WO2010125932A1 - Ensemble contenant une protéine de fusion, procédé de production de l'ensemble, et procédé de dosage au moyen de l'ensemble - Google Patents

Ensemble contenant une protéine de fusion, procédé de production de l'ensemble, et procédé de dosage au moyen de l'ensemble Download PDF

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WO2010125932A1
WO2010125932A1 PCT/JP2010/056843 JP2010056843W WO2010125932A1 WO 2010125932 A1 WO2010125932 A1 WO 2010125932A1 JP 2010056843 W JP2010056843 W JP 2010056843W WO 2010125932 A1 WO2010125932 A1 WO 2010125932A1
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protein
fluorescent dye
enzyme
fusion protein
target
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PCT/JP2010/056843
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English (en)
Japanese (ja)
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法明 山本
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コニカミノルタホールディングス株式会社
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Priority to JP2011511368A priority Critical patent/JP5660035B2/ja
Publication of WO2010125932A1 publication Critical patent/WO2010125932A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

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  • the present invention relates to an aggregate mainly composed of proteins, a production method thereof, and an assay method using the aggregate. 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).
  • SPFS surface plasmon excitation enhanced fluorescence spectroscopy
  • 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.
  • ATR total reflection
  • the amount of photons in the laser beam is increased to several tens to several hundreds times (the 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.
  • 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.
  • Patent Document 1 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.
  • Patent Document 2 discloses a method in which an antibody against an antigen held by blood cells or the like is used to agglutinate the blood cells, trap them with a filter, and detect them.
  • Patent Document 2 is a method of aggregating relatively large cells such as blood cells in which the target substance holds 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.
  • 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.
  • the present inventors have found that a protein (A) specific to the site ⁇ of the analyte and a protein (B) specific to the site ⁇ of the analyte having the site ⁇ . ) 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.
  • 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 ⁇ possessed by a target substance, target virus or target cell, and a fluorescent dye or enzyme, and a target material having the site ⁇
  • a fusion protein-containing assembly comprising at least a total of two or more proteins (B) that specifically associate with one or more sites ⁇ 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
  • each of the proteins (A) and (B) is an antibody, a Fab fragment, a Fab ′ fragment or a F (ab ′) 2 fragment. body.
  • 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 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.
  • fusion protein-containing assembly according to any one of items 1 to 5, wherein the enzyme is alkaline phosphatase, ⁇ -galactosidase, ⁇ -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 ⁇ of the target substance, target virus, or target cell, and one or more sites ⁇ of the target substance, target virus, or target cell having the site ⁇ 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.
  • each of the proteins (A) and (B) is an antibody, a Fab fragment, a Fab ′ fragment, or a F (ab ′) 2 fragment.
  • 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).
  • Item 10 The method for producing a fusion protein-containing assembly according to any one of Items 7 to 10 above, wherein the enzyme is alkaline phosphatase, ⁇ -galactosidase, ⁇ -glucosidase, or glucose oxidase. .
  • 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).
  • the metal thin film is formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum.
  • 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 ).
  • the assembly of the present invention 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.
  • FIG. 1 Schematic diagram of an embodiment of a fusion protein (I) containing a fluorescent dye
  • FIG. 1 Schematic diagram of one embodiment of fusion protein (I) containing an enzyme
  • FIG. 1 Schematic diagram of one embodiment of a fusion protein (II) containing a fluorescent dye
  • FIG. 1 Schematic diagram of an embodiment of a fusion protein (I) containing an enzyme
  • FIG. 1 Schematic diagram of an embodiment of a fusion protein (I) containing an enzyme
  • the fluorescent substrate 13 is hydrolyzed by the enzyme 3b fix
  • the fusion protein-containing assembly of the present invention comprises a protein (A) that specifically associates with one or more sites ⁇ possessed by a target substance, target virus, or target cell, and a target substance, target virus, or target cell having the site ⁇ .
  • a fusion protein comprising a total of two or more proteins (B) that specifically associate with one or more sites ⁇ 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 ⁇ possessed by a target substance, target virus or target cell, and a fluorescent dye or enzyme, and a target material having the site ⁇ , It is characterized by containing at least a total of two or more proteins (B) that specifically associate with one or more sites ⁇ of the target virus or target cell and a fusion protein containing a fluorescent dye or an enzyme.
  • 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 ⁇ , 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 ⁇ of a target cell and a fluorescent dye or enzyme, wherein the protein (A) And (B) each having one or more.
  • 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.
  • 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.
  • the antibody and protein A, protein G, or protein A / G are immobilized, 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 parts ⁇ and ⁇ may be the same.
  • the enzyme is preferably alkaline phosphatase, ⁇ -galactosidase, ⁇ -glucosidase or glucose oxidase.
  • the method for producing a fusion protein-containing assembly of the present invention includes the steps (a), (b) and (c).
  • each of the proteins (A) and (B) is preferably an antibody, a Fab fragment, a Fab ′ fragment or a F (ab ′) 2 fragment.
  • the method for producing a fusion protein-containing assembly of the present invention is characterized by including at least one of steps (d) and (e) and (f).
  • the sites ⁇ and ⁇ may be the same.
  • the enzyme is preferably alkaline phosphatase, ⁇ -galactosidase, ⁇ -glucosidase or glucose oxidase.
  • the assay method of the present invention is characterized by including the following steps (g) to (l).
  • the immune reaction field in steps (g) and (h) and the SPFS detection field in steps (j) and (k) are independent of 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 ).
  • the fusion protein-containing assembly of the present invention comprises a protein (A) that specifically associates with one or more sites ⁇ possessed by a target substance, target virus, or target cell, and a target substance, target virus, or target cell having the site ⁇ .
  • At least a fusion protein comprising a total of two or more proteins (B) that specifically associate with one or more sites ⁇ 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 ⁇ possessed by a target substance, target virus or target cell, and a fluorescent dye or enzyme, and a target material having the site ⁇ , It is characterized by containing at least a total of two or more proteins (B) that specifically associate with one or more sites ⁇ of the target virus or target cell and a fusion protein containing a fluorescent dye or an enzyme.
  • the aggregate of the present invention includes “a protein (A) that specifically associates with one or more sites ⁇ 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 ⁇ of target cells and “fluorescent dye” or “enzyme” And having one or more of each of the proteins (A) and (B).
  • 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.
  • 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, more preferably 100 nM or more, particularly preferably 1 ⁇ M 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, still 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 total number contained in the aggregate of the protein (A) and the protein (B) is constant.
  • each of “target substance”, “target virus” and “target cell” 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 single substance or a compound) or a virus, or may be a single-celled or multi-cellular organism, and is not particularly limited Absent.
  • target substance examples 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 ( ⁇ -fetoprotein), a tumor marker, a signaling substance, a hormone, or the like, and is not particularly limited.
  • AFP ⁇ -fetoprotein
  • target virus examples 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.
  • viruses that infect animals such as influenza virus, hepatitis virus, human immunodeficiency virus (HIV), viruses that infect plants, or bacteriophages that infect bacteria.
  • HIV human immunodeficiency virus
  • 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.
  • the target substance, target virus, or target cell can specifically associate with (combine with) the protein (that is, one or more sites ⁇ and the sites). is one or more sites ⁇ that are completely different from ⁇ .) or the sites ⁇ and ⁇ are the same. Note that “same” includes not only completely identical but also partially identical (partially overlapping).
  • protein (A) specifically associated with one or more sites ⁇ 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 ⁇ , 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.
  • protein includes polypeptides and oligopeptides.
  • a protein (B) that specifically associates with one or more sites ⁇ of the target substance, target virus, or target cell having the site ⁇ (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 ⁇ and one or more sites ⁇ and specifically associates (bonds) via the sites ⁇ . It is not limited, and specific examples include the same kind as the protein (A).
  • 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 type of 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, which will be described later, and may be any known fluorescent dye.
  • 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.
  • fluorescent dyes examples 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 Co., Ltd.), Coumarin family fluorescent dye (Invitrogen Corp.), Rhodamine family fluorescent dye (GE Healthcare Bioscience Co., Ltd.), Cyanine family fluorescent dye, Indocarbocyanine family fluorescent dyes, oxazine family fluorescent dyes, thiazine family fluorescent dyes, squaraine family fluorescent dyes, chelated lanthanide dyes Millie's fluorescent dye, BODIPY (registered trademark) family fluorescent dye (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
  • 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.
  • rare earth complex fluorescent dyes such as Eu and Tb can also be used as fluorescent dyes in the present invention.
  • 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.
  • a commercially available rare earth complex fluorescent dye is ATBTA-Eu 3+ .
  • BFP blue fluorescent protein
  • CFP cyan fluorescent protein
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • DsRed red fluorescent protein
  • APC allophycocyanin
  • Fluorescent fine particles such as latex protein and latex that can be used as the fluorescent dye in the present invention.
  • 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.
  • 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.
  • a fluorescent dye having a maximum fluorescence wavelength of 400 nm or more it is desirable to use.
  • These fluorescent dyes may be used alone or in combination of two or more.
  • An enzyme (D) that catalyzes a reaction to be converted is used.
  • C examples include alkaline phosphatase (ALP), peroxidase (POD), galactosidase (GAL) and the like.
  • ALP alkaline phosphatase
  • POD peroxidase
  • GAL galactosidase
  • 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”.
  • those used for the enzyme reaction (D-1) include, for example, ⁇ -galactosidase ( ⁇ GAL), ⁇ -glucosidase ( ⁇ Glu), alkaline phosphatase (ALP), and the like.
  • examples of those used in D-2) include glucose oxidase (GOD).
  • fluorescent substrate “fluorescent dye (E)”, “quenching agent substrate” and “quenching agent” will be described later.
  • 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.
  • 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.
  • 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.
  • the antibody which fusion protein (I) 10 shown in FIG.1 and FIG.2 has two proteins (A) 1 and two proteins (B) 2,
  • this invention is limited to this aspect.
  • an embodiment having only four proteins (A) is also included.
  • avidin includes streptavidin and neutravidin.
  • 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.
  • 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
  • protein G or protein A / G may be used in addition to protein A.
  • an association which may be a protein (A) and protein (B) is a total of 2 to a 5.
  • And is intended to include.
  • 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.
  • protein A and protein G may be natural or recombinant.
  • the fusion protein having a larger total number of proteins (A) and proteins (B) (that is, a higher concentration) is preferable.
  • 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).
  • 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 an antibody (immunoglobulin), Fab fragment, Fab ′ fragment or F (ab ′) fragment.
  • Step (a) includes “a protein (A) that specifically associates with one or more sites ⁇ of target substance, target virus or target cell” (protein (A)), and “target substance having the site ⁇ .
  • 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 ⁇ of a target virus or target cell, protein (B) Is a step of immobilizing.
  • 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.
  • 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.
  • 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 ".
  • the step (b) is a “protein (A) that specifically associates with one or more sites ⁇ 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 ⁇ of the target substance, target virus or target cell having the site ⁇ ”
  • 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.
  • 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 is 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.
  • Examples of a method for immobilizing biotin on a protein include an amine coupling method, a thiol coupling method, and an aldehyde coupling method.
  • an enzyme is immobilized on either a protein or biotin.
  • the present invention is not limited to these methods.
  • proteins (A) and (B) are antibodies
  • biotin is preferably immobilized on constant regions (C H 1 and C L ), more preferably Fc fragments
  • 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 ).
  • biotinylated protein (A) and the protein (B) immobilized with biotin are also referred to as “biotinylated protein (A)” and “biotinylated protein (B)”, respectively.
  • 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.
  • 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, even 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.
  • the aggregate can be stably stabilized. It is preferable because it can be formed.
  • 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.
  • 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.
  • Step (d) includes “an antibody (A) that specifically associates with one or more sites ⁇ possessed by the target substance, target virus or target cell” and “target substance, target virus or target cell having the site ⁇ ”.
  • a “fluorescent dye” or an “enzyme” is immobilized on each of the “antibody (B) specifically associated with one or more sites ⁇ ”.
  • the method for immobilizing the fluorescent dye or enzyme to each of the antibodies (A) and (B) is the same as the method for immobilizing a protein described in the above 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.
  • 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”.
  • 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)
  • 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.
  • the step (f) includes at least one antibody (A) and (B), preferably 9: 1 to 1: 9, more preferably 8: 2 to 2: 8, and 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).
  • 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.
  • 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.
  • 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).
  • 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) 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.
  • the assay method of the present invention comprises the following steps (g) to (l).
  • 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.
  • samples 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.
  • the temperature is usually 4 to 50 ° C., preferably 10 to 40 ° C.
  • the time is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.
  • 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) is a step of isolating only the aggregate by passing the “reactant” obtained through the step (g) through a “filter”.
  • the “reactant” is obtained through the above step (g).
  • the specimen is the target cell.
  • 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 (II) was associated with 12 to form an aggregate 21, so that only the target cells were specifically removed from the specimen.
  • 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.
  • a commercially available product for example, “Anopore, pore size: 0.02 ( ⁇ m)” 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.
  • 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 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 to 8 can be used.
  • the metal thin film included in the following “plasmon excitation sensor” is formed of a metal including gold, No. 2 in Table 2 from the viewpoint of gold transmittance and excitation wavelength. 7, 1,3-diculoro-9,9-dimethyl-acid-2-one-7-yl phosphate (DDAO phosphate) (manufactured by 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) 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 it does not receive, there is no restriction
  • Table 2 shows examples of fluorescent substrates used in the present invention.
  • ⁇ GAL ⁇ -galactosidase
  • ⁇ -glucosidase catalyzes the reaction of desorbing ⁇ Glu from TG- ⁇ Glu, which is a “quenching substrate”, and generating TG, which is a “quenching agent”.
  • free TG is a fluorescent dye (F) because it causes an excitation wavelength of 490 nm and a fluorescence dye (F) having a fluorescence wavelength of 475 to 495 nm and Fluorescence Resonance Energy Transfer (FRET).
  • Fluorescence such as fluorescence of terbium (Tb) chelate (fluorescence wavelength: 495 nm) or enhanced cyan fluorescent protein (Enhanced Cyan Fluorescence Protein; ECFP) (fluorescence wavelength: 475 nm) can be quenched.
  • TG is 2-Me TG represented by the following formula
  • Alkaline phosphatase catalyzes a reaction that hydrolyzes the “Quencher Substrate” AttoPhos® substrate, and BBT (2 ′-[2-benzthiazoyl] -6′-hydroxyl ”“ Quencher ”. -Benzthiazole).
  • BBT excitation wavelength: 482 nm
  • F fluorescent dye
  • Tb terbium
  • Glucose oxidase which can be used in the “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 of OMe TG or the like can be reduced (that is, quenched).
  • preferred combinations of the quencher substrate, the enzyme, the quencher and the 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 ⁇ g / mL, more preferably 1 to 1,000 ⁇ g / mL.
  • the temperature, time, and flow rate at which the solution is circulated are the same as those in the above step (g).
  • 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
  • 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).
  • 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”.
  • 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.
  • 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].
  • the size (length ⁇ width) is not particularly limited as long as the thickness is preferably 1.40 to 2.20 and 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 preferable from the viewpoint of optical properties and detergency.
  • the transparent flat substrate is preferably cleaned with acid and / or plasma before forming metal protrusions on the surface.
  • the cleaning treatment with 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.
  • a plasma dry cleaner (“PDC200” manufactured by Yamato Scientific Co., Ltd.) for 0.1 to 30 minutes.
  • 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.
  • Examples of methods 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.
  • gold 20-70 nm
  • silver 20-70 nm
  • aluminum 10-50 nm
  • copper 20-70 nm
  • platinum 20-70 nm
  • alloys thereof 10-70 nm
  • chromium The thickness of the thin film is more preferably 1 to 3 nm.
  • the thickness of the metal thin film is within the above range because surface plasmons are easily generated. Moreover, if it is a metal thin film which has such thickness, a magnitude
  • 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 is 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 formation method of the spacer layer made of a dielectric material 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.
  • 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.
  • SAM Self-Assembled Monolayer
  • 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.
  • 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.
  • 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.
  • the amount of the fluorescent dye (F) that can be immobilized is large, and the strength of the obtained layer is preferable. .
  • 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.
  • Tb terbium
  • ECFP enhanced cyan fluorescent protein
  • fluorescent dyes (F) have high water solubility.
  • 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.
  • 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.
  • SP solubility parameter
  • These fluorescent dyes (F) may be used alone or in combination of two or more.
  • 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; polyethylene, polyolefin, etc., and silicon rubber, Teflon (registered trademark), polyethylene, polypropylene in the chemical solution delivery part. Etc. are used.
  • the cross section of the flow path of the plasmon excitation sensor part is preferably independently about 100 nm to 1 mm in length and width.
  • 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 the 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 together.
  • a method of fixing with a closing tool such as a screw is preferred.
  • a gold substrate is formed on an integrally molded plastic product or a separately manufactured gold substrate is fixed to the gold surface.
  • 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.
  • PBS phosphate buffered saline
  • TBS Tris buffered saline
  • 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 aggregates during the feeding may be 100 ⁇ g / mL to 0.001 pg / mL.
  • the total amount of the liquid delivery that is, the volume of the flow path is usually 0.001 to 20 mL, preferably 0.1 to 1 mL.
  • the liquid flow rate is usually 1 to 2,000 ⁇ L / min, preferably 5 to 500 ⁇ L / min.
  • 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.
  • 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.
  • the surface plasmon is generated on the surface of the metal thin film by the laser light irradiation under the total reflection attenuation condition (ATR). Due to the electric field enhancement effect of surface plasmons, the fluorescent dye is excited by photons that are increased by several tens to several hundred times the amount of photons irradiated. Note that the amount of photon increase due to 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, but is usually about 10 to 20 times that of gold.
  • the fluorescent dye 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.
  • the resonance wavelength is determined by the metal species used in the metal thin film
  • a semiconductor having a wavelength of 0.01 to 100 mW. A laser etc. are mentioned.
  • the “prism” is intended to allow 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.
  • 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.
  • 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”.
  • optical filter examples include a neutral density (ND) filter and a diaphragm lens.
  • ND neutral density
  • 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 convert the laser light into 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 “condensing lens” is intended to efficiently collect the fluorescent signal on the detector, and may be an arbitrary condensing system.
  • 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.
  • the “SPFS detector” is preferably a photomultiplier (a photomultiplier manufactured by Hamamatsu Photonics) from the viewpoint of ultra-high 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.
  • 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).
  • 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.
  • Example 1 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)
  • a step (a) anti-alpha fetoprotein (AFP) monoclonal antibody 1D5 and anti-AFP monoclonal antibody 6D2 (2.5 mg / mL, manufactured by Japan Medical Laboratory) were added to “Monoclonal Antibody Labeling Kit” (Molecular Probe). Alexa Fluor (registered trademark) 647 was immobilized respectively.
  • AFP anti-alpha fetoprotein
  • the fluorescently 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).
  • 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.
  • a fusion protein (I) -containing assembly was produced.
  • 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).
  • 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.
  • 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.
  • a sample containing 100 ⁇ L of the fusion protein (I) -containing assembly (1 mg / mL) produced in Example 1 and 800 ⁇ L of AFP (prepared as a 1 ng / mL TBS solution) as a target antigen For 25 minutes.
  • step (h) the reaction product obtained through the above step (g) is added as a filter to “Vector Spin Micro (pore size: 0.02 ⁇ m)” manufactured by Whatman Co., Ltd. until the solution on the filter disappears. Aggregates were isolated by centrifugation. Thereafter, as a washing step, 1,000 ⁇ L of TBS containing 0.05% by mass of Tween 20 was added to the filter, and the aggregate was washed again by centrifuging until there was no solution on the filter.
  • Vector Spin Micro pore size: 0.02 ⁇ m
  • the aggregate trapped on the filter through the above step (h) is resuspended in 50 ⁇ L 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.
  • the prism SIGMA KOKI Co., Ltd. product
  • 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.
  • 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 designated as “blank signal”.
  • the assay signal was evaluated by the following formula from the measurement result obtained in the step (j).
  • Assay 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.
  • Examplementation of assay method The assay method was performed in the same manner as in Example 3 except that 100 ⁇ L of the fusion protein (II) -containing assembly solution (1 mg / mL) obtained in Example 2 was used. Table 4 shows the obtained results.
  • the substrate thus obtained is immersed in an ethanol solution containing 1 mM 10-carboxy-1-decanethiol for 24 hours or more to form a SAM (Self Assembled Monolayer) on one side of the 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 arranged 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.
  • 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 ⁇ L / min. And equilibrated the surface.
  • MES 25 ° C., pH 5.0
  • the solution was replaced with PBS, 0.8 mL of a PBS solution containing 1 ng / mL of AFP was added and circulated for 25 minutes.
  • Washing was performed by circulating TBS containing 0.05% by mass of Tween 20 for 5 minutes as a solution.
  • 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.
  • 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.
  • step (a) 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))
  • AFP anti-alpha fetoprotein
  • 6D2 2.5 mg / mL, manufactured by Japan Medical Laboratory
  • “Alkaline Phosphatase Labeling Kit” (stock) Enzyme-labeled antibodies 1D5 and 6D2 were produced by immobilizing alkaline phosphatase using Dojindo Laboratories).
  • 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.
  • 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.
  • a fusion protein (I) -containing assembly was produced.
  • Example 2 Method for producing fusion protein (II) -containing assembly (iv)
  • 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.
  • 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.
  • 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.
  • a sample containing 100 ⁇ L of the fusion protein (I) -containing assembly (1 mg / mL) produced in Example 1 and 800 ⁇ L of AFP (prepared as a 1 ng / mL TBS solution) as a target antigen For 25 minutes.
  • step (h) the reaction product obtained through the above step (g) is added as a filter to “Vector Spin Micro (pore size: 0.02 ⁇ m)” manufactured by Whatman Co., Ltd. until the solution on the filter disappears. Aggregates were isolated by centrifugation. afterwards, As a washing step, 1,000 ⁇ L 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.
  • Vector Spin Micro pore size: 0.02 ⁇ m
  • step (i) the aggregate trapped on the filter through (h) is used as a fluorescent substrate (1,3-dicilo-9,9-dimethyl-acidine-2-one-7-yl phosphate (DDAO).
  • Phosphorate solution (Molecular Probes) (50 ⁇ L) was reacted to produce DDAO as the fluorescent dye (E) on the filter.
  • 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).
  • the prism SIGMA KOKI Co., Ltd. product
  • 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 ⁇ W), 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 designated as “blank signal”.
  • step (l) the assay signal was evaluated by the following formula from the measurement result obtained in step (k).
  • Assay 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.
  • Examplementation of assay method The assay method was performed in the same manner as in Example 3 except that 100 ⁇ L 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.
  • the substrate thus obtained is immersed in an ethanol solution containing 1 mM 10-carboxy-1-decanethiol for 24 hours or more to form a SAM (Self Assembled Monolayer) on one side of the 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 arranged 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.
  • 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 ⁇ L / min. And equilibrated the surface.
  • MES 25 ° C., pH 5.0
  • the solution was replaced with PBS, 0.8 mL of a PBS solution containing 1 ng / mL of AFP was added and circulated for 25 minutes.
  • Washing was performed by circulating TBS containing 0.05% by mass of Tween 20 for 5 minutes as a solution.
  • 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.
  • 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.
  • the fusion protein-containing assembly of the present invention When 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.

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Abstract

Cette invention concerne : un procédé de dosage ayant une sensibilité élevée, une précision élevée et une promptitude considérablement améliorée; un ensemble contenant une protéine de fusion, qui peut être utilisé dans le procédé de dosage; et un procédé de production dudit ensemble. L'ensemble contenant une protéine de fusion est caractérisé en ce qu'il comprend au moins une protéine de fusion qui comprend une protéine (A), une protéine (B), et un colorant fluorescent ou une enzyme. La protéine (A) peut se lier spécifiquement à au moins un site α dans une substance cible, un virus cible ou une cellule cible, la protéine (B) peut se lier spécifiquement à au moins un site β dans la substance cible, le virus cible ou la cellule cible qui contient le site α, et au moins deux molécules au total de la protéine (A) et de la protéine (B) sont contenues dans la protéine de fusion.
PCT/JP2010/056843 2009-04-28 2010-04-16 Ensemble contenant une protéine de fusion, procédé de production de l'ensemble, et procédé de dosage au moyen de l'ensemble WO2010125932A1 (fr)

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WO2012133048A1 (fr) * 2011-03-31 2012-10-04 富士フイルム株式会社 Dispositif de détection de biomolécules et procédé de détection associé
US9927432B2 (en) 2014-06-13 2018-03-27 Konica Minolta Laboratory U.S.A., Inc. Biosensor having decoupled capture chamber and detection chamber, using particle aggregation and size-separation
JPWO2019044202A1 (ja) * 2017-08-31 2020-08-20 コニカミノルタ株式会社 Ｂ型肝炎ウイルス表面抗原の検出方法および検出キット
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JP2012215472A (ja) * 2011-03-31 2012-11-08 Fujifilm Corp 生体分子検出装置および生体分子検出方法
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JPWO2019044202A1 (ja) * 2017-08-31 2020-08-20 コニカミノルタ株式会社 Ｂ型肝炎ウイルス表面抗原の検出方法および検出キット
CN112255061A (zh) * 2020-10-13 2021-01-22 南开大学 一种通过免疫沉淀分离和检测蛋白质的方法

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