JP2010091527A - Assay method using surface plasmon - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an assay method, apparatus, and kit using surface plasmon. <P>SOLUTION: The assay method includes the steps of: (a) bringing particles with ligands fixed onto a surface thereof into contact with a specimen; (b) reacting a conjugation of enzyme and ligands identical with or different from the above ligands on the particles obtained in (a); (c) further reacting an enzyme fluorescent substrate on the particles obtained in (b) to generate a fluorochrome; (d) isolating the fluorochrome obtained in (c); (e) bringing the fluorochrome obtained in (d) into contact with a thin metal film surface of a plasmon excitation sensor having at least a transparent planar substrate and the thin metal film formed on one surface of the substrate; (f) irradiating the plasmon excitation sensor obtained in (e) with a laser beam via a prism from the other surface of the substrate having no thin metal film to measure a fluorescence amount emitted from the excited fluorochrome; and (g) calculating an amount of analyte in the specimen out of a measurement result obtained in (f). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an assay method using surface plasmon, the assay device, and the assay kit. More specifically, the present invention relates to an assay method utilizing surface plasmon, the assay device, and the assay kit based on the principle of surface plasmon-excited fluorescence spectroscopy (SPFS; Surface 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 that the laser beam has is increased to several tens to several hundreds times (electric field enhancement effect of surface plasmon), and by this, the fluorescent dye in the vicinity of the gold thin film is efficiently excited, so that a trace amount and / or extremely low concentration can be obtained. 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, in the detection of trace analytes (target antigens), the amount of fluorescent dye in the conjugate associated with the antigen in the assay is also extremely small, which becomes a bottleneck in the amount of fluorescence generated, and therefore the plasmon electric field Even if enhancement is used, the amount of fluorescence signal does not increase, and it is difficult to improve assay sensitivity.

Patent Document 2 examines signal amplification and non-specific reaction reduction by complexly combining a reaction with an apoenzyme or holoenzyme and an immune reaction on a sensor substrate.
However, in order to establish such a measurement system, extremely precise molecular orientation technology is premised. Therefore, when the apo / holoenzyme reaction is preferential or dominant over the immune reaction, the measurement system itself There is a high risk that
Japanese Patent No. 3294605 JP 2008-139245 A

  The present invention relates to an assay method using a surface plasmon, which is highly sensitive and highly accurate by independence of an immune reaction field and a detection field, and has excellent specificity essential for an immunoassay, and the assay device And an assay kit.

  As a result of diligent research to solve the above-mentioned problems, the present inventors can completely separate the immune reaction field and the detection field by using a secondary antibody labeled with an enzyme. The present invention has been completed by discovering that both the fluorescence emission corresponding to the amount of photons and the specificity can be compatible even with the target antigen of.

That is, the assay method of the present invention is characterized by comprising at least the following steps (a) to (g).
Step (a): a step of contacting a specimen with particles having a ligand immobilized on the surface thereof,
Step (b): reacting a particle obtained through the step (a) with a conjugate of a ligand and an enzyme, which may be the same as or different from the ligand,
Step (c): a step in which the particles obtained through the step (b) are further reacted with an enzyme fluorescent substrate to produce a fluorescent dye,
Step (d): a step of isolating the fluorescent dye obtained through the step (c),
Step (e): The fluorescent dye obtained through the step (d) is brought into contact with the thin film surface of a plasmon excitation sensor having at least a transparent flat substrate and a metal thin film formed on one surface of the substrate. Process,
Step (f): The plasmon excitation sensor obtained in step (e) 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 (g): a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (f).

It is preferable that the immune reaction field in the steps (a) to (d) and the detection field in the steps (e) to (g) are independent from each other.
The specimen may be at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.

  The enzyme may be at least one enzyme selected from the group consisting of alkaline phosphatase (ALP), peroxidase (POD), and galactosidase (GAL).

The metal thin film is preferably formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper and platinum.
The plasmon excitation sensor further includes a spacer layer, and the spacer layer is preferably formed on the other surface of the metal thin film that is not in contact with the transparent flat substrate.

The apparatus of the present invention is used for the step (f).
The kit of the present invention is characterized by being used in the assay method of the present invention.

  Since the present invention can remove particles, that is, completely separate the immune reaction field and the detection field, the immune reaction conditions and the detection conditions can be optimized, and is less susceptible to scattering noise. Since it has three sensitivity amplification mechanisms, that is, immune reaction optimization, chemical amplification and physical amplification, it is possible to provide an extremely sensitive and highly accurate assay method.

Hereinafter, the present invention will be specifically described.
<Assay method>
The assay method of the present invention comprises at least the following steps (a) to (g), and preferably further comprises a washing step.

Step (a): a step of contacting a specimen with particles having a ligand immobilized on the surface thereof,
Step (b): reacting a particle obtained through the step (a) with a conjugate of a ligand and an enzyme, which may be the same as or different from the ligand,
Step (c): a step in which the particles obtained through the step (b) are further reacted with an enzyme fluorescent substrate to produce a fluorescent dye,
Step (d): a step of isolating the fluorescent dye obtained through the step (c),
Step (e): The fluorescent dye obtained through the step (d) is brought into contact with the thin film surface of a plasmon excitation sensor having at least a transparent flat substrate and a metal thin film formed on one surface of the substrate. Process,
Step (f): The plasmon excitation sensor obtained in step (e) 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 (g): a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (f).

(Process (a))
The step (a) is a step of bringing the specimen having the ligand immobilized on the surface thereof into contact with the specimen.

  A “particle” is a water-insoluble carrier used in the present invention to detect an analyte contained in a specimen. The material that forms water-insoluble particles may be insoluble in water. The term “water-insoluble” specifically means a solid phase that does not dissolve in water or any other aqueous solution. The particles may be any known support or matrix that is currently widely used and proposed for applications such as fixation and separation.

  The material forming the water-insoluble particles includes an inorganic compound, a metal, a metal oxide, an organic compound, or a composite material combining these. If the ligand immobilized on the surface of the particle can bind the analyte contained in the specimen, the material, shape and size of the particle are not particularly limited, but preferably the amount of ligand immobilized is increased. From the viewpoint, it is a material that can provide a large surface area.

  The material used as particles is not particularly limited, but generally synthetic organic polymers such as polystyrene, polypropylene, polyacrylate, polymethyl methacrylate, polyethylene, polyamide, latex; glass, silica, silicon dioxide, nitriding It may be an inorganic substance such as silicon, zirconium oxide, aluminum oxide, sodium oxide, calcium oxide, magnesium oxide, zinc oxide, iron oxide or chromium oxide; or a metal such as stainless steel or zirconia. These materials generally have a porous irregular surface, and may be, for example, fibers, webs, sintered bodies, porous bodies, and the like.

  Examples of the shape of the particles include a sphere shape, an ellipsoid shape, a cone shape, a cube shape, and a rectangular parallelepiped shape. Of these, spherical particles are preferable because they are easy to produce and easy to rotate and agitate the particles during use.

The particle size, that is, the average particle diameter is preferably 0.5 to 10 μm, and 2 to 6 μm.
m is more preferable. When the average particle size is less than 0.5 μm, when the particles are magnetic particles,
Sufficient magnetic responsiveness is not exhibited, it takes a considerably long time to separate the magnetic particles, and a very large magnetic force is required to separate them. On the other hand, when the average particle diameter exceeds 10 μm, the particles are likely to settle in the aqueous solution, and thus an operation of stirring the medium is required when contacting the specimen. Further, since the surface area of the particle body is small, it may be difficult to capture the analyte contained in the specimen.

  In addition to the aspect in which the entire particle including the surface is composed of the same material, it may be composed of a hybrid body composed of a plurality of materials as required. For example, in order to be able to cope with the automation of analysis, the core portion is made of a magnetically responsive material such as iron oxide or chromium oxide, and the surface thereof is coated with an organic synthetic polymer.

  As such a “particle”, the surface immobilization density of the following ligand can be easily adjusted, and from the viewpoint of easy B / F separation and further solid-liquid separation, magnetic particles are preferable, and the step (d) shown in FIG. The magnetic particles contain a magnetic material such as a paramagnetic material, a strong paramagnetic material, or a ferromagnetic material in that the magnetic particles can be easily (solid-liquid) separated and recovered by the magnetic force of the magnet. What is formed is preferable, and what contains a paramagnetic substance and / or a strong paramagnetic substance is more preferable. In particular, it is preferable to use a strong paramagnetic substance in that there is no or little residual magnetization.

Specific examples of such a “magnetic material” include triiron tetroxide (Fe ₃ O ₄ ), γ-heavy sesquioxide (γ-Fe ₂ O ₃ ), various ferrites, iron, manganese, cobalt, chromium, and the like. And various alloys such as cobalt, nickel, and manganese, and among these, triiron tetroxide is particularly preferable. Cobalt and nickel have an affinity for the histidine tag.

  The “magnetic material” used for the particles is a bead made of particles having a small particle diameter, has excellent magnetic separation (ie, ability to separate in a short time by magnetism), and is operated by a gentle up-and-down shaking operation. It is preferable that it can be redispersed.

  The content ratio of the magnetic substance in the magnetic particles is 70% by weight or less, preferably 20 to 70% by weight, more preferably 30 to 30% because the content ratio of the non-magnetic organic substance or the like is 30% by weight or more. 70% by weight is desirable. When such a content is less than 20% by weight, sufficient magnetic responsiveness is not exhibited, and it may be difficult to separate particles in a short time by a required magnetic force. On the other hand, when the content exceeds 70% by weight, the amount of the magnetic substance exposed on the surface of the particle main body increases, so that elution of constituent components of the magnetic substance, for example, iron ions occurs. The material may be adversely affected, and the particle body may become brittle and a practical strength may not be obtained.

A commercial item can also be used as such a magnetic particle, for example, Dynabeads series (made by Dynal Biotech ASA) etc. are mentioned.
A “ligand” is a molecule or molecular fragment that can specifically recognize (or be recognized) and bind to an analyte contained in a specimen, and as such a “molecule” or “molecular fragment” For example, nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), or nucleosides, nucleotides and their modified molecules, which may be single-stranded or double-stranded), proteins ( Polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules and complexes thereof are not particularly limited.

  Examples of the “protein” include antibodies and the like, specifically, anti-α-fetoprotein (AFP) monoclonal antibody (available from Japan Medical Laboratory), anti-carcinoembryonic antigen (CEA) ) Monoclonal antibody, anti-CA19-9 monoclonal antibody, anti-PSA monoclonal antibody and the like.

In the present invention, the term “antibody” includes polyclonal antibodies or monoclonal antibodies, antibodies obtained by gene recombination, and antibody fragments.
As a method for immobilizing a ligand on the particle surface, an optimum crosslinking method can be selected in accordance with the particle surface terminal functional group. In addition, depending on the properties of the particle surface, a method of directly adsorbing directly to the surface can also be mentioned as an effective means.

Examples of the “specimen” include blood, serum, plasma, urine, nasal fluid, saliva, stool, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.) and the like, and appropriately diluted with a desired solvent, buffer solution, etc. May be used. Of these samples, blood, serum, plasma, urine, nasal fluid and saliva are preferred. These may be used alone or in combination of two.

  The “analyte” contained in the specimen is a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding to a ligand immobilized on the particle surface. “Molecules” or “molecular fragments” include, for example, nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., which may be single-stranded or double-stranded, or nucleosides, nucleotides And modified molecules thereof), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules and complexes thereof. Specifically, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signal transmitter, a hormone, etc. It is not particularly limited.

  As a condition for contacting the specimen with the ligand immobilized on the surface of the ligand, 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.

(Washing process)
The washing step is preferably included before and / or after the following step (b), and is a step of washing the surface of the particles obtained in the above step (a) or the particles obtained in the following step (b). is there.

  As the washing solution used in the step, a surfactant such as Tween 20 or Triton X100 is dissolved in the same solvent or buffer used in the reaction of steps (a) and (b), preferably 0.00001 to Those containing 1% by weight are desirable.

The temperature and flow rate at which the cleaning liquid is circulated are preferably equal to the “temperature and flow rate at which the liquid feed is circulated” in step (a).
The time for circulating the cleaning liquid is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.

(Process (b))
Step (b) refers to a ligand and an enzyme that may be the same as or different from the ligand used in step (a) on the particles obtained through step (a), preferably the washing step. And a conjugate reaction.

  The ligand used in step (b) is a molecule or molecular fragment capable of specifically recognizing and binding to the analyte contained in the specimen. Examples of such “molecule” or “molecular fragment” include, for example, , Nucleic acids (DNA that may be single-stranded or double-stranded, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., or nucleosides, nucleotides and modified molecules thereof), proteins (polypeptides , Oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules or complexes thereof, etc. It may be the same as or different from the ligand used in the above step (a).

  Examples of the “protein” include an antibody, and the antibody is not particularly limited regardless of whether it is a monoclonal antibody or a polyclonal antibody. However, when the “ligand” immobilized on the particle surface (step (a)) is a monoclonal antibody, the “ligand” used in step (a) is recognized by the “ligand” used in step (b). It is preferable to recognize parts other than the part to be performed.

  Examples of the “enzyme” include alkaline phosphatase (ALP), peroxidase (POD), galactosidase (GAL) and the like, and have a molecular size that hardly affects the immune reaction, that is, the reaction between the ligand and the analyte. In view of the above, ALP, POD and GAL may be used, but the present invention is not particularly limited to these enzymes. These enzymes can be used alone or in combination of two or more.

  A “conjugate of a ligand and an enzyme” is a ligand labeled with an enzyme. Examples of the method for labeling an enzyme with a ligand include a method in which a streptavidinized enzyme is reacted with a biotinylated ligand. A commercially available product may be used as the streptavidinated enzyme, and examples thereof include phosphatase-labeled streptavidin (manufactured by KPL).

The concentration of the “ligand labeled with an enzyme” thus prepared is preferably 0.001 to 10,000 μg / mL, more preferably 1 to 1,000 μg / mL.
As reaction conditions, the temperature and time may be the same as those in the above step (a).

(Process (c))
The step (c) is a step in which a fluorescent dye is generated by further reacting an enzyme fluorescent substrate with the particles obtained through the step (b), preferably the washing step.

The “enzyme fluorescent substrate” is a substance that can generate a fluorescent dye by being hydrolyzed by the “enzyme”, and examples thereof include 1 to 8 listed in Table 1.
The “fluorescent dye” is a general term for substances that emit fluorescence by irradiating predetermined excitation light in the present invention, or excited by using an electric field effect. Including luminescence.

In Table 1, POD represents peroxidase; βGlu represents β glucosidase; GAL represents galactosidase; ALP represents alkaline phosphatase.
Among these enzyme fluorescent substrates, when the metal thin film included in the “plasmon excitation sensor” described below is formed of a metal including gold, 1,3-dicroro-9,9 is used from the viewpoint of gold transmittance and excitation wavelength. -Dimethyl-acid-2-one-7-yl phosphate (DDAO phosphate) (manufactured by Molecular Probes) is preferred.

  The autofluorescence wavelength of the enzyme fluorescent substrate is such that the greater the difference between the autofluorescence wavelength and the fluorescence wavelength of the fluorescent dye, the easier it is to avoid the influence of the background signal, and high-accuracy measurement is possible. Absent.

(Process (d))
Step (d) is a step of isolating the fluorescent dye obtained through the step (c).
As a method of isolating the fluorescent dye, for example, when the particle is a magnetic particle, a method of solid-liquid separation of the solution containing the fluorescent dye and the particle by magnetic force or centrifugation may be used. In the case of a body, a porous body or a substrate, a solution and particles containing a fluorescent dye can be isolated without using a solid-liquid separation method.

(Process (e))
In step (e), the fluorescent dye obtained through step (d) is applied to the surface of the plasmon excitation sensor having at least a transparent flat substrate and a metal thin film formed on one surface of the substrate. It is a process of making it contact.

  The “plasmon excitation sensor” includes a transparent flat substrate and a metal thin film formed on one surface of the substrate, and preferably further includes a spacer layer, and the spacer layer is formed of the transparent metal thin film. It is desirable to form on the other surface not in contact with the flat substrate.

  Such a plasmon excitation sensor has a substrate and a gold thin film, such as a sensor chip used in a Biacore system manufactured by GE Healthcare Biosciences, and a spacer layer is formed on the gold thin film. Also included.

  The “transparent flat substrate” may be made of glass or plastic such as polycarbonate (PC) or cycloolefin polymer (COP), and the refractive index [nd] is preferably 1.40-2. As long as it is 20 and the thickness is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm, the size (length × width) is not particularly limited.

The transparent glass substrate made of glass is commercially available from BK7 (refractive index [nd] 1.52) and LaSFN9 (refractive index [nd] 1.85) manufactured by SCHOTT AG, manufactured by Sumita Optical Glass Co., Ltd. K-PSFn3 (refractive index [nd] 1.84), K-LaSFn17 (refractive index [nd] 1.88) and K-LaSFn22 (refractive index [nd] 1.90), manufactured by OHARA INC. -LAL10 (refractive index [nd] 1.72) or the like is preferable from the viewpoint of optical characteristics and detergency.

The transparent flat substrate is preferably cleaned with acid and / or plasma before forming a metal thin film 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.
The “metal thin film” is formed on one surface of the above “transparent flat substrate”, preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably It is preferably made of gold and may be an alloy of these metals. Such metal species are preferable because they are stable against oxidation and increase in electric field due to surface plasmons increases.

  In addition, only when a glass flat substrate is used as the “transparent flat substrate”, the glass and the metal thin film can be bonded more firmly, so that a thin film of chromium, nickel chromium alloy or titanium is formed in advance. Is preferred.

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 enhancing 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.

  The “spacer layer” is formed on the other surface of the metal thin film not in contact with the “transparent flat substrate” for the purpose of preventing metal quenching of the fluorescent dye by the “metal thin film”. For example, a SAM (Self Assembled Monolayer) or a dielectric material may be used.

  As a single molecule contained in “SAM”, usually a carboxyalkanethiol having about 4 to 20 carbon atoms (for example, available from Dojindo Laboratories Co., Ltd., Sigma Aldrich Japan Co., Ltd.), particularly preferably 10 -Carboxy-1-decanethiol is used. Carboxyalkanethiol having 4 to 20 carbon atoms has properties such as little optical influence of SAM formed using it, that is, high transparency, low refractive index, and thin film thickness. Therefore, it is preferable.

  The method for forming the SAM is not particularly limited, and a conventionally known method can be used. As a specific example, there is a method of immersing a glass flat substrate having a metal thin film formed on the surface thereof in an ethanol solution containing 10-carboxy-1-decanethiol (manufactured by Dojindo Laboratories). In this way, the thiol group of 10-carboxy-1-decanethiol binds to the metal and is immobilized, and self-assembles on the surface of the gold thin film to form a SAM.

As the “dielectric”, various inorganic substances that are optically transparent, or natural or synthetic polymers can be used. From the viewpoint of chemical stability, production stability, and optical transparency, silicon dioxide (SiO ₂ ) Or titanium dioxide (TiO ₂ ).

  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 a 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 with a spin coater.

As a method of bringing the fluorescent dye obtained through the step (d) into contact with the spacer layer side surface of such a “plasmon excitation sensor”, a method of dropping, spraying, or applying a solution containing the fluorescent dye Is mentioned. Further, there may be mentioned a method in which the following flow path is formed on the plasmon excitation sensor and a solution containing the fluorescent dye is brought into contact with the surface of the plasmon excitation sensor.

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

  As the material, the plasmon excitation sensor part consists of a homopolymer or copolymer, polyethylene, polyolefin, etc. containing methyl methacrylate, styrene or the like as a raw material, and silicon rubber, Teflon (registered trademark), polyethylene, polypropylene, etc. Etc. are used.

  In the plasmon excitation sensor unit, from the viewpoint of increasing the contact efficiency with the specimen and shortening the diffusion distance, it is preferable that the cross section of the channel of the plasmon excitation sensor unit is independently about 100 nm to 1 mm in length and width.

  As a method of fixing the plasmon excitation sensor to the flow path, in a small-scale lot (laboratory level), first, on the surface on which the metal thin film of the plasmon excitation sensor is formed, A dimethylsiloxane (PDMS) sheet 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 closed with screws or the like. A method of fixing with a tool is preferred.

  In large lots (factory level) manufactured industrially, as a method of fixing the plasmon excitation sensor to the flow path, a gold substrate is formed on a plastic integrally molded product, or a separately manufactured gold substrate is fixed, and the gold surface is fixed. Further, after the dielectric layer, the fluorescent dye layer, and the ligand are immobilized, it can be manufactured by covering with a plastic integrally formed 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 analyte contained in the specimen being sent 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.
It is.
(Process (f))
In step (f), the plasmon excitation sensor obtained in step (e) 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.

It is desirable to adjust the energy and photon amount immediately before the laser light enters the prism through an optical filter and a polarizing 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. The amount of increase in photons due to the electric field enhancement effect depends on the refractive index of the glass serving as the substrate, the metal species and the film thickness of the metal thin film, but is usually about 10 to 20 times that of gold.

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

  As the “laser light”, an LD laser having a wavelength of 200 to 900 nm and 0.001 to 1,000 mW, or a semiconductor laser having a wavelength of 230 to 800 nm and 0.01 to 100 mW is preferable.

  The “prism” is intended to allow the laser light through various filters to efficiently enter the plasmon excitation sensor, and the refractive index is preferably the same as that of the “transparent flat substrate”. 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” 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 various types of noise light such as scattered light generated by the influence of structures or deposits on the surface of the excitation sensor), autofluorescence of the enzyme fluorescent substrate, and examples thereof include interference filters and color filters. It is done.

  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 (manufactured by Nikon Corporation or Olympus Corporation) used in a microscope or the like may be diverted. 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.

  In Table 2, plasmon excitation using Alexa Fluor (registered trademark) 647 (in Table 2, conditions 1 to 3) and HiLyte Fluor (registered trademark) 647 (in Table 2, conditions 4 to 6) as fluorescent dyes, respectively. The SPFS fluorescence signal by a sensor is shown.

  In Table 2, the difference between the fluorescence signal value at the time of CCD observation under the condition in which the concentration-adjusted fluorescent dye solution was fed to the plasmon excitation sensor and the fluorescence signal value at the time of CCD observation under the condition in which MilliQ water was fed. The SPFS signal of the fluorescent dye adjusted to each concentration is used.

  From Table 2, it can be seen that highly sensitive measurement can be carried out even in a solution with a very small amount of fluorescent dye. This result indicates that the measurement is possible even under the condition that the fluorescent dye produced from the enzyme fluorescent substrate by the enzyme reaction is present at least in a trace amount as shown in Table 2. That is, in the assay method of the present application using an enzyme fluorescent substrate, it is shown that a highly sensitive measurement can be realized as an immunoassay result.

  Table 3 also shows that the plasmon excitation sensor has a large ratio between “Signal” and “Noise”, and the value of “Signal” that changes depending on the amount of the fluorescent dye is relatively large compared to “Noise”. It is shown that it is possible to measure easily.

  When MilliQ water was sent to the plasmon excitation sensor, the signal value when observed from the CCD was “Noise” (plasmon scattering noise), and a 10 nM Alexa Fluor (registered trademark) 647 aqueous solution was sent. The numerical value of the fluorescence signal when observed from the CCD is “Signal”.

The plasmon excitation sensor 1 in Table 2 is manufactured as follows.
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 has a thickness of 1 to 3 nm, and the gold thin film has a thickness of 44 to 52 nm.

The plasmon excitation sensor 2 is manufactured in the same manner as the plasmon excitation sensor 1 except that the resistance heating vapor deposition method is used instead of sputtering in the method of manufacturing the plasmon excitation sensor 1, and the plasmon excitation sensor 1 further increases plasmon scattering. The plasmon excitation sensor 3 drops a salt concentration adjusting solution in which polystyrene fine particles (manufactured by Polysciences Inc.) having an average particle diameter of about 100 nm are dispersed on the surface of the plasmon excitation sensor 2, and is allowed to stand for several minutes. By washing with MilliQ water, the sensor surface has the fine particles on the surface.

  From Table 3, it can be seen that the ratio of Signal to Noise (S / N ratio) decreases with increasing Noise. That is, it is apparent that a plasmon excitation sensor with as small a plasmon scattering noise as possible is suitable for realizing a highly sensitive measurement.

  In the plasmon excitation sensor 3, Noise is significantly increased by particles fixed in a metal foil film shape. That is, in the fluorescence measurement using the plasmon enhancement field, it is apparent that it is difficult to realize high-sensitivity measurement because noise increases if fine particles or the like are present on the sensor surface. Therefore, in the fluorescence measurement using the plasmon enhancement field, complete separation of the immune reaction field and the detection field is one solution for realizing high-sensitivity measurement.

(Process (g))
The step (g) is a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (f).

  More specifically, a calibration curve is created by performing measurement with a target antigen or target antibody at a known concentration, and the target antigen amount or target antibody amount in the sample to be measured is measured based on the created calibration curve. This is a step of calculating from the signal.

<Device>
The apparatus of the present invention is used in the step (f).
That is, the apparatus of the present invention is for carrying out the assay method of the present invention using the plasmon excitation sensor.

  The “apparatus” includes at least a light source, various optical filters, a prism, a cut filter, a condenser lens, and an SPFS detection unit. It is preferable to have a liquid feeding system combined with a plasmon excitation sensor when handling a sample liquid, a washing liquid, a labeled antibody liquid, or the like. As the liquid feeding system, for example, a microchannel device connected to a liquid pump may be used.

In addition, a surface plasmon resonance (SPR) detection unit, that is, a photodiode as a light receiving sensor dedicated to SPR, an angle variable unit for adjusting the optimum angle of SPR and SPFS (to determine total reflection attenuation (ATR) conditions with a servomotor) The angle of 45 to 85 ° can be changed by synchronizing the photodiode and the light source with a resolution of 0.01 ° or more).
A computer for processing information input to the SPFS detection unit may also be included.

Preferred embodiments of the light source, the optical filter, the cut filter, the condensing lens, and the SPFS detection unit are the same as those described above.
As the “liquid feed pump”, for example, a micro pump suitable for a small amount of liquid feed, a syringe pump with high feed accuracy and low pulsation, which is preferable but cannot be circulated, a simple and excellent handleability but a small amount of liquid feed For example, a tube pump may be difficult.

<Kit>
The kit of the present invention is characterized by being used in the assay method of the present invention. In performing the assay method of the present invention, a primary antibody, a ligand such as an antigen, a specimen, and a secondary antibody are used. It is preferable to include everything needed.

  For example, by using the kit of the present invention, blood or serum as a specimen, and an antibody against a specific tumor marker, the content of the specific tumor marker can be detected with high sensitivity and high accuracy. From this result, the presence of a preclinical noninvasive cancer (carcinoma in situ) that cannot be detected by palpation or the like can be predicted with high accuracy.

  Specifically, such a “kit” includes a plasmon excitation sensor in which a metal thin film is formed on one surface of a transparent flat substrate; a solution or dilution liquid for dissolving or diluting the specimen; a plasmon excitation sensor and a specimen; These include various reaction reagents and washing reagents for reacting, and can also include various equipment or materials required for carrying out the assay method of the present invention and the above-mentioned “device”.

  Further, the kit element may include a standard material for preparing a calibration curve, instructions, a necessary set of equipment such as a microtiter plate capable of simultaneously processing a large number of samples, and the like.

Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these.
[Production 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 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.

[Preparation Example 2] (Preparation of secondary antibody labeled with alkaline phosphatase)
An anti-α-fetoprotein (AFP) monoclonal antibody (available from Nippon Medical Laboratory, Inc.) as a secondary antibody was biotinylated using a biotinylation kit (manufactured by Dojindo Laboratories). The procedure followed the protocol attached to the kit.

  Next, the obtained biotinylated anti-AFP monoclonal antibody solution and a streptavidin-labeled alkaline phosphatase (ALP) solution (Phosphatase-labeled streptavidin (KPL)) solution are mixed and stirred at 4 ° C. for 60 minutes. It was made to react by doing.

  Finally, the unreacted antibody and the unreacted enzyme were purified using a molecular weight cut filter (manufactured by Nippon Millipore) to obtain an alkaline phosphatase-labeled anti-AFP monoclonal antibody solution. The obtained antibody solution was stored at 4 ° C. after protein quantification.

[Preparation Example 3] (Preparation of Alexa Fluor (registered trademark) 647-labeled secondary antibody)
The biotinylated anti-AFP monoclonal antibody solution obtained in Preparation Example 2 and the streptavidin-labeled Alexa Fluor (registered trademark) 647 (Molecular Probes) solution are mixed and reacted by stirring at 4 ° C. for 60 minutes. I let you.

  Next, the unreacted antibody and the unreacted enzyme were purified using a molecular weight cut filter (manufactured by Nippon Millipore) to obtain an Alexa Fluor (registered trademark) 647-labeled anti-AFP monoclonal antibody solution. The obtained antibody solution was stored at 4 ° C. after protein quantification.

[Example 1]
As step (a), first, anti-α-fetoprotein (AFP) monoclonal antibody (obtained from Nippon Medical Laboratory) is immobilized on Dynabeads (manufactured by Dynal Biotech ASA) as magnetic particles. Turned into. The immobilization method was in accordance with the protocol attached to Dynabeads.

  A specimen containing AFP (prepared in a 1 ng / mL TBS solution) as a target antigen is brought into contact with 100 μL of magnetic particles (prepared in a 0.015 wt% TBS solution) on which the anti-AFP monoclonal antibody is immobilized. The reaction was allowed for 10 minutes.

As the washing step, the particles obtained through the step (a) were collected with a magnet for solid-liquid separation, and only the liquid of the reaction solution after the step (a) was discarded. To the remaining particles, 300 μL of TBS containing 0.05% by weight of Tween 20 was dispensed, stirred for 1 minute, and collected by a magnet. Such a washing process was repeated three times.

In step (b), 200 μL of the alkaline phosphatase-labeled anti-AFP monoclonal antibody (TBS solution prepared at 1,000 ng / mL) obtained in Preparation Example 2 was added to the particles obtained through the washing step. The reaction was allowed for 10 minutes.

As the washing step, the particles obtained through the step (b) were collected with a magnet for solid-liquid separation, and only the liquid of the reaction solution after the step (b) was discarded. To the remaining particles, 300 μL of TBS containing 0.05% by weight of Tween 20 was dispensed and stirred for 1 minute, and then the particles were collected by a magnet. Such a washing process was repeated three times.

  As the step (c), the particles obtained through the washing step described above were added to an enzyme fluorescent substrate solution (1,3-dicilo-9,9-dimethyl-acid-2-one-7-yl phosphate (DDAO) prepared with TBS. 100 μL of phosphate (manufactured by Molecular Probes) was dispensed and allowed to react for 5 minutes after stirring.

As the step (d), the reaction solution obtained through the step (c) was subjected to solid-liquid separation by collecting particles with a magnet and isolated as a fluorescent dye solution.
As the step (e), the fluorescent dye solution obtained through the step (d) was brought into contact with the surface of the plasma excitation sensor obtained in Production Example 1 by liquid feeding.

  As the step (f), the prism (Sigma Kogyo Co., Ltd. product) is applied to the plasmon excitation sensor obtained in the step (e) from the other surface of the glass transparent flat substrate on which the gold thin film is not formed. ) Was irradiated with laser light (640 nm, 40 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 signal”.

The SPFS measurement signal when AFP was 0 ng / mL was defined as “assay noise signal”.
As the step (g), from the measurement results obtained in the step (f), the sensitivity was evaluated by evaluating the assay S / N ratio by the following formula, and the accuracy was evaluated by calculating the CV value. As the CV value, the value of 100 percent of the standard deviation with respect to the average value was calculated from the result of six measurements under the same conditions.

Assay S / N ratio = | (assay fluorescence signal) | / | (assay noise signal) |
That is, if the value of the fluorescent signal that changes depending on the amount of fluorescent dye proportional to the amount of antigen is large from the assay S / N ratio, and the assay noise signal is sufficiently small relative to the assay fluorescent signal, the reliability of the immunoassay measurement can be improved. I understand that it is expensive.

Table 4 shows the obtained results.
[Comparative Example 1]
First, the plasmon excitation sensor obtained in Production Example 1 was fixed to a flow path, and ultrapure water was fed as a liquid for 10 minutes, and then PBS was circulated for 20 minutes with a peristaltic pump at room temperature and a flow rate of 500 μL / min. Was equilibrated.

Subsequently, 5 mL of PBS containing 50 mM N-hydroxysuccinimide (NHS) and 100 mM water-soluble carbodiimide (WSC) was fed and circulated for 20 minutes, followed by anti-α-fetoprotein (AFP) monoclonal. By circulating 2.5 mL of antibody (1D5, 2.5 mg / mL, manufactured by Nippon Medical Laboratory) for 30 minutes, SAM
The primary antibody was immobilized on the top. In addition, the nonspecific adsorption | suction prevention process was performed by circulating 30 minutes by PBS buffer physiological saline containing 1weight% bovine serum albumin (BSA).

Instead of PBS, 0.5 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 weight of Tween 20 for 10 minutes.

Secondary antibody labeled with Alexa Fluor (registered trademark) 647 (1,000 ng / m
2.5 mL of PBS solution prepared to be L) was added and circulated for 20 minutes.
Then, it was cleaned by circulating TBS containing 0.05% by weight of Tween 20 for 20 minutes.

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

Table 4 shows the obtained results.
[Comparative Example 2]
First, the plasmon excitation sensor obtained in Production Example 1 was fixed to the flow path, and ultrapure water was fed as a liquid for 10 minutes, and then PBS was circulated for 20 minutes using a peristaltic pump at room temperature at a flow rate of 500 μL / min. Was equilibrated.

Subsequently, 5 mL of PBS containing 50 mM N-hydroxysuccinimide (NHS) and 100 mM water-soluble carbodiimide (WSC) was fed and circulated for 20 minutes, followed by anti-α-fetoprotein (AFP) monoclonal. By circulating 2.5 mL of antibody (1D5, 2.5 mg / mL, manufactured by Nippon Medical Laboratory) for 30 minutes, SAM
The primary antibody was immobilized on the top. In addition, the nonspecific adsorption | suction prevention process was performed by circulating 30 minutes by PBS buffer physiological saline containing 1% bovine serum albumin (BSA).

Instead of PBS, 0.5 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 weight of Tween 20 for 10 minutes.

Alkaline phosphatase-labeled anti-AFP monoclonal antibody obtained in Preparation Example 2 (1,
2.5 mL of PBS solution prepared to be 000 ng / mL) was added and circulated for 20 minutes.

Washing was carried out by circulating TBS containing 0.05% by weight of Tween 20 for 10 minutes.
100 μL of an enzyme fluorescent substrate solution (1,3-dicilo-9,9-dimethyl-2-one-7-yl phosphate (DDAO phosphate) (manufactured by Molecular Probes)) prepared with TBS was introduced into the plasmon excitation sensor. The reaction was allowed for 5 minutes.

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

  Table 4 shows the obtained results.

  From Table 4, Example 1 which is a fluorometric immunoassay using plasmon excitation in which an immune reaction field and a detection field are completely separated from each other has a higher fluorescence signal value and assay S / N. The ratio was achieved, and it was found that measurement with extremely high sensitivity and a wide dynamic range is possible.

  Further, in Comparative Example 2, which is an immunoassay result obtained by enzyme amplification on the sensor substrate, the signal was amplified as compared with Comparative Example 1. However, the noise rises as well, and the amplification reaction on the same substrate cannot find an advantage in the assay S / N ratio.

  On the other hand, in Example 1, each reaction system can be optimized by completely separating each of the immune reaction (including the washing step), the amplification reaction, and the detection reaction, and highly sensitive measurement is possible. It became. Also, regarding the accuracy, the CV value result of Example 1 was better than that of Comparative Examples 1 and 2. In particular, significance was recognized with respect to the time of AFP (1 ng / mL) signal, and it was found that the present invention is a highly sensitive and highly accurate measurement method.

  Since the assay method of the present invention can be detected with high sensitivity and high accuracy, for example, even a very small amount of tumor marker contained in blood can be detected. The presence of pre-clinical non-invasive cancer (carcinoma in situ) that cannot be detected by, for example, can be predicted with high accuracy.

FIG. 1 shows that, in an immune reaction field, a primary antibody 2 immobilized on the surface of a particle 1 with a target antigen 3 contained in a specimen is recognized and bound to a secondary antibody 4 labeled with an enzyme, Further, by adding a fluorescent substrate (not shown), the fluorescent dye 10 is generated, and the isolated fluorescent dye 10 is converted into a glass transparent flat substrate 6 as a detection field, and one surface of the substrate 6. A surface of the plasmon excitation sensor having a gold thin film 7 formed on the substrate and a spacer layer (not shown) formed on the other surface of the thin film 7 not in contact with the substrate. Then, laser light is irradiated from the other surface of the substrate 6 where the thin film 7 is not formed via a prism (not shown), and the fluorescent dye 10 is excited by the surface plasmon and emits light. , Shows a schematic diagram of the assay method of the present invention 2 shows steps (a) to (c): a primary antibody 2 in which a target antigen 3 contained in a specimen is immobilized on the surface of magnetic particles 1 in a test tube 12 which is an immune reaction field. And the secondary antibody 4 labeled with the enzyme are recognized and bound to each other, and a fluorescent substrate (not shown) is further added to produce a fluorescent dye 10. Step (d): outside of the test tube 12 The fluorescent dye 10 is isolated by bringing the magnet 11 closer to the surface, and the steps (e) and (f): a transparent flat substrate 6 made of glass, which is a detection field, and gold formed on one surface of the substrate 6 On the surface of the plasmon excitation sensor having a thin film 7 and a spacer layer (not shown) made of a dielectric formed on the other surface of the thin film 7 not in contact with the substrate, The separated fluorescent dye 10 is contacted to form the thin film 7 of the substrate 6 From no other surface, through the prism is irradiated with a laser beam (not shown), a fluorescent dye 10 is emitting light when excited by the surface plasmon, a schematic view of the assay of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Particle 2 ... Primary antibody 3 ... Target antigen contained in a specimen 4 ... Secondary antibody 5 ... Ruthenium complex 6 ... Transparent flat substrate made of glass 7. -Gold thin film 9 ... Enzyme 10 ... Fluorescent dye 11 ... Magnet 12 ... Test tube

Claims (8)

An assay method comprising at least the following steps (a) to (g).
Step (a): a step of contacting a specimen with particles having a ligand immobilized on the surface thereof,
Step (b): reacting a particle obtained through the step (a) with a conjugate of a ligand and an enzyme, which may be the same as or different from the ligand,
Step (c): a step in which the particles obtained through the step (b) are further reacted with an enzyme fluorescent substrate to produce a fluorescent dye,
Step (d): a step of isolating the fluorescent dye obtained through the step (c),
Step (e): The fluorescent dye obtained through the step (d) is brought into contact with the thin film surface of a plasmon excitation sensor having at least a transparent flat substrate and a metal thin film formed on one surface of the substrate. Process,
Step (f): The plasmon excitation sensor obtained in step (e) 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 (g): a step of calculating the amount of analyte contained in the specimen from the measurement result obtained in the step (f).   The assay method according to claim 1, wherein the immunoreaction field in steps (a) to (d) and the detection field in steps (e) to (g) are independent of each other.   The assay method according to claim 1 or 2, wherein the specimen is at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.   The assay method according to any one of claims 1 to 3, wherein the enzyme is at least one enzyme selected from the group consisting of alkaline phosphatase (ALP), peroxidase (POD) and galactosidase (GAL).   The assay method according to any one of claims 1 to 4, 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 plasmon excitation sensor further has a spacer layer,
The assay method according to claim 1, wherein the spacer layer is formed on the other surface of the metal thin film that is not in contact with the transparent flat substrate.   The apparatus used for the process (f) of Claim 1.   A kit for use in the assay method according to claim 1.