JP5720248B2 - Immunoassay using surface plasmon and fluorescence resonance energy transfer - Google Patents

Description

  The present invention relates to an immunoassay method utilizing surface plasmon and fluorescence resonance energy transfer, and a system and kit for the assay. More specifically, the present invention relates to an immunoassay method using surface plasmon based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) and applying FRET technology to fluorescence detection.

  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 surface of the metal thin film. The amount of photons contained in the laser light is increased to several tens to several hundreds times (the electric field enhancement effect of surface plasmons), and this effectively excites the fluorescent dye in the vicinity of the metal thin film, resulting in a trace amount and / or extremely low concentration of the analog. It is a method that can detect light.

  As an example related to a biosensor or biochip based on the principle of SPFS, Patent Document 1 discloses an electric field enhanced by surface plasmon by disposing a primary antibody-immobilized film using carboxymethyldextran on the surface of a metal substrate. Discloses a method for detecting a fluorescent dye associated with an antigen. However, in the detection of a very small amount of analyte (for example, a target antigen), the amount of fluorescent dye in the complex associated with the antigen in the assay is also extremely small, and this becomes a bottleneck of the amount of fluorescence generated. Even if plasmon electric field enhancement is used, the amount of fluorescence signal is not enhanced, and it is difficult to improve assay sensitivity.

  In Patent Document 2, a reaction by an apoenzyme or holoenzyme and an immune reaction are combined in a complicated manner on a sensor substrate, and signal amplification and nonspecific reaction reduction are studied. In order to establish such a measurement system, extremely precise molecular orientation technology is a prerequisite, and if the apo / holoenzyme reaction is preferential or dominant over the immune reaction, the risk that the measurement system itself cannot be established High nature.

  On the other hand, a technique for detecting molecules interacting closely with each other with high sensitivity using the fluorescence resonance energy transfer (FRET) phenomenon is known, and two molecules forming a specific binding pair are used as fluorescent probes. FRET technology has been established (see Patent Documents 3 and 4, Non-Patent Document 1 for details of fluorescence resonance energy transfer). In FRET, energy from an excited fluorophore (donor) is transferred to another fluorophore (acceptor) via intermolecular dipole-dipole coupling. This is possible only when the distance between the donor and the acceptor is short (10-100Å) and the donor spectrum and the acceptor absorption spectrum are partially overlapping fluorescence. The energy transfer is then detected as a change in fluorescence.

Japanese Patent No. 3294605 JP 2008-139245 A JP-T-2006-505775 JP-T-2006-506634

Szollosi, J., Damjanovich, S., Matyus, L. (1998) Application of Fluorescence Resonance Energy Transfer in the Clinical Laboratory: Routine and Research. Communications in Clinical Cytometry, 34: 159-179.

  When the present inventors detect the problem of the conventional sandwich immunoassay method (FIG. 1), that is, when detecting a very small amount of an analyte (for example, an antigen), the immune complex itself is inevitably generated even if the ordinary sandwich immunoassay method is performed. In order to solve the problem that the amount of fluorescent probe is often not suitable for its detection capability, and therefore it is not satisfactory in terms of sensitivity. As a result, in place of the detection method in the conventional sandwich immunoassay method (FIG. 1), a method of detecting an immune complex formed by an antigen-antibody reaction by surface plasmon excitation enhanced fluorescence analysis (SPFS) is adopted, and FRET By realizing the luminescence enhancement based on the technology, the assay method of the present invention (FIG. 2) constituted by a mechanism in which substantially only the immune complex emits light was completed.

  The present invention has excellent specificity that is indispensable for an immunoassay, and preferably combines surface plasmon and fluorescence resonance energy transfer with high sensitivity and high accuracy by independently combining an immune reaction field and a detection field. It is an object to provide an assay method, system and kit for the assay.

The present invention, which is a highly sensitive assay method capable of specifically enhancing and measuring only the fluorescence of the immune complex even with a very small amount of target antigen, has the following constitution. That is,
An immunoassay method for measuring the amount of antigen in a specimen using a primary antibody bound with a fluorescent dye as a donor molecule and a secondary antibody bound with a fluorescent dye as an acceptor molecule, comprising at least
Step (a): causing an antigen-antibody reaction with the antigen contained in the sample and both antibodies of said primary and secondary antibodies, immune complexes antigen is sandwiched by both antibodies, plasmon excitation sensor Immobilizing on a metal thin film substrate,
Step (b): Based on the surface plasmon excitation fluorescence, fluorescence resonance energy transfer from the immune complex with a laser beam is irradiated to plasmon excitation sensor immobilized, excited donor molecule fluorescent dye of the primary antibody ( Measuring the amount of fluorescence of the acceptor molecule of the secondary antibody emitted by FRET),
It is characterized by including.
The secondary antibody is preferably obtained by binding a fluorescent dye serving as the acceptor molecule to an antibody fragment.
Moreover, it is preferable that the said secondary antibody couple | bonds the fluorescent pigment | dye used as the said acceptor molecule through SH group which appeared in the reduced type antibody.
Furthermore, the primary antibody and the secondary antibody are preferably those into which a binding reactive group capable of regulating the spatial structure of FRET by a binding mode other than the antigen-antibody reaction is introduced. As the binding reaction group, biotin and avidin are preferable. The binding reactive group is also preferably a DNA or polynucleotide and its complementary strand. In this case, the primary antibody binds the DNA or polynucleotide bound with the fluorescent dye serving as the donor molecule to the antibody. The secondary antibody is obtained by binding the complementary chain to which the fluorescent dye serving as the acceptor molecule is bound to the antibody.

In the step (a), the contacting with said a specimen secondary antibody, then the secondary antibody conjugated with an antigen, by contacting the metal thin film substrate immobilized the primary antibody, the immune complex It is desirable to fix it on a metal thin film substrate. Alternatively, in the step (a), the said primary antibody immobilized is contacted with the metal thin film substrate and the sample, by contacting a secondary antibody to the next the substrate, the immune complex on the metal thin film substrate It may be fixed. Or in the step (a), the said contacting the primary antibody and the secondary antibody and the analyte to form the immune complex, then the immune complexes are contacted to the metal thin film substrate wherein a metal thin film on a substrate You may fix to.

The donor and acceptor molecules are, Alexa Fluor (registered trademark) 647 / Cy5.5, HiLyte Fluor647 / Cy5.5, fluorescein isothiocyanate (FITC) / tetramethylrhodamine isothiocyanate (TRITC), and R- phycoerythrin (R- It is preferably a FRET pair selected from a pair group consisting of PE) / allophycocyanin (APC).

Another aspect of the present invention is a kit for the assay, at least, plasmon excitation sensor, a primary antibody bound to a fluorescent dye comprising the antibody and a by donor molecules which are immobilized on the surface of the metal thin film substrate In the above immunoassay method, comprising the fluorescent dye, the primary antibody and the reaction reagent for preparation, and the fluorescent dye, the secondary antibody and the reaction reagent for producing a secondary antibody to which the fluorescent dye as the acceptor molecule is bound. The kit is used to carry out a step of forming an immune complex by causing a sandwich-type antigen-antibody reaction between the primary antibody, the antigen in the specimen, and the secondary antibody.
As in the above assay, also in the kit, the secondary antibody is preferably an antibody fragment in which a fluorescent dye serving as the acceptor molecule is bound. Moreover, it is preferable that the said secondary antibody couple | bonds the fluorescent pigment | dye used as the said acceptor molecule through SH group which appeared in the reduced type antibody. Furthermore, the primary antibody and the secondary antibody are preferably those into which a binding reactive group capable of regulating the spatial structure of FRET by a binding mode other than the antigen-antibody reaction is introduced. As the binding reaction group, biotin and avidin are preferable. The binding reactive group is also preferably a DNA or polynucleotide and its complementary strand. In this case, the primary antibody has the DNA or polynucleotide bound with the fluorescent dye serving as the donor molecule bound to the antibody. The secondary antibody is obtained by binding the complementary chain to which the fluorescent dye serving as the acceptor molecule is bound to the antibody.

  When detecting an extremely small amount of analyte, the conventional sandwich immunoassay method has a small amount of fluorescence signal and relatively increases background noise, so that the S / N ratio is deteriorated. Even when SPFS is introduced into the detection system, a mismatch occurs between the surface plasmon electric field enhancement (example of electric field enhanced photon amount) and the amount of fluorescent dye (example of fluorescent photon amount) of a very small amount of analyte, resulting in a sensitivity limit. There was a possibility.

On the other hand, in the immunoassay method of the present invention, a mechanism in which only the immune complex emits light by providing luminescence enhancement based on the FRET technique in detection with a plasmon excitation sensor (SPFS) is provided. Furthermore, it is desirable to make the immune reaction field and the detection field independent. This increases the difference between the excitation wavelength and the fluorescence wavelength and increases the fluorescence signal emitted from the immune complex. On the other hand, fluorescence such as secondary antibodies that are not involved in the immune reaction is not detected, so background noise is generated. Reduced to allow for high S / N ratio assays. Specifically, in the assay of the present invention, from a specimen containing an analyte (eg, target antigen) at a concentration of 10 ⁻¹⁸ mol (1 amol / L) to 10 ⁻¹² mol (1 pmol / L) per liter, The analyte can be detected with high sensitivity and high accuracy.

  When the FRET pairs are separated from each other by more than 10 nm, the FRET disappears and the acceptor emission on the secondary antibody is significantly attenuated. Therefore, only the fluorescence derived from the immune complex is substantially detected through FRET that is expressed when the donor / acceptor pair is at an appropriate distance, and a washing operation (B / F separation) is not required. .

  In the assay of the present invention, since the immune reaction field and the fluorescence detection field can be completely separated, the immune reaction conditions and the detection conditions can be optimized separately, and are hardly affected by scattering noise and background noise. Furthermore, since it has three sensitivity amplification mechanisms, namely, immune reaction optimization, chemical amplification and physical amplification, it is possible to provide an extremely sensitive and highly accurate SPFS / FRET immunoassay.

FIG. 1 schematically shows a conventional sandwich immunoassay. The fluorescent dye (50) is labeled only on the secondary antibody (40, 41, 42). In this case, the secondary antibody (42) non-specifically bound by excitation (100) and the fluorescent dye (50) of the free secondary antibody (41) also emit light, increasing noise (300). For this reason, a cleaning operation is required. FIG. 2 is a diagram schematically showing a sandwich immunoassay using SPFS / FRET of the present invention. The primary antibody (20) and the secondary antibody (40, 41, 42) are labeled with a fluorescent dye (60) serving as a donor molecule and a fluorescent dye (70) serving as an acceptor molecule, respectively. The secondary antibody (40) oriented at an appropriate distance from the excited (100) fluorescent dye (60) of the primary antibody (20) only when the immune complex (500) to be detected is formed The energy is transferred to the fluorescent dye (70) and emits fluorescence (FRET (400)). By measuring this fluorescence, only the immune complex (500) can be detected (200). On the other hand, no light is emitted from the free secondary antibody (41) and the non-specifically bound secondary antibody (42). Further, the fluorescence emitted from the fluorescent dye (60) of the primary antibody (20), which becomes noise (300), can be separated by a filter in this case.

  In this specification, “analyte” is a substance to be assayed, that is, a test substance, and refers to a relatively large molecule mainly designated as “antigen”. On the other hand, “analyte” refers to drugs, metabolites, various environmental hazards. Low molecular weight compounds (haptens) such as substances can also be targeted. In addition, the “fluorescent dye” is a general term for substances that emit fluorescence by irradiating predetermined excitation light or using the electric field effect in the present invention. Including luminescence.

  Surface plasmon excitation enhanced fluorescence spectroscopy or surface plasmon-field enhanced fluorescence spectroscopy may be referred to as SPFS, and fluorescence resonance energy transfer may be referred to as FRET.

<Immunoassay method>
The immunoassay method of the present invention comprises:
An immunoassay method for measuring the amount of antigen in a specimen using a primary antibody bound with a fluorescent dye as a donor molecule and a secondary antibody bound with a fluorescent dye as an acceptor molecule, comprising at least
Step (a): An antigen-antibody reaction is caused between the antigen contained in the specimen and both the primary antibody and the secondary antibody, and the immune complex sandwiched by the two antibodies is used as a metal thin film substrate. Immobilizing on the top,
Step (b): The formed immune complex is irradiated with laser light by a surface plasmon excitation fluorescence method, and light is emitted by fluorescence resonance energy transfer (FRET) from the donor molecule fluorescent dye excited by the primary antibody. Measuring the fluorescence amount of the acceptor molecule,
It is characterized by including.

  When an antigen-antibody reaction occurs between the antigen in the specimen, the primary antibody, and the secondary antibody, a specific immune complex is formed in which the antigen is sandwiched by both antibodies. The second half of the assay is to detect the immune complex thus formed with high sensitivity. That is, the fluorescent dye of the donor molecule is excited by SPFS for the purpose of specifically detecting only a small amount of the immune complex. From the other surface of the substrate where the thin film is not formed, the immune complex is irradiated with a laser beam via a prism, and the FRET phenomenon occurs between the fluorescent dye and the acceptor molecule of the excited donor molecule. To express. The fluorescence of the acceptor molecule of the secondary antibody excited by this FRET is detected by a plasmon excitation sensor, and the amount of emitted fluorescence is obtained.

Antigen-antibody reaction The immunoassay method of the present invention employs a method in which the immune complex formed as described above is detected with high sensitivity by SPFS / FRET, but a primary dye conjugated with a fluorescent dye serving as a donor molecule for FRET expression. A secondary antibody to which an antibody and a fluorescent dye as an acceptor molecule are bound is used.

  The primary antibody used in the assay of the present invention is an antibody that can recognize and bind to an analyte (for example, a target antigen) contained in a specimen. At the same time, the primary antibody binds a fluorescent dye that becomes the FRET donor molecule. Further, the primary antibody is immobilized on the surface of a metal thin film substrate constituting the plasmon excitation sensor, preferably a spacer layer, for example, SAM (Self Assembled Monolayer), or is immobilized on the substrate during fluorescence detection. (FIG. 2).

  In the present invention, the secondary antibody is a specific antibody against the same target antigen as the primary antibody, and binds a fluorescent dye serving as the FRET acceptor molecule. The secondary antibody recognizes and binds to an epitope of the same target antigen (however, an epitope different from that of the primary antibody). In this way, the primary antibody and the secondary antibody specifically bind to different sites of the same antigen to form a sandwich-type immune complex, and this is immobilized on the surface of the metal thin film substrate constituting the plasmon excitation sensor. This is the first half of the immunoassay. The secondary antibody may bind non-specifically to the primary antibody but not the target antigen, but in this case it is necessary to prevent FRET from being expressed.

  In the present invention, the term “antibody” includes polyclonal or monoclonal antibodies, antibodies obtained by gene recombination, and antibody fragments. For example, anti-alpha fetoprotein (AFP) monoclonal antibody (available from Japan Medical Laboratory), anti-carcinoembryonic antigen (CEA) monoclonal antibody, anti-CA19-9 monoclonal antibody, anti-PSA monoclonal antibody, etc. It is done.

  When the primary antibody immobilized on the plasmon excitation sensor is a polyclonal antibody, the secondary antibody may be a monoclonal antibody or a polyclonal antibody. When the primary antibody is a monoclonal antibody, the secondary antibody is preferably a monoclonal antibody that recognizes an epitope that the primary antibody does not recognize, or a polyclonal antibody. For example, when the primary antibody immobilized on the plasmon excitation sensor is an AFP monoclonal antibody, the secondary antibody that binds to it is a monoclonal antibody that can recognize and bind to an antigen that competes with AFP contained in the specimen. Requires an antibody or polyclonal antibody.

  Preferable examples include immunoglobulins (antibodies) each consisting of a pair of H and L chains, their Fabs (antigen-binding fragments), or similar protein fragments showing effective epitope affinity. .

  When the analyte is a low molecular weight substance (such as a hapten) that is difficult to be recognized as an epitope having a different antigen, it is desirable to use Fab, scFv, or the like as a binding partner instead of the antibody. Details of such a non-competitive immunoassay are disclosed in US Pat.

  “Specimen” refers to a biological sample, environmental sample, or the like that has been pretreated as required for this assay. Examples of the sample include blood, serum, plasma, urine, nasal fluid, saliva, feces, body cavity fluid (eg, cerebrospinal fluid, ascites fluid, pleural effusion), and the like, and appropriately diluted with a desired solvent, buffer solution, etc. May be. Of these samples, blood, serum, plasma, urine, nasal fluid and saliva are preferred. These may be used alone or in combination of two.

  Antigen contained in the specimen is a molecule or molecular fragment that can be specifically recognized (or recognized) and bound by the secondary antibody and the primary antibody immobilized (or immobilized) on the substrate surface. It is. Examples of such “molecules” or “molecular fragments” 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 modifications thereof Examples thereof include molecules, complexes, and the like. Specifically, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signal transducing substance, a hormone, and the like, and is not particularly limited.

  In the antigen-antibody reaction in the immunoassay method of the present invention, an antigen-antibody reaction is first caused by contacting the specimen with an antibody. In this case, as a condition for incubating the reaction product, 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. In the case of a large number of specimens, when a positive control and a negative control are also performed at the same time, it is convenient to perform on a multi-well plate.

-Formation of immune complex In the step (a) of immobilizing the formed immune complex on the plasmon excitation sensor metal thin film substrate, there are the following three modes for causing an antigen-antibody reaction. First, as two aspects of generating immune complexes in advance,
In the step (a), the specimen is brought into contact with the secondary antibody, and then the secondary antibody bound with the antigen is brought into contact with the metal thin film substrate (of the plasmon excitation sensor) on which the primary antibody is immobilized. A method of immobilizing the immune complex on a metal thin film substrate (for a plasmon excitation sensor);
In the step (a), the primary antibody and the secondary antibody are brought into contact with a specimen, and the formed immune complex is brought into contact with a metal thin film substrate (of a plasmon excitation sensor) to thereby form the immune complex. This is a method of immobilizing on a metal thin film substrate (for a plasmon excitation sensor). In this second mode, the immune complex formed is immobilized by some interaction that occurs between the primary antibody and the binding partner on the metal thin film substrate (of the plasmon excitation sensor) or on the spacer layer. The interaction may be, for example, a gold-S bond, biotin- (strept) avidin, or an antibody against the primary antibody.

  The above-mentioned “contact” is a state in which the surface on which the primary antibody of the plasmon excitation sensor is immobilized is immersed in a liquid (reaction solution) that has been applied or sent. This refers to bringing the immune complex contained in the liquid into contact with the metal thin film substrate or spacer layer of this plasmon excitation sensor. The specific method will be described in detail in the section “Plasmon Excitation Sensor” below. In the step (b), the “contact” between the reaction solution and the plasmon excitation sensor includes, for example, an immune complex formed in the liquid supply circulating in the flow path, and the primary antibody of the plasmon excitation sensor is immobilized. In a state where only one surface is immersed in the liquid feeding, a mode in which the plasmon excitation sensor and the reaction liquid in the step (a) are brought into contact with each other is preferable. This is easily realized by fixing the plasmon excitation sensor to the flow path so that the surface on which the primary antibody is fixed becomes a part of the flow path.

  The first method should be adopted when the primary antibody needs to be stably immobilized on the substrate surface. In the second method, when the surface of the substrate can easily bind the primary antibody that forms an immune complex with the secondary antibody, or when the spacer described below is installed and in such a state Can be adopted. Since the primary antibody, the secondary antibody and the antigen are in a free state in the solution and cause an antigen-antibody reaction, there is a feature that there is no interference such as steric hindrance.

  Further, both of these methods are characterized in that the immune reaction field and the detection field of the formed immune complex are independent from each other. Therefore, there is an advantage that a washing operation for the purpose of B / F (binding / free) separation is not required. It greatly contributes to the reduction of fluorescence background noise.

Another remaining format is similar to the conventional sandwich immunoassay format, where the primary antibody is a solid phase antibody,
A method in which the immune complex is immobilized on a metal thin film substrate of a plasmon excitation sensor by contacting the specimen with a metal thin film substrate on which the primary antibody is immobilized, and then applying a secondary antibody to the substrate. is there. The secondary antibody to which the fluorescent dye of the acceptor molecule is bound is applied to the plasmon excitation sensor metal thin film substrate in contact with the specimen. “Contact” in the case of “contacting the metal thin film substrate on which the primary antibody is immobilized and the specimen, and then applying the secondary antibody to the substrate” is as described above. That is, a solution containing the secondary antibody is brought into contact with the substrate by liquid feeding or the like.

A sample solution is applied to the substrate (or spacer layer), the substrate is washed with a washing buffer, and then a secondary antibody solution is added in a predetermined amount to the surface of the substrate on which the primary antibody is immobilized. For a certain period of time. An antigen-antibody reaction occurs between the primary antibody and the secondary antibody that bind to the antigen contained in the specimen. The washing operation is an operation of washing the surface after applying the specimen to the substrate surface having the primary antibody. Therefore, the washing operation is preferably included before and / or after the application of the secondary antibody. As the cleaning solution to be used, a surfactant such as Tween (registered trademark) 20, Triton (registered trademark) X100 is dissolved in the same solvent or buffer, and preferably contains 0.00001 to 1% by weight. .

  This embodiment should be employed when the primary antibody needs to be stably immobilized on the surface of the substrate and it is preferable to first bind the primary antibody and the antigen.

  As a condition for bringing the substrate having the primary antibody immobilized on the surface thereof into contact with the specimen, the temperature is usually 4 to 50 ° C., preferably 10 to 40 ° C., and the time is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.

  In the step (a) of immobilizing the formed immune complex on the plasmon excitation sensor metal thin film substrate, the immune complex is immobilized on the metal thin film surface of the metal thin film substrate. When a spacer layer is installed as described later, it may be fixed on the surface of the spacer layer. The plasmon excitation sensor includes a metal thin film and a transparent flat substrate, and a spacer layer may be formed on the other surface of the metal thin film that is not in contact with the transparent flat substrate.

  Various conventional techniques may be used to bind a desired fluorescent dye to a primary antibody or a secondary antibody and to immobilize the primary antibody on a substrate. As a method for immobilizing the primary antibody on the substrate surface, an optimal crosslinking method according to the terminal functional group on the substrate surface can be selected. Further, depending on the properties of the substrate surface, a method of directly adsorbing directly to the surface can also be mentioned as an effective means.

  For the labeling of the fluorescent dye on the antibody, a conventionally used fluorescent labeling method for proteins (see, for example, JP-A-6-109732) is used. The fluorescent dye molecule and an appropriate cross-linking agent such as silane are used. What is necessary is just to make it couple | bond with the antibody molecule which reacted the coupling agent etc. and activated this. Examples of the crosslinking agent include N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) and derivatives thereof, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester and derivatives thereof, and the like.

  Specifically, the carboxyl group of the fluorescent dye is converted into water-soluble carbodiimide (WSC) (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)) and N-hydroxysuccinimide ( NHS) and then esterifying the carboxyl group and the amino group of the antibody protein by dehydration using water-soluble carbodiimide; immobilizing the antibody protein and fluorescence having an isothiocyanate and amino group, respectively. A method in which a dye is reacted and immobilized; a method in which an antibody having a sulfonyl halide and an amino group and a fluorescent dye are reacted and immobilized; a method in which an antibody having an iodoacetamide and a thiol group and a fluorescent dye are reacted and immobilized; Be mentioned

  For example, using an immunoglobulin as a primary antibody and a secondary antibody, and utilizing the above-mentioned cross-linking substance having simultaneously a site capable of binding to the Fc region and a labeled fluorescent dye, the desired fluorescent dye is converted into the primary antibody or the secondary antibody. What is necessary is just to couple | bond with an antibody.

FRET
Fluorescence resonance energy transfer is a phenomenon in which a dye (referred to as “donor”) is excited by a light source in a system that detects molecules (donors and acceptor molecules) that interact in close proximity. Later, the energy is transferred to another dye (referred to as an “acceptor”). For energy transfer, the emission spectrum of the donor dye significantly overlaps with the excitation spectrum of the acceptor. The extremely close juxtaposition of the two dyes is generally closer to 100 angstroms (Å), preferably closer to 50 angstroms, more preferably closer to 10 angstroms (1 angstrom is 0.1 nanometer) equal.). Essential for energy transfer between donor / acceptor pairs. FRET is usually based on the interaction between a donor dye and an acceptor dye, both fluorescent dyes.

  In order to achieve resonance energy transfer as FRET, the donor dye molecule must absorb light and transfer it to the second dye molecule, the acceptor, through resonance of the excited electrons. In order to perform energy transfer, the wavelength of the donor's fluorescence emission must be lower than the excitation wavelength of the acceptor, ie the process must be energetically “downhill”.

Examples of suitable fluorescent dyes used as donor molecules, acceptor molecules are fluorescein labels, for example 5- (and 6) -carboxyfluorescein, 5- or 6-carboxyfluorescein, 6- (fluorescein)- 5- (and 6) - carboxamide (carboxamide) hexanoic acid (hexanoic acid) and fluorescein isothiocyanate (fluorescein isothiocyanate), Alexa Fluor (registered trademark) dyes, cyanine dyes, e.g. Cy2, Cy3, Cy5, Cy7, optionally substituted Coumarin, R-phycoerythrin, allophycoerythrin, Texas Red and Princeston Red, and R-phycoerythrin and, for example, Cy5 or Texas Red It is a body. Further, it may be a lanthanide complex, for example, a chelate fluorescent material such as terbium or eurobium.

The donor molecule and acceptor molecule are preferably a FRET pair. Any FRET pair may be used as the FRET pair in the present invention. Preferred FRET pairs (donor molecule / acceptor molecule), for example, Alexa Fluor (registered trademark) 647 / Cy5.5, HiLyte Fluor 647 / Cy5.5, fluorescein isothiocyanate (FITC) / tetramethylrhodamine isothiocyanate (TRITC), R- It is selected from the group of phycoerythrin (R-PE) / allophycocyanin (APC) pairs. The R-PE / APC pair is very promising because 90% FRET efficiency can be achieved despite the R-PE and APC dimensions. Furthermore, this FRET pair can be easily detected utilizing two common lasers in structures emitting at 488 and 632 nm. However, the FRET pair in the present invention is not limited to these examples.

  A LANCE assay combining time-resolved fluorescence technology with Wallac fluorescent lanthanide chelate and FRET technology is known. In LANCE TR-FRET using time-resolved fluorescence immunoassay technology, the donor label (Eurobium chelate) and the acceptor label (Allophycocyanin) are based on proximity to each other. The excited energy of the eurobium chelate transfers energy to the acceptor at a short distance by non-radiative resonance energy transfer. Since the lanthanide that is a fluorescent substance is in an excited state for a long time, nonspecific background due to short-lived fluorescence that can be generated by direct excitation of the acceptor by excitation light can be avoided.

  When the metal thin film included in the following “plasmon excitation sensor” is formed of a metal including gold, 1,3-diculoro-9,9-dimethyl-acidine-2- One-7-yl phosphate (DDAO phosphate) (Molecular Probes) is preferred.

  As for the autofluorescence wavelength of the donor molecule, the greater the difference between the autofluorescence wavelength and the fluorescence wavelength of the acceptor fluorescent dye based on FRET, the easier it is to avoid the influence of the background signal, and as a result, highly accurate measurement is possible. There is no particular limitation.

Step (b): SPFS / FRET
The formed immune complex is qualitatively or quantitatively measured by exciting the fluorescence of the primary antibody constituting it with SPFS and then measuring the fluorescence development of the acceptor molecule of the secondary antibody enhanced by FRET. Can be assayed.

  By bringing the fluorescent dye obtained through the step (a) into contact with the thin film surface of the plasmon excitation sensor having at least the transparent flat substrate and a metal thin film formed on one surface of the substrate, The donor molecule fluorescent dye bound to the antibody is excited. Such a detection field is completely separated from the immune reaction field in a preferred embodiment. This corresponds to the first method and the second method in the antigen-antibody reaction. Specifically, the antigen-antibody reaction solution containing the formed immune complex is used as a thin film surface or spacer layer surface of the plasmon excitation sensor substrate. Or pass through. Accordingly, by appropriately providing a flow path system described later, it is possible to rapidly assay a large number of specimens including standard analytes of known concentrations, controls, and specimen specimens. Moreover, as a mode for high-throughput processing of the assay, a μ-TAS (Micro total Analysis System) form may be taken.

・ Surface plasmon excitation fluorescence method (SPFS)
SPFS generates several dense waves (surface plasmons) on the metal thin film surface under the condition that the irradiated laser light undergoes total reflection attenuation (ATR) on the gold thin film surface, thereby doubling the photon amount of the irradiated laser light by several tens. Increased to several hundred times (electric field enhancement effect of surface plasmon), thereby enabling highly sensitive fluorescence measurement by efficiently exciting the fluorescent dye in the vicinity of the gold thin film.

  The “plasmon excitation sensor” includes a transparent flat substrate and a metal thin film formed on one surface of the substrate, and further preferably has a spacer layer, and the spacer layer is the transparent flat plate of the metal thin film. It is desirable to form on the other surface which is not in contact with the 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 [n _d ] is preferably 1.40-2. And the thickness is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm, and 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, it is preferable that a group having affinity for gold as the surface metal thin film is bound to the primary antibody. For example, mercapto group (—SH), dithio group (—SS—), sulfo group (—SO ₃ H), thiocarboxy group (—C (O) SH), dithiocarboxy group (—CSSH), amino group (—NH) ₂ ) or a hydroxyl group (—OH). A group having a particularly high affinity for gold is a mercapto group, a dithio group, a sulfo group, a thiocarboxy group, or a dithiocarboxy group, and particularly preferably a mercapto group.

  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. Further, in order to capture the immune complex on the surface of the plasmon excitation sensor via the primary antibody constituting the complex, streptavidin may be bound to the spacer layer or a streptavidinized portion may be included. Biotin bound to the primary antibody and its streptavidin specifically interact with each other to achieve “immobilization of the primary antibody by capture”.

  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 immune complex obtained through the above step (a) into contact with the surface of the metal thin film substrate or the spacer layer side surface of such a “plasmon excitation sensor”, a solution containing the fluorescent dye is dropped. , Spraying, coating, and the like. 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. A polymer such as

  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 a screw 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 for diluting the specimen, 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.

・ Fluorescence measurement The second half of this assay consists of the plasmon excitation sensor with the above-mentioned immune complex immobilized on the other surface of the substrate on which the thin film is not formed, and laser light via a prism. , And the amount of fluorescence emitted from the excited secondary antibody fluorescent dye is measured. From the measurement result obtained in the above step, the antigen amount of the analyte contained in the specimen is calculated.

  More specifically, the laser light irradiation by the surface plasmon excitation fluorescence method is performed by transmitting the laser light to the immune complex via a prism from the other transparent substrate surface on which the metal thin film of the plasmon excitation sensor is not formed. Irradiation is performed, and thereby the fluorescence amount of the acceptor molecule emitted by the fluorescence resonance energy transfer (FRET) phenomenon from the excited donor molecule fluorescent dye of the primary antibody is determined. A calibration curve can be created by performing measurement with a target antigen at a known concentration, and the amount of target antigen in the sample can be calculated from the measured fluorescence amount based on the created calibration curve.

  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 donor 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.

Table 1, as a fluorescent dye, Alexa Fluor (R) 647 (in Table 1, Condition 1-4), respectively (in Table 1, Condition 5-6) and HiLyte Fluo r6 47 using, SPFS by plasmon excitation sensor A fluorescent signal is shown.

  In Table 1, 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 1, it can be seen that highly sensitive measurement can be carried out even in a solution containing a very small amount of fluorescent dye. This result shows that the measurement is possible even at least under the condition that the fluorescent dye is present in an extremely small amount as shown in Table 1. That is, the immunoassay method of the present invention shows that highly sensitive measurement is possible through measurement of the formed immune complex.

  Table 2 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 fluorescent dye is relatively large compared to “Noise”, and has high sensitivity. 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. The fine particles are adhered to the surface of the sensor by washing with MilliQ water.

  From Table 2, 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 exist 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.

  According to the immunoassay of the present invention, in a detection field by a plasmon excitation sensor, a fluorescent dye possessed by a free secondary antibody or a secondary antibody non-specifically bound to the primary antibody by fluorescence measurement using a plasmon enhancement field, Since FRET does not appear, it does not emit fluorescence or remains very weak (FIG. 2). Therefore, the fluorescence background noise is at a low level. Since only fluorescence derived from the formed immune complex can be detected, even a very small amount of immune complex can be detected with high sensitivity by enhanced fluorescence emission. In particular, when the reaction field that forms an immune complex and the detection field based on fluorescence emission are separated as described above, optimization of various conditions and suppression of noise and background levels can be achieved very effectively. In this sense, the mode 1 or mode 2 in the step (a) is preferable.

  On the other hand, in the conventional sandwich immunoassay, detection is performed by labeling a secondary antibody with a fluorescent dye and exciting it. Originally, even if we wanted to detect only the fluorescence derived from the immune complex, it was inevitable that the fluorescent dye of the non-specifically bound or free secondary antibody would actually emit light. It becomes higher (Fig. 1). In order to sufficiently reduce the noise level, it is necessary to perform B / F separation by performing a cleaning operation.

<Immunoassay system>
The system of the present invention is a set of apparatuses for performing the assay method of the present invention using the plasmon excitation sensor. That is,
at least,
A primary antibody bound to a fluorescent dye serving as a donor molecule, which is immobilized on a metal thin film substrate of a plasmon excitation sensor,
A secondary antibody to which a fluorescent dye serving as an acceptor molecule is bound,
A plasmon excitation sensor, a light source for surface plasmon excitation fluorescence method, and a fluorescence detector, and cause a sandwich-type antigen-antibody reaction between both the primary antibody and the secondary antibody and the antigen contained in the specimen. The plasmon excitation sensor emits light by fluorescence resonance energy transfer from the fluorescent dye of the primary antibody excited by irradiating the immune complex immobilized on the metal thin film substrate with laser light by the surface plasmon excitation fluorescence method. An immunoassay system characterized in that the amount of the antigen is measured by measuring the amount of fluorescence of the acceptor molecule.

  The “apparatus” of the system of the present invention 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 a liquid feeding system, for example, a microchannel device (for example, μ-TAS) connected to a liquid feeding 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 photodiode and the light source are synchronized with each other so that the angle can be changed by 45 to 85 ° (the resolution is preferably 0.01 ° or more).) Data processing for processing information input to the SPFS detector A computer may also be included.

  Preferred embodiments of the light source, the optical filter, the cut filter, the condenser 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 in that it is used for the immunoassay of the present invention, and in addition to the primary antibody and the secondary antibody, all the necessary antibodies are required for carrying out the assay of the present invention. It is preferable to include. As a preferred kit,
Including at least a plasmon excitation sensor, a primary antibody bound to a fluorescent dye serving as a donor molecule, which is an antibody immobilized on the surface of the metal thin film substrate, and a secondary antibody bound to a fluorescent dye serving as an acceptor molecule, In this immunoassay method, a kit used for carrying out the step of forming an immune complex by causing a sandwich-type antigen-antibody reaction between the primary antibody, the antigen in the specimen, and the secondary antibody is shown. It is.

  The metal thin film that is a member of the plasmon excitation sensor is desirably formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum. A more preferable metal thin film is a gold thin film. Furthermore, the plasmon excitation sensor is composed of the metal thin film and the transparent flat substrate, and further has a spacer layer, and 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 embodiment is preferred. The configuration, installation method, significance, and the like of such a spacer layer are as described above. Such a spacer layer is preferably a SAM (Self Assembled Monolayer).

  As a component of the “kit” of the present invention, specifically, in addition to a plasmon excitation sensor in which a metal thin film is formed on one surface of a transparent flat substrate, a lysis solution or dilution solution for dissolving or diluting a specimen; antibody Various reaction reagents and washing reagents for reacting the analyte with the analyte, and various equipment or materials required for carrying out the assay method of the present invention can also be included. Further, as a kit element, a standard material for preparing a calibration curve, a manual, a necessary set of equipment such as a microtiter plate capable of simultaneously processing a large number of samples may be included.

Problems of FRET In the present invention, the FRET technique is applied by labeling two types of antibodies (primary and secondary antibodies that are binding partners) with fluorescent dyes that form a FRET donor-acceptor pair. When the specimen is brought into contact with the primary antibody and the secondary antibody, an immune complex is formed between the antigen and the antibody in the specimen. If the binding partner and the analyte are relatively small in such an immune complex, the fluorophores may come close to each other, resulting in the desired measurable FRET signal. Be expected.

  In order to allow the donor / acceptor pair to induce a detectable FRET fluorescence signal, a short distance (1-10 nm) between the donor molecule and the acceptor molecule is required as described above. . However, in order to detect the extremely small amount of analyte targeted by the present invention with extremely high sensitivity, the distance between the donor molecule and the acceptor molecule present in the formed immune complex is further set to about 10 nm. And the fluorescent dyes of the two antibodies need to be oriented (or juxtaposed) with each other so that they are suitable for FRET. A device for realizing such precise molecular orientation is required.

-Optimization of FRET effect In the assay of the present invention, as described above, it is a precondition for detecting an immune complex that a donor / acceptor pair induces a measurable FRET fluorescence signal.

  A complete antibody is a large Y-type protein molecule, and due to its length (30-40 nm) and the flexibility of the antibody hinge region, antibody molecules will be conjugated at relatively large intervals. Antibody flexibility may reduce the possibility of energy transfer between a pair of dyes bound to side-by-side antibodies. Also, the size of the antibody or fluorescent dye may interfere with energy transfer through the negative steric effect exerted. Furthermore, when a fluorescent dye is bound to an antibody, it is generally not easy to control the complexing site. That is, in the case of antibody conjugates, the fluorescent dye moiety can be attached to a different part of the antibody molecule. Depending on such sites, the spatial orientation of the dye in the antibody molecule can be advantageous or inconvenient with respect to energy transfer efficiency. Thus, close juxtaposition of molecules that induce FRET is a requirement for energy transfer between donor / acceptor pairs, while in molecules that are part of interacting molecules or complexes, the parallel May not necessarily be sufficient to achieve effective energy transfer between the dyes attached to the prepared antibody, eg, between the fluorescent dye-conjugated antibodies.

  Of the initial conditions for energy transfer to occur as FRET, one is that the donor and acceptor molecules must be in close proximity, typically 0.2 to 10 nm. In view of the above-mentioned problems assumed, the following measures can be taken as a measure for obtaining the FRET signal as a desired intensity.

  1. The antibody is fragmented (Fab) and the molecular size is reduced to reduce the fluctuation of the molecular structure, thereby reducing FRET inhibition. In addition, the distance between the labeled fluorescent dyes can be shortened.

  2. The antibody is reduced, and fluorescent labeling is performed using the SH group appearing in the Fc part of the antibody. In this case, the labeling rate per molecule may be lower than the method using amino groups, but since the position of the SH group is fixed, the labeling position can be determined (if there is another free SH) This is also labeled). Furthermore, since the fluorescent dye can be labeled near the center of the antibody molecule, the distance between the dyes can be shortened.

  3. Stabilization of parallelism between antibodies by introducing a binding reactive group that can regulate the spatial structure of FRET in the antibody molecule so as to increase the possibility of the energy transfer and / or increase the energy transfer efficiency. And / or can be enhanced.

  Such binding reactive groups make it possible to adjust the alignment between the antibodies so as to increase the possibility of energy transfer between the dyes. Use of such an antibody set may dramatically increase the probability of energy transfer compared to the use of an antibody set without a binding reactive group. Spatial structure here refers not only to the spacing between the dyes but also to their relative orientation. Modulating the spatial structure between antibodies includes modulating and stabilizing the spatial structure of the dye. The conjugating reactive groups can bring the dyes into an interval within 10 nm of each other, more preferably within an interval of within 5 nm, and most preferably within an interval of within 2 nm of each other. Thus, preferably the linking reactive group is small, for example smaller than 10 kilodalton (kDa), preferably smaller than 5 kDa, more preferably smaller than 2 kDa, or most preferably smaller than 1 kDa (US Pat. ).

3-1. Method using biotin- (strept) avidin system Biotin- (strept) avidin system is preferred as an example of the above-mentioned binding reaction group for adjusting the spatial arrangement of fluorescent dyes added to an antibody, and has such a small size. This conjugation reactive group is advantageous to achieve close spacing between the fluorescent dye pairs. Biotin can bind proteins with small molecules having a molecular weight of only 244 daltons (Da). Various standard procedures for preparing biotin conjugates are known to those skilled in the art. For example, when streptavidin is bound to a secondary antibody and biotin is bound to a primary antibody, when a complex of both antibodies is formed via a common antigen, the binding reaction group of these antibodies, biotin and streptoid Avidin binds. Spatial rearrangement based on the bond formation results in a close orientation between the fluorescent dye pairs of both antibodies. Furthermore, it is also desirable to attach a binding group for anchoring to the metal thin film or spacer layer of the plasmon excitation sensor on the Fc base of the primary antibody (in this case, the primary antibody is immobilized in advance). May be)

  Alternatively, DNA or polynucleotide of an appropriate size may be introduced as a binding reaction group as described below, and hybridization thereof may be used.

3-2. Method 1 using DNA 1) A primary antibody is labeled with a DNA-donor dye, and a secondary antibody is labeled with a complementary strand DNA-acceptor molecule.
2) Immobilize the primary antibody on the substrate.
3) The primary antibody is reacted with an antigen and then a secondary antibody to form an immune complex, which is immobilized on the substrate via the primary antibody. Alternatively, the immune complex may be immobilized by bringing a reaction solution of the secondary antibody and the specimen (a reaction solution containing a complex of the antigen and the secondary antibody) into contact with the substrate.

At this time, under normal conditions, DNA and complementary strand DNA may hybridize regardless of the presence or absence of immune complex formation. Therefore, as a first condition, conditions (conditions such as salt concentration and temperature) that “immune complexes are formed but DNA hybridization does not occur” are set. For example, when the salt concentration is low, hybridization does not occur.
4) Remove unreacted secondary antibody with wash buffer.
5) Next, conditions (for example, salt concentration, temperature) for “maintaining immune complexes and allowing the DNA to hybridize” are set and the conditions are applied. If the salt concentration is high, hybridization occurs, but if it is too high, the immune reaction may be inhibited. Therefore, the salt concentration is preferably about 150 mM.
6) Hybridization occurs between the primary antibody DNA-donor dye and the secondary antibody complementary-strand DNA-acceptor dye, and FRET is expressed between the donor dye and the acceptor dye.

  S / N improvement is expected by this method. This is because the higher the dye modification rate of DNA, the more signal improvement can be expected. DNA-complementary strand DNA hybridization need not be specific (for example, as in SNP analysis), and may be within a certain distance from the nearby one.

As a modified example of the above method, 1) and 2) are the same, but the steps after 3) may be changed as follows.
3) The primary antibody is reacted with an antigen and then a secondary antibody to form an immune complex, which is immobilized on the substrate via the primary antibody. Alternatively, the immune complex may be immobilized by bringing a reaction solution of the secondary antibody and the specimen (a reaction solution containing a complex of the antigen and the secondary antibody) into contact with the substrate. At that time, the DNA and the complementary strand DNA hybridize regardless of the presence or absence of immune complex formation.
4) Set conditions (for example, salt concentration, temperature) that “maintain immune complexes and dissociate double-stranded DNA” and apply the conditions. Unreacted secondary antibody not forming an immune complex is removed with a washing buffer. At this time, the hybridization between the DNA forming the immune complex and the complementary strand DNA is once dissociated.
5) Set conditions (for example, salt concentration, temperature) for “maintaining immune complexes and allowing DNA to hybridize”, and apply the conditions.
6) The DNA and the complementary strand DNA hybridize with the nearest neighbors.

  That is, the primary antibody and the DNA labeled with the secondary antibody that are closer to each other due to the formation of the immune complex hybridize preferentially, and FRET is expressed between the donor dye and the acceptor dye.

  In this case, there is an advantage that B / F separation is not required with the improvement of S / N.

Moreover, as another aspect of the modified example, the above 1) to 4) are the same, but the steps after 5) are as follows.
5) A buffer solution containing a protein denaturant (such as urea) and having a high salt concentration is developed on the substrate. As a result, the protein is denatured and dissolved in a state where the structure is broken. The DNA on the substrate and the complementary strand DNA are not affected by the three-dimensional structure and hybridize with high probability. The primary antibody does not come off if it is immobilized on the substrate by a covalent bond. Also, DNA is not detached from the substrate even under denaturing conditions as long as it is covalently bound to the antibody molecule. In contrast, the secondary antibody may be released. Further, the complementary strand DNA that has been bound to the secondary antibody may be removed from the antibody molecule.
6) The primary antibody DNA-donor dye immobilized on the substrate and the secondary antibody-derived complementary DNA-acceptor dye are hybridized, and FRET is expressed between the donor dye and the acceptor dye.

  In this case, improvement in FRET efficiency can be expected, and it is not affected by the kinetics of the immune reaction. It is necessary to set protein denaturation conditions so that the DNA is stable and hybridizes with the complementary strand DNA.

  The primary antibody, specimen and secondary antibody are reacted in advance, and the formed immune complex is then contacted with a (plasmon excitation sensor) metal thin film substrate to bind the primary antibody of the immune complex onto the substrate. In the method of finally immobilizing on the substrate, conditions (for example, salt concentration, temperature) are set after “immobilization of the immune complex and DNA is hybridized” after the immobilization. do it.

Preferred Embodiment A preferred form of the assay of the present invention is an embodiment in which an immune reaction field and a fluorescence detection field are separated. That is, in the immune reaction field, the primary antibody labeled with the donor molecule recognizes and binds to the target antigen contained in the specimen, and the secondary antibody labeled with the acceptor molecule also recognizes another epitope of the antigen. Binding, resulting in the formation of a sandwich-type immune complex.

  The reactants of these antigens and both antibodies are then applied to a plasmon excitation sensor. The binding group of the primary antibody Fc base is captured by the tethering binding group provided on the surface of the metal thin film substrate, and the sandwich-type immune complex is immobilized on the metal thin film substrate (the above step (a )). Here, if necessary, a washing operation with a washing buffer may be performed.

  The detection field of the assay includes a glass transparent flat substrate, a metal thin film formed on one surface of the substrate, and a spacer layer formed on the other surface of the thin film that is not in contact with the substrate. Is a plasmon excitation sensor. The sandwich-type immune complex that is immobilized in contact with the surface on the spacer layer side is irradiated with a laser beam via a prism from the other surface of the substrate on which the thin film is not formed. To do. Primary antibody donor molecules emit light when excited by surface plasmons. A fluorescent dye (acceptor molecule) bound to the secondary antibody by FRET expression is excited to emit light. Since only the immune complex thus formed emits enhanced fluorescence, this is detected and the amount of fluorescence is measured (step (b) above).

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the examples. Note that the device name, the material used, the concentration of the material used, the amount used, the numerical conditions such as the processing time and the processing temperature, the processing method, etc. are only suitable examples within the scope of the present invention. Absent.

[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 disposed so that the SAM surface is inside the flow path (however, the silicon rubber spacer is used for liquid feeding). The pressure-sensitive adhesive sheet was pressed from the outside of the flow path, and the flow path sheet and the plasmon excitation sensor were fixed with screws.

[Preparation Example 2] (Preparation of Alexa Fluor (registered trademark) 647-labeled primary antibody)
As a primary antibody, an anti-α fetoprotein (AFP) monoclonal antibody (available from Nippon Medical Laboratory, Inc.) 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 the streptavidin-labeled Alexa Fluor (registered trademark) 647 (Molecular Probes) solution are mixed and reacted by stirring at 4 ° C. for 60 minutes. It was. Subsequently, 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 the protein amount was quantified.

[Production Example 3] (Production of Cy5.5-labeled secondary antibody)
A solution of biotinylated anti-AFP monoclonal antibody obtained in Preparation Example 2 and streptavidin-labeled Cy5. 5 ( Molecular Probes) solution was mixed and reacted at 4 ° C. for 60 minutes with stirring.

Next, the unreacted antibody and the unreacted enzyme are purified using a molecular weight cut filter (manufactured by Nippon Millipore) to obtain Cy5. 5 to give the target識抗AFP monoclonal antibody solution. The obtained antibody solution was stored at 4 ° C. after protein quantification.

[Example 1]
(Production of plasmon excitation sensor)
A plasmon excitation sensor is prepared in the same manner as in [Preparation Example 1], and the primary antibody is immobilized.

First, ultrapure water was circulated with a peristaltic pump at room temperature and a flow rate of 500 μL / min for 10 minutes and then PBS for 20 minutes. 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, and then the method of [Preparation Example 2] Then, 2.5 mL of Alexa Fluor (registered trademark) 647-labeled primary antibody solution labeled with α-fetoprotein (AFP) monoclonal antibody (1D5, 2.5 mg / mL, manufactured by Japan Medical Laboratory) was circulated for 30 minutes. By immersing, the primary antibody was immobilized on the SAM. Finally, a non-specific adsorption prevention treatment is performed by circulating the solution for 30 minutes in PBS buffered saline containing 1% bovine serum albumin (BSA), and a plasma excitation sensor is prepared using SPFS. The assay was performed.

(Implementation of assay method)
Step (i): The solution was replaced with PBS, 2.5 mL of PBS containing 1 ng / mL of AFP was added and circulated for 25 minutes.

Washing step: Washing was performed by circulating TBS containing 0.05% by weight of Tween (registered trademark) 20 for 20 minutes. Here, the blank fluorescence is used as a light source, an LD laser is used as a light source, a laser beam having a wavelength of 635 nm is adjusted with an optical filter: (Sigma Kogyo Co., Ltd.), and the amount of photons is adjusted. -LAL10 (refractive index [n] = 1.72)) is used to irradiate the plasmon excitation sensor fixed to the flow path, a cut filter (Sigma Kogyo Co., Ltd.), and a 20 × objective lens as a condenser lens It was detected by a CCD image sensor (manufactured by Texas Instruments) using Nikon Corporation.

  Step (ii): Cy5.5-labeled secondary antibody labeled with α-fetoprotein (AFP) monoclonal antibody (6D2, 2.5 mg / mL, manufactured by Japan Medical Laboratory) by the method of [Preparation Example 3] 2.5 mL of PBS containing 1,000 ng / mL was added and circulated for 20 minutes.

  Step (iii): The signal value when observed from the CCD 20 minutes after the start of liquid feeding 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.

  Step (iv): The assay S / N ratio in the plasmon excitation sensor of the present invention was evaluated by the following equation. That is, the reliability of the immunoassay measurement is high when the numerical value of the fluorescent signal that changes with the amount of the fluorescent dye proportional to the amount of antigen is large and the assay noise signal is numerically sufficiently small relative to the assay fluorescent signal.

Assay S / N ratio = | (assay fluorescence signal) | / | (assay noise signal) |
The obtained results are shown in Table 3.

[Example 2]
(Production of plasmon excitation sensor)
It was produced in the same manner as in Example 1.

(Implementation of assay method)
Step (i): The solution was replaced with PBS, 2.5 mL of PBS containing 1 ng / mL of AFP was added and circulated for 25 minutes.

Washing step: Washing was performed by circulating TBS containing 0.05% by weight of Tween (registered trademark) 20 for 20 minutes. Here, the blank fluorescence is used as a light source, an LD laser is used as a light source, a laser beam having a wavelength of 635 nm is adjusted with an optical filter: (Sigma Kogyo Co., Ltd.), and the amount of photons is adjusted. -LAL10 (refractive index [n] = 1.72)) is used to irradiate the plasmon excitation sensor fixed to the flow path, a cut filter (Sigma Kogyo Co., Ltd.), and a 20 × objective lens as a condenser lens It was detected by a CCD image sensor (manufactured by Texas Instruments) using Nikon Corporation.

  Step (ii): Cy5.5-labeled secondary antibody labeled with α-fetoprotein (AFP) monoclonal antibody (6D2, 2.5 mg / mL, manufactured by Japan Medical Laboratory) by the method of [Preparation Example 3] 2.5 mL of PBS containing 1,000 ng / mL was added and circulated for 20 minutes.

Washing step: Washing was performed by circulating TBS containing 0.05% by weight of Tween (registered trademark) 20 for 10 minutes.

  Step (iii): The signal value observed from the CCD 10 minutes after the start of washing 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.

  Step (iv): The assay was evaluated by calculating the same assay S / N ratio as in Example 1.

  The obtained results are shown in Table 3.

[Comparative Example 1]
(Production of plasmon excitation sensor)
A plasmon excitation sensor was prepared in the same manner as in [Preparation Example 1], and the primary antibody was immobilized.

  First, ultrapure water was circulated with a peristaltic pump at room temperature and a flow rate of 500 μL / min for 10 minutes and then PBS for 20 minutes. 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, and then anti-α-fetoprotein (AFP) monoclonal. The primary antibody was solid-phased on the SAM by circulating 2.5 mL of an antibody (1D5, 2.5 mg / mL, manufactured by Japan Medical Clinical Laboratory Laboratories) solution for 30 minutes. Finally, a non-specific adsorption prevention treatment is performed by circulating the solution for 30 minutes in PBS buffered saline containing 1% bovine serum albumin (BSA), and a plasma excitation sensor is prepared using SPFS. The assay was performed.

(Implementation of assay method)
Step (i): The solution was replaced with PBS, 2.5 mL of PBS containing 1 ng / mL of AFP was added and circulated for 25 minutes.

Washing step: Washing was performed by circulating TBS containing 0.05% by weight of Tween (registered trademark) 20 for 20 minutes. Here, the blank fluorescence is used as a light source, an LD laser is used as a light source, a laser beam having a wavelength of 635 nm is adjusted with an optical filter: (Sigma Kogyo Co., Ltd.), and the amount of photons is adjusted. -LAL10 (refractive index [n] = 1.72)) is used to irradiate the plasmon excitation sensor fixed to the flow path, a cut filter (Sigma Kogyo Co., Ltd.), and a 20 × objective lens as a condenser lens It was detected by a CCD image sensor (manufactured by Texas Instruments) using Nikon Corporation.

Step (ii): Alexa Fluor (registered trademark) 647 labeled with α-fetoprotein (AFP) monoclonal antibody (6D2, 2.5 mg / mL, manufactured by Japan Medical Laboratory) by the method of [Preparation Example 2] 2.5 mL of PBS containing 1,000 ng / mL of the labeled secondary antibody solution was added and circulated for 20 minutes.

Washing step: Washing was performed by circulating TBS containing 0.05% by weight of Tween (registered trademark) 20 for 10 minutes.
Step (iii): The signal value observed from the CCD 10 minutes after the start of washing 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.

  Step (iv): The assay was evaluated by calculating the same assay S / N ratio as in Example 1.

  The obtained results are shown in Table 3.

  From Table 3, it was found that in the present invention, a very high assay S / N ratio was achieved as compared with the conventional fluorescence-labeled SPFS measurement, and an extremely sensitive measurement method was possible.

  Example 2 was obtained by adding a washing operation to the assay process of Example 1, but the assay signal was slightly lower than that of Example 1 due to the addition of the washing operation. However, since the assay signal noise hardly changes, it is considered that there is almost no increase in the assay signal due to nonspecific adsorption in Example 1. From this, it is considered that the decrease in the assay signal originates from the fact that the equilibrium state of the immune complex changes due to the replacement of the reaction solution by the washing operation, and the secondary antibody gradually dissociates. .

  In addition, the assay signal is reduced by about 20% compared to the assay signal of the fluorescence labeled SPFS measurement shown in Comparative Example 1. This is considered to be the effect of insufficient FRET efficiency, and further improvement can be expected by optimizing the FRET efficiency.

  Since the assay method of the present invention is a method capable of detecting a target antigen with high sensitivity and high accuracy, for example, even a trace amount of tumor marker contained in blood can be detected, 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.

DESCRIPTION OF SYMBOLS 10 ... Metal thin film substrate 20 ... Primary antibody 30 ... Antigen 40 ... Secondary antibody 41 ... Free secondary antibody 42・ ・ ・ ・ ・ ・ Non-specific secondary antibody 50 ・ ・ ・ ・ ・ ・ Fluorescent dye 60 ・ ・ ・ ・ ・ ・ Fluorescent dye 70 as donor molecule ・ ・ ・ ・ ・ ・ Fluorescent dye 100 as acceptor molecule ・... Excitation 200 ... Detection 300 ... Noise 400 ... FRET
500 ... Detection target (immunocomplex)

Claims (16)

An immunoassay method for measuring the amount of antigen in a specimen using a primary antibody bound with a fluorescent dye as a donor molecule and a secondary antibody bound with a fluorescent dye as an acceptor molecule, comprising at least
Step (a): An antigen-antibody reaction is caused between an antigen contained in a specimen and both the primary antibody and the secondary antibody, and an immune complex sandwiched by the two antibodies is used as a plasmon excitation sensor. Immobilizing on a metal thin film substrate,
Step (b): Based on the surface plasmon excitation fluorescence method, the plasmon excitation sensor on which the immune complex is immobilized is irradiated with laser light, and fluorescence resonance energy transfer from the donor molecule fluorescent dye excited with the primary antibody ( Measuring the amount of fluorescence of the acceptor molecule of the secondary antibody emitted by FRET),
An immunoassay method comprising the steps of:   The immunoassay method according to claim 1, wherein the secondary antibody is obtained by binding a fluorescent dye serving as the acceptor molecule to an antibody fragment.   The immunoassay method according to claim 1, wherein the secondary antibody is obtained by binding a fluorescent dye serving as the acceptor molecule via an SH group appearing in a reduced antibody.   The immunoassay according to any one of claims 1 to 3, wherein the primary antibody and the secondary antibody are those into which a binding reactive group capable of regulating the spatial structure of FRET is introduced by a binding mode other than an antigen-antibody reaction. Method.   The immunoassay method according to claim 4, wherein the binding reactive group is biotin and avidin.   The binding reactive group is a DNA or polynucleotide and a complementary strand thereof, and the primary antibody is obtained by binding the DNA or polynucleotide to which a fluorescent dye serving as the donor molecule is bound to an antibody, and the secondary antibody. The immunoassay method according to claim 4, wherein the antibody is obtained by binding the complementary chain, to which the fluorescent dye serving as the acceptor molecule is bound, to the antibody.   In the step (a), the specimen and the secondary antibody are brought into contact, and then the secondary antibody bound with the antigen is brought into contact with the metal thin film substrate on which the primary antibody is immobilized. The immunoassay method according to claim 1, wherein the immunoassay method is immobilized on a metal thin film substrate.   In step (a), the immune complex is immobilized on the metal thin film substrate by contacting the specimen with the metal thin film substrate on which the primary antibody is immobilized, and then bringing the secondary antibody into contact with the substrate. The immunoassay method according to any one of claims 1 to 6, wherein   In the step (a), the primary antibody and the secondary antibody are brought into contact with a specimen to form the immune complex, and then the immune complex is brought into contact with a metal thin film substrate to be placed on the metal thin film substrate. The immunoassay method according to claim 1, which is immobilized. The donor and acceptor molecules are, Alexa Fluor (registered trademark) 647 / Cy5.5, HiLyte Fluor647 / Cy5.5, fluorescein isothiocyanate (FITC) / tetramethylrhodamine isothiocyanate (TRITC), and R- phycoerythrin (R- The immunoassay method according to any one of claims 1 to 9, which is a FRET pair selected from a pair group consisting of PE) / allophycocyanin (APC).   At least a plasmon excitation sensor, an antibody immobilized on the surface of the metal thin film substrate, and the fluorescent dye, primary antibody and reaction reagent, and acceptor molecule for producing a primary antibody bound with a fluorescent dye serving as a donor molecule The immunoassay method according to any one of claims 1 to 10, comprising the fluorescent dye, a secondary antibody and a reaction reagent for producing a secondary antibody to which a fluorescent dye to be bound is formed. A kit used for carrying out a step of forming an immune complex by causing a sandwich-type antigen-antibody reaction between the antigen and the secondary antibody.   The kit according to claim 11, for producing a secondary antibody in which a fluorescent dye to be the acceptor molecule is bound to an antibody fragment.   The kit according to claim 11, wherein the secondary antibody is prepared by binding a fluorescent dye serving as the acceptor molecule via an SH group appearing in a reduced antibody.   The said primary antibody and a secondary antibody for producing what introduce | transduced the coupling | bonding reaction group which can adjust the spatial structure of FRET by coupling | bonding modes other than an antigen antibody reaction. Kit.   The kit according to claim 14, wherein the binding reactive group is biotin and avidin.   The binding reactive group is a DNA or polynucleotide and its complementary strand, and the primary antibody is a DNA or polynucleotide bound with a fluorescent dye serving as the donor molecule bound to an antibody. The kit according to claim 14, wherein the next antibody is obtained by binding the complementary strand to which the fluorescent dye serving as the acceptor molecule is bound to the antibody.

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