. As a result, a fusion protein (I) -containing assembly was produced.

[Example 2]
(Method for producing fusion protein (II) -containing assembly (iv))
As a step (d), by immobilizing alkaline phosphatase to each of anti-AFP monoclonal antibody 1D5 and anti-AFP monoclonal antibody 6D2, using “Alkaline Phosphatase Labeling Kit” (manufactured by Dojindo Laboratories) Labeled antibodies 1D5 and 6D2 were produced.

  As the step (f), the enzyme-labeled antibody 1D5, the enzyme-labeled antibody 6D2, and the protein A / G obtained in the step (d) are mixed at a molar ratio of 1D5: 6D2: protein A / G of 1: 1: By mixing so as to be 1, a fusion protein (II) -containing assembly was produced.

[Production Example 1]
(Preparation of Alexa Fluor (registered trademark) 647-labeled secondary antibody)
Alexa Fluor (registered trademark) 647 was immobilized on the anti-AFP monoclonal antibody 6D2 using “Monoclonal Antibody Labeling Kit” (Molecular Probe).

[Example 3]
(Production of plasmon excitation sensor)
A glass transparent flat substrate (“S-LAL 10” manufactured by OHARA INC.) Having a refractive index [nd] of 1.72 and a thickness of 1 mm is plasma-cleaned, and a chromium thin film is formed on one surface of the substrate by sputtering. After that, a gold thin film was further formed on the surface by a sputtering method. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.

A spacer layer made of silicon dioxide (SiO ₂ ) as a dielectric was formed by sputtering on one side of the gold thin film that was not in contact with the chromium thin film. The spacer layer had a thickness of 15 nm.

(Implementation of assay method)
As a step (g), a sample containing 100 μL of the fusion protein (I) -containing assembly (1 mg / mL) produced in Example 1 and 800 μL of AFP (prepared as a 1 ng / mL TBS solution) as a target antigen For 25 minutes.

As the step (h), the reaction product obtained through the above step (g) is added as a filter to “Vector Spin Micro (pore size: 0.02 μm)” manufactured by Whatman Co., Ltd. until the solution on the filter disappears. Aggregates were isolated by centrifugation. after that,
As a washing step, 1,000 μL of TBS containing 0.05% by mass of Tween 20 was added to the filter, and the aggregate was washed by centrifuging again until there was no solution on the filter.

  In step (i), the aggregate trapped on the filter through (h) above is used as a fluorescent substrate (1,3-dicilo-9, dimethyl-acid-2-one-7-yl phosphate (DDAO). Phosphorate solution (manufactured by Molecular Probes) (50 μL) was reacted to produce DDAO as the fluorescent dye (E) on the filter.

  As the step (j), the solution obtained on the filter through the step (i) was brought into contact with the surface of the plasma excitation sensor obtained in the above (production of the plasma excitation sensor).

  As the step (k), the prism (SIGMA KOKI Co., Ltd. product) is applied to the plasmon excitation sensor obtained in the step (j) from the other surface of the glass transparent flat substrate on which the gold thin film is not formed. ) Was irradiated with laser light (633 nm, 10 μW), and the signal value when the amount of fluorescence emitted from the excited fluorescent dye (E) was observed from the CCD was measured and used as an “assay measurement signal”.

  The SPFS measurement signal when AFP was 0 ng / mL was defined as “blank signal”.

As step (l), the assay signal was evaluated by the following formula from the measurement result obtained in step (k).
Assay signal = | (assay measurement signal) − (blank signal) |
The results obtained are shown in Table 5.

[Example 4]
(Production of plasmon excitation sensor)
A plasmon excitation sensor was produced in the same manner as in Example 3.

(Implementation of assay method)
The assay method was performed in the same manner as in Example 3 except that 100 μL of the fusion protein (II) -containing assembly solution (1 mg / mL) obtained in Example 2 was used. The results obtained are shown in Table 5.

[Comparative Example 1]
(Production of plasmon excitation sensor)
A glass transparent flat substrate (S-LAL 10 manufactured by OHARA INC.) Having a refractive index [nd] of 1.72 and a thickness of 1 mm is plasma-cleaned, and a chromium thin film is formed on one surface of the substrate by sputtering. A gold thin film was further formed on the surface by sputtering. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.

  The substrate thus obtained is immersed in an ethanol solution containing 1 mM of 10-carboxy-1-decanethiol for 24 hours or more to form a SAM (Self Assembled Monolayer) on one surface of a gold thin film. did. The substrate was removed from the solution, washed with ethanol and isopropanol, and then dried with an air gun.

  A polydimethylsiloxane (PDMS) sheet having a flow path height of 0.5 mm is provided on the surface of the SAM, and the substrate is disposed so that the SAM surface is inside the flow path (however, the silicon rubber spacer is used for liquid feeding). The pressure-sensitive adhesive sheet was pressed from the outside of the flow path, and the flow path sheet and the plasmon excitation sensor were fixed with screws.

(Implementation of assay method)
The obtained plasmon excitation sensor is fixed to the flow path, and ultrapure water is supplied as a liquid for 10 minutes, and then MES (25 ° C., pH 5.0) is supplied for 20 minutes by a peristaltic pump at room temperature (25 ° C.), a flow rate of 500 μL / min. And equilibrated the surface.

  Subsequently, 5 mL of MES (25 ° C., pH 5.0) containing 25 mg / mL of N-hydroxysuccinimide (NHS) and 25 mg / mL of water-soluble carbodiimide (WSC) was supplied and circulated for 20 minutes. After the solution was added, 30 mL of an Acetate solution (25 ° C., pH 6.0) containing 30 ml of anti-α-fetoprotein (AFP) monoclonal antibody (1D5, 2.5 mg / mL, manufactured by Japan Medical Laboratory) was added. By circulating the solution for 1 minute, the primary antibody was immobilized on the SAM. Subsequently, 5 mL of 50 mM Tris (25 ° C., pH 7.4) was circulated for 15 minutes to block unreacted activated ester groups, and the PBS buffered saline containing 1% by mass bovine serum albumin (BSA) was used. Then, non-specific adsorption prevention treatment was performed by circulating the solution for 30 minutes.

  Instead of PBS, 0.8 mL of a PBS solution containing 1 ng / mL of AFP was added and circulated for 25 minutes.

  Washing was carried out by circulating TBS containing 0.05% by mass of Tween 20 for 5 minutes as a solution.

  0.8 mL of the secondary antibody (PBS solution prepared to be 1,000 ng / mL) labeled with Alexa Fluor (registered trademark) 647 obtained in Production Example 1 was added and circulated for 20 minutes.

  Then, it was cleaned by circulating TBS containing 0.05% by mass of Tween 20 for 10 minutes.

  The signal value observed from the CCD was measured and used as an assay measurement signal. The SPFS measurement signal when AFP was 0 ng / mL was used as a blank signal. The assay was evaluated by calculating the same assay signal as in Example 1.

  Table 5 shows the measurement results of the assay method of the present invention.

  From Table 5, the assay method of the present invention is an extremely high number of orders of magnitude compared with the conventional heterogeneous SPFS measurement of Comparative Example 1 by applying the homogeneous assay system using the assembly of the present invention to the SPFS measurement. It was found that sensitive measurement is possible.

  When the fusion protein-containing assembly of the present invention is used in the assay method of the present invention, it is a highly sensitive and highly accurate method with dramatically improved rapidity. Thus, the presence of a preclinical non-invasive cancer (carcinoma in situ) that cannot be detected by palpation or the like can be predicted with high accuracy.

1 Protein that specifically associates with one or more sites α of target substance, target virus or target cell (A)
2 Protein (B) that specifically associates with one or more sites α of the target substance, target virus or target cell
3a fluorescent dye 3b enzyme 4 biotin 5 avidin 6 protein A / G
7 Target substance, target virus or target cell 8 Filter 9 Transparent flat substrate, metal thin film formed on one surface of the substrate, and formed on the other surface of the metal thin film not in contact with the substrate Plasmon excitation sensor having at least a spacer layer made of a dielectric material 10 Fusion protein (I)
11 Fusion protein (II)
12 Fusion protein (II)
13 Fluorescent substrate 14 Fluorescent dye (E)
20 aggregate 21 aggregate

Claims (16)

Protein (A) that specifically associates with one or more sites α of target substance, target virus or target cell, and specifically associates with one or more sites β of target substance, target virus or target cell having the site α Or at least a fusion protein comprising two or more proteins (B) and a fluorescent dye or enzyme, or
A fusion protein comprising a total of two or more proteins (A) that specifically associate with one or more site α possessed by a target substance, target virus or target cell, and a fluorescent dye or enzyme, and a target material having the site α, A fusion protein-containing assembly comprising at least a total of two or more proteins (B) that specifically associate with one or more sites β of a target virus or target cell and a fusion protein containing a fluorescent dye or an enzyme body.   Biotin is immobilized on each of the proteins (A) and (B), and a fluorescent dye or enzyme is immobilized on at least one of the protein (A), protein (B), biotin, and avidin, and the fusion protein 2. The fusion protein-containing assembly according to claim 1, comprising a total of at least one of the proteins (A) and (B) and one avidin. The fusion protein-containing assembly according to claim 1 or 2, wherein each of the proteins (A) and (B) is an antibody, a Fab fragment, a Fab 'fragment or a F (ab') ₂ fragment. .   The proteins (A) and (B) are both antibodies, and the fluorescent dye or enzyme is immobilized only on the antibody, protein A, protein G or protein A / G alone, Alternatively, the antibody is immobilized on protein A, protein G or protein A / G, and the fusion protein comprises at least one of the proteins (A) and (B) in total 2 to 5, protein A, protein The fusion protein-containing assembly according to any one of claims 1 to 3, comprising G or protein A / G.   The fusion protein-containing assembly according to any one of claims 1 to 4, wherein the sites α and β are the same.   The fusion protein-containing assembly according to any one of claims 1 to 5, wherein the enzyme is alkaline phosphatase, β-galactosidase, β-glucosidase, or glucose oxidase. A method for producing a fusion protein-containing assembly comprising the following steps (a), (b) and (c):
Step (a): a protein (A) that specifically associates with one or more sites α of the target substance, target virus, or target cell, and one or more sites β of the target substance, target virus, or target cell having the site α Immobilizing a fluorescent dye or enzyme on at least one of a protein consisting of a protein (B) that specifically associates with biotin, biotin and avidin;
Step (b): Biotin by immobilizing biotin to which the fluorescent dye or enzyme may be immobilized on each of the proteins (A) and (B) to which the fluorescent dye or enzyme may be immobilized. Steps for obtaining modified proteins (A) and (B) and step (c): mixing so as to contain one or more biotinylated proteins (A) and (B) to which fluorescent dyes or enzymes may be immobilized, respectively. And
By further mixing the biotinylated protein (A) and (B) with avidin in which a fluorescent dye or an enzyme may be immobilized so as to have a molar ratio of 4: 1 or more, Obtaining a fusion protein-containing assembly. The method for producing a fusion protein-containing assembly according to claim 7, wherein each of the proteins (A) and (B) is an antibody, a Fab fragment, a Fab 'fragment, or a F (ab') ₂ fragment. The method for producing a fusion protein-containing assembly according to claim 7 or 8, comprising at least one of the following steps (d) and (e) and (f).
Step (d): antibody (A) that specifically associates with one or more sites α of the target substance, target virus or target cell, and one or more sites β of the target substance, target virus or target cell having the site α Immobilizing a fluorescent dye or an enzyme on each of the antibodies (B) that specifically associate with
Step (e): Protein A, Protein G or Protein A / G is immobilized with a fluorescent dye or enzyme, and Step (f): The fluorescent dye or enzyme may be immobilized with the antibody (A) and ( B) are mixed so that each contains at least one, and the total of the antibodies (A) and (B) may be combined with protein A, protein G or protein A / G (However, when the fluorescent dye or enzyme is not immobilized on the antibodies (A) and (B), the fluorescent dye or enzyme is immobilized on the protein A, protein G, or protein A / G used.) Are further mixed so that the molar ratio is 2: 1 or more to obtain an aggregate.   The said site | part (alpha) and (beta) are the same, The manufacturing method of the fusion protein containing assembly as described in any one of Claim 7- Claim 9 characterized by the above-mentioned.   The said enzyme is alkaline phosphatase, (beta) -galactosidase, (beta) -glucosidase, or glucose oxidase, The manufacturing method of the fusion protein containing assembly as described in any one of Claim 7-10 characterized by the above-mentioned. An assay method comprising the following steps (g) to (l);
Step (g): by bringing the fusion protein-containing assembly according to any one of claims 1 to 6 into contact with the specimen, the target substance, target virus or target cell contained in the specimen is brought into contact with the specimen. Forming an aggregate via
Step (h): a step of isolating only the aggregate by passing the reaction product obtained through the step (g) through a filter,
Step (i): a step of reacting the aggregate obtained through the step (h) with a fluorescent substrate or a quencher substrate to produce a fluorescent dye (E) or a quencher, respectively (however, the above step (g) , Only when a fusion protein-containing assembly comprising the enzyme according to any one of claims 1 to 6 is used).
Step (j): Spacer layer comprising a transparent flat substrate, a metal thin film formed on one surface of the substrate, and a dielectric formed on the other surface of the metal thin film not in contact with the substrate Or the surface of the spacer layer or the surface of the fluorescent dye (F) layer of a plasmon excitation sensor having at least a fluorescent dye (F) layer formed on the other surface not in contact with the metal thin film. A step of contacting the aggregate obtained through (h) or the fluorescent dye (E) obtained through the above step (i) or a quencher;
Step (k): The plasmon excitation sensor obtained in step (j) was excited by being irradiated with laser light from the other surface of the substrate on which the thin film was not formed via a prism. A step of measuring the amount of fluorescence emitted from the fluorescent dye, and a step (l): a step of calculating the amount of the target substance, the amount of the target virus or the amount of the target cell from the measurement result obtained in the step (k) .   The assay method according to claim 12, wherein the immunoreaction field in steps (g) and (h) and the SPFS detection field in steps (j) and (k) are independent of each other.   The assay method according to claim 12 or 13, wherein the metal thin film is formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper and platinum.   The assay method according to claim 14, wherein the metal is gold. The assay method according to any one of claims 12 to 15, wherein the dielectric material includes silicon dioxide (SiO ₄ ) or titanium dioxide (TiO ₂ ).