JP2016205994A - Method for detection or quantitative determination and device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To promote combination and aggregation, for example, of an antigen-antibody reaction while suppressing damages on a sample such as thermal denaturation of protein.SOLUTION: The present invention provides a method for detection or quantitative determination of a detection target 50. The method includes the steps of: mixing a first combined material of a first affinic material 12 with the detection target 50 and a first resonating structure 11 and the sample; resonating the first resonating structure 11 by imparting a wave with a vibration frequency specific to the resonating structure 11; and making a determination for a complex including the first combined material 10 and the detection target 50.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a detection or quantification method and apparatus.

  Conventionally, a latex agglutination method has been performed as a method for detecting a detection target in a specimen. For example, in the case of detecting an antigen in a fluid such as a biological sample, the latex agglutination method is a method of mixing a fluid with a latex carrying an antibody or a fragment thereof that specifically binds to the antigen. This is a method of detecting or quantifying an antigen by measuring the degree (see, for example, Patent Document 1).

  According to this latex agglutination method, an antigen added as a specimen crosslinks a plurality of latex-bound antibodies to promote latex agglutination. However, when the amount of the antigen is very small, the crosslinking is difficult to occur, so that the latex does not aggregate sufficiently, and even if it is aggregated, it cannot be detected because it is below the detection sensitivity. For this reason, it was difficult to rapidly detect a trace amount of antigen.

  Therefore, methods utilizing enzyme substrate reactions such as ELISA and CLEIA are also widely adopted. In these methods, for example, a primary antibody that specifically binds to an antigen is bound to the antigen, and a secondary antibody having an enzyme is bound to the primary antibody. Here, the antigen is detected or quantified by adding the enzyme substrate and measuring the degree of reaction catalyzed by the enzyme. According to these methods, for example, when a coloring reagent or a luminescent reagent is used as a substrate, the detection sensitivity of coloring or luminescence after the addition of the substrate is high.

  In order to shorten the stirring time for reacting the specimen and the reagent, a stirring apparatus has been proposed that generates an acoustic flow by radiating sound waves in the resonance frequency band to a liquid sample containing the specimen and the reagent ( For example, see Patent Document 2). However, this sound wave is generated from a sound generating portion attached to the wall surface of the reaction vessel, and requires a high frequency of about several MHz to several hundred MHz in order to stir the entire liquid sample in the vessel. Therefore, there is a possibility that the measurement object such as a protein is thermally denatured. In order to minimize damage to the liquid sample such as heat denaturation of the protein, it is necessary to instantaneously suppress the generation time of the sound wave, and there is a possibility that the stirring becomes insufficient.

  In addition, in the ELISA method, it has been proposed to use resonance in order to quickly contact the capture antibody immobilized on the inner wall of the tip / cone with the antigen in the specimen (see, for example, Patent Document 3). . However, this resonance also occurs in the entire solution. Further, it is necessary to perform a suction / discharge operation of the reagent in the well facing the chip cone at a frequency causing resonance, and a special device is required.

Japanese Patent Publication No.58-ll575 JP 2010-91306 A Special table 2013-532834 gazette

  The present invention has been made in view of the above circumstances, and an object thereof is to promote binding, aggregation, and the like in an antigen-antibody reaction and the like while suppressing damage to a specimen such as heat denaturation of a protein.

  In the reaction field where bonding, aggregation, and the like are performed, the present inventors can resonate a structure that is not a dispersion medium or a solvent but a dispersoid, thereby increasing the chance of contact of the reactant by vibration, and as a result. The present inventors have found that binding, aggregation and the like can be promoted, and have completed the present invention. Specifically, the present invention is as follows.

[1] A method for detecting or quantifying a detection target in a specimen,
Mixing the first binding substance in which the first affinity substance for the detection target and the first resonant structure are bound, and the specimen;
Resonating the first resonant structure by applying a wave having a frequency unique to the first resonant structure;
A method comprising a step of determining a complex including the first bound substance and the detection target.
[2] Mixing the first binding substance, the specimen, and a second affinity substance for the detection target;
The method according to [1], wherein the first affinity substance and the second affinity substance are capable of simultaneously binding to the detection target at different sites of the detection target.
[3] The second affinity substance is combined with at least the second resonant structure to form a second combined substance,
The second resonant structure has the same or different natural frequency as the first resonant structure,
In the method, after the second affinity substance is mixed with the specimen and before the determination, the second resonance property is applied by applying a wave having a specific frequency to the second resonance structure. The method according to [2], further comprising resonating the structure.
[4] The first affinity substance or the second affinity substance is bound to a labeling enzyme,
The method according to [2] or [3], wherein the method further comprises reacting the labeling enzyme present in the complex with its substrate before or when performing the determination. .
[5] The first resonant structure includes solid particles made of a single substance, hollow particles that may have a structure in a part of a hollow portion, and particles made of a metal and an organic polymer. The method according to any one of [1] to [4], which is a particle selected from the group consisting of:
[6] The first combined substance and / or the second combined structure further includes a magnetic substance,
The method further includes separating the agglomerated magnetic substance by applying a magnetic force after the resonance, before performing the determination, or when performing the determination. The method according to claim 1.
[7] An apparatus for detecting or quantifying a detection target in a specimen,
The apparatus includes a facility for installing a container including a first binding substance in which a first affinity substance for the detection target, a first resonant structure is bound, and the specimen,
Means for applying a wave having a frequency unique to the first resonant structure; and
An apparatus comprising means for determining a complex including the first bound substance and the detection target.
[8] A method for allowing a substance A to interact with an affinity substance for the substance A,
Mixing the substance A and the binding substance in which the affinity substance and the resonant structure are bound,
And resonating the resonant structure by applying a wave having a natural frequency to the resonant structure.
[9] A binding material including an affinity substance for a detection target and a resonance structure-containing body,
The resonant structure-containing body contains a resonant structure in a part thereof,
The resonance structure resonates by applying a wave having a frequency unique to the resonance structure.
[10] A method of using the resonant structure, wherein a resonant structure that is bonded to an affinity substance for a detection target is resonated by applying a wave having a frequency unique to the resonant structure.

  In the present specification, the “first resonant structure” and the “second resonant structure” may be collectively referred to as “resonant structure”. In addition, the “first affinity substance” and the “second affinity substance” are sometimes collectively referred to as “affinity substances”. Furthermore, “first kit” and “second kit” described later may be collectively referred to as “kit”.

  According to the present invention, the affinity substance is vibrated by binding the resonance structure to the affinity substance for the detection target and resonating the resonance structure in the reaction field between the detection target and the affinity substance. The vibration can be transmitted to the reaction field. If a detection target exists in the reaction field, vibration can be transmitted to the detection target. These vibrations can increase the chances of contact between the detection target and the affinity substance. As a result, the binding between the detection target and the affinity substance is promoted. By promoting the binding, a complex including the detection target and the affinity substance is easily formed, and aggregation of the complex is also promoted.

  Further, according to the present invention, the wave applied to resonate the resonant structure requires low energy. Therefore, there is no damage to the specimen such as heat denaturation of the protein. Further, since it is sufficient to apply a low-energy wave, the wave can be applied for a relatively long time as compared with the conventional resonance stirring method, so that a sufficient vibration can be given to promote bonding and aggregation.

Therefore, according to the present invention, the detection accuracy can be improved even if the detection target is very small. Further, it is possible to shorten the time for bonding, aggregation and the like. Furthermore, since binding, aggregation, etc. can be promoted even in a very narrow reaction field, not only for conventional cells and wells, but also for reactions in microchemical processes, microchannels, microreactors, etc. Can also be used.
The present invention is suitable for detection and quantification of a detection target in a specimen such as a biological sample using an antigen-antibody reaction.

It is the schematic of one Embodiment of the 1st detection or quantification method which makes an antigen a detection target. It is the schematic of one Embodiment of the 1st detection or quantification method which makes an antibody a detection target. It is the schematic of one Embodiment of the 2nd detection or quantification method which makes an antigen a detection target. It is the schematic of one Embodiment of the 2nd detection or quantification method which makes an antibody a detection target. It is a typical perspective view of one embodiment of a cell and a wave generator with which a device for detecting or quantifying is provided. It is a schematic plan view of one embodiment of a part of a discrete clinical automatic biochemical analyzer equipped with a wave generator.

  Hereinafter, embodiments of the present invention will be described.

<Method of detection or quantification>
The detection or quantification method of the present invention is included in the first binding substance by mixing the specimen and at least the first binding substance, and applying a wave having a frequency unique to the first resonant structure. The method includes resonating the first resonant structure and determining a complex including the first combined object and the detection target.

  In the present specification, the terms “bond”, “bonded” and the like include, in principle, directly or indirectly bonded or states thereof unless otherwise specified. In this specification, for example, with respect to A and B, terms such as “directly bond” and “directly bond” indicate that A and B are combined without any intervening or their state. means. In this specification, for example, with respect to A and B, terms such as “indirectly bound” and “indirectly bound” mean that A and B intervene with other molecules. Means the state of being connected.

  The first binding substance is a combination of the first affinity substance for the detection target and at least the first resonant structure. The first affinity substance and the first resonant structure may be directly coupled.

(Resonant structure)
In the present invention, the resonant structure is not particularly limited as long as it resonates by applying a wave having a frequency unique to the resonant structure, and may be, for example, a solid particle made of a single substance. In the present specification, “solid particles” are particles that are filled with contents. The solid particles preferably do not have a hollow portion in the hollow particles described later. The resonant structure may also be a hollow particle composed of a shell portion and a hollow portion surrounded by the shell portion, a composite particle composed of a metal and an organic polymer, or the like. Examples of the former hollow particles include those made of an organic polymer such as polystyrene. The structure may have, for example, a rod-like or mesh-like structure in a part of the hollow portion. Examples of the latter composite particles include those composed of a metal such as iron and an organic polymer such as polystyrene. The structure includes, for example, a structure in which particles composed of a metal are coated with an organic polymer, metal A core-shell structure consisting of a core portion consisting of and a shell portion surrounding the core portion and consisting of an organic polymer, and other structures corresponding to a structure in which a part of particles consisting of an organic polymer replaces a metal, etc. The former may be a hollow particle made of an organic polymer and a composite particle made of a metal such as a rod-like or mesh-like structure existing in a part of the hollow part.

In the present specification, the resonant structure does not necessarily have a spherical shape or a shape similar to a sphere, and can have various forms.
Since the resonant structure is a structure as described above, for example, the resonant structure is resonated by imparting a wave having a specific frequency to the resonant structure, and vibration caused by the resonance is continuously transmitted to the reaction field. Can do.

The lower limit of the average particle diameter of the first resonant structure is preferably 0.001 μm, more preferably 0.01 μm, still more preferably 0.05 μm, still more preferably 20 μm, and the upper limit is preferably 500 μm, More preferably, it is 400 micrometers, More preferably, it is 300 micrometers, More preferably, it is 280 micrometers.
The first resonant structure in the present invention has an average particle diameter within the above numerical range, and is usually so large that the average particle diameter of the affinity substance is negligible. Therefore, the first resonant structure is unique to the first resonant structure. The energy efficiency with respect to applying a wave having a vibration frequency is good, and resonance can also be caused by applying a wave with a relatively low energy.

  Since the resonant structure is relatively large as described above, it is preferable to have a low specific gravity in order to prevent the resonance structure from sinking and being unable to transmit vibration to the reaction field. As described above, the resonant structure is preferably a structure having a hollow portion or a composite structure having both a metal portion and an organic polymer portion as described above, and reacts with vibration caused by resonance. In the case of the former hollow structure, it is more preferable that the shell portion is thinner, and in the case of the latter composite structure, it is more preferable that there are more organic polymer portions relative to the metal portion as long as it can be transmitted to the field.

In the present invention, the first resonant structure may be a solid phase that binds the first affinity substance. The solid phase as the resonant structure is preferably one that can flow in a test solution or the like rather than a container such as a well or one that is coupled to the container in that the vibration due to resonance is widely transmitted to the reaction field.
The solid phase is not particularly limited, and examples thereof include latex particles, magnetic particles, and beads. Examples of beads include cellulose beads, porous glass beads, silica gel, low and high cross-linked polystyrene beads optionally cross-linked with divinylbenzene, grafted co-poly beads, poly- Acrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N, N-bis-acryloyl ethylene diamine, and glass particles coated with a hydrophobic polymer, etc. It may contain various compositions including alkanethiolate-derived gold, polyamide, acrylic copolymer, nylon, dextran, polyacrolein, etc., polystyrene beads such as low and high cross-linked polystyrene beads, poly -Acrylamide beads are preferred Ku, polystyrene beads is more preferable.

In the present specification, a detection or quantification method that does not use a second affinity substance described later may be referred to as a first detection or quantification method.
In the first detection or quantification method, the first resonance structure is preferably bound to the first affinity substance separately from the latex particles. The first detection or quantification method is also suitable for the latex agglutination method because the first resonant structure is bound to the first affinity substance.

(Affinity substance)
The detection or quantification method of the present invention may further use a second affinity substance for the detection target.
The first affinity substance and the second affinity substance can be simultaneously bound to the detection target at different sites of the detection target.
In the present specification, the detection or quantification method using the second affinity substance may be referred to as a second detection or quantification method.

For example, when the detection target is an antigen, the first affinity substance and the second affinity substance may be antibodies.
The antibody used herein may be any type of immunoglobulin molecule, and may be an immunoglobulin molecule fragment having an antigen binding site such as Fab. The antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody that recognizes different antigenic determinants of the antigen.
In the second detection or quantification method of the present invention, the first affinity substance and the second affinity substance are two kinds of monoclonal antibodies having different antigen recognition sites for one kind of antigen to be detected. It is preferable.

  When the detection target is an antibody, the first affinity substance may be an antigen having an antigenic determinant recognized by the antibody. In the second detection or quantification method of the present invention, the second affinity substance is usually a second antibody that can bind to the antibody to be detected.

(Labeling enzyme)
In the second detection or quantification method of the present invention, the first affinity substance or the second affinity substance may be bound to a labeling enzyme. Examples of the labeling enzyme include those used in methods utilizing an enzyme substrate reaction such as ELISA and CLEIA, and specific examples include alkaline phosphatase and horseradish peroxidase.
The labeling enzyme may be directly bound to the first affinity substance or the second affinity substance, but may be indirectly bound via a resonance structure.

(Magnetic substance)
In the second detection or quantification method of the present invention, the first affinity substance or the second affinity substance may further be bound to a magnetic substance. The magnetic substance is preferably in the form of fine particles. In the second detection or quantification method of the present invention, by using a magnetic substance, by adding a magnetic force, the aggregated magnetic substance can be separated, and the detection accuracy can be improved.

The second affinity substance may be bound to the second resonant structure.
As the second resonant structure, for example, the solid phase described above as the first resonant structure can be used. Further, the second resonant structure may be a linker or the like interposed between the second affinity substance and the labeling enzyme.

  In the present specification, a second binding substance is formed by combining a second binding substance with at least one selected from the group consisting of a labeling enzyme, a magnetic substance, and a second resonant structure. Sometimes it is a thing.

  The second detection or quantification method of the present invention is suitable for a method using an enzyme substrate reaction such as an ELISA method or a CLEIA method (chemiluminescence enzyme immunoassay).

(Method for producing the above components used in the detection or quantification method of the present invention)
The first binding substance is produced by binding the first affinity substance and the first resonant structure. Further, when the second affinity substance constitutes the second binding substance, the second affinity substance is produced by binding the second affinity substance and another constituent of the second binding substance.

For example, when the affinity substance is an antibody or an antigen and is directly bound to a solid phase, a conventional method such as a physical adsorption method or a chemical binding method can be used. In addition, for example, when an affinity substance and a labeling enzyme are directly bound, a conventional method can be used. Furthermore, when binding an affinity substance and a magnetic substance, although not particularly limited, for example, a chemical bonding method for a special functional group can be used, specifically, both the affinity substance and the magnetic substance, Substances having an affinity for each other (for example, avidin and biotin, glutathione and glutathione S transferase) may be bound, and the affinity substance and the magnetic substance may be indirectly bound via these substances.
The resonant structure can be produced by a conventional method such as a physical adsorption method or a chemical bonding method.

(Detection method)
According to the detection method of the present invention, the specimen and at least the first combined substance are mixed, the first resonant structure is resonated by applying a wave having a specific frequency to the first resonant structure, and the first resonant structure is resonated. A step of determining the presence or absence of a complex including the one binding substance and the detection target.

(mixture)
First, the first binding substance in which the first affinity substance is bound to the first resonant structure and the specimen are mixed in a container.
Further, a second affinity substance may be mixed. The second affinity substance may constitute a second binding substance. In the mixing, for example, the specimen can be put into a container holding a test solution containing the first binding substance and the second affinity substance.

(Aggregation)
A wave having a frequency unique to the first resonant structure is imparted to the obtained mixture, thereby causing the first resonant structure to resonate. When the second resonant structure is present in the mixture, a wave having a specific frequency may be further imparted to the second resonant structure. The wave having a frequency unique to the first resonant structure and the wave having a frequency unique to the second resonant structure may be the same or different.

  By applying a wave having a frequency unique to the first resonant structure, the vibration is also transmitted to the first affinity substance coupled to the first resonant structure. Since the mixture is a liquid, vibrations can also be transmitted to the reaction field surrounding the first affinity substance. If a detection target exists in the reaction field, vibration can be further transmitted to the detection target. When the detection target exists in the specimen, these vibrations increase the chance of contact between the detection target and the first affinity substance, and as a result, the binding between the detection target and the first affinity substance is promoted. The By this binding, a complex including the first binding substance and the detection target is formed and aggregates.

  When the second resonant structure is present in the mixture, the second resonant structure can be obtained by applying a wave having a specific frequency to the second resonant structure in addition to the resonance of the first resonant structure. The vibration is also transmitted to the second affinity substance bonded to the structure. And a vibration can also be transmitted to the reaction field surrounding the second affinity substance. When the detection target exists, these vibrations increase the chances of contact between the detection target and the first affinity substance and the second affinity substance, and as a result, the detection target and the first affinity. Binding of the substance and the second affinity substance is facilitated. The complex containing the first bound substance and the detection target further contains the second affinity substance and aggregates.

  The above phenomenon will be described with reference to the drawings. In addition, the embodiment of the present invention shown in each drawing is merely one embodiment, and is not limited thereto.

As one embodiment of the first detection or quantification method of the present invention, for example, as shown in FIG. 1, when the detection target 50 is an antigen 51, the first conjugate 10 has a first resonance structure. Latex particles as the body 11 and the antibody 12 as the first affinity substance for the antigen 51 are combined.
As another embodiment of the first detection or quantification method of the present invention, for example, as shown in FIG. 2, when the detection target 50 is an antibody 52, the first conjugate 10 has the first resonance. Latex particles as the sexual structure 11 and the antigen 13 as the first affinity substance for the antibody 52 are combined.

  In FIGS. 1 and 2, after a sample is put into each of the embodiments of the first detection or quantification method of the present invention and mixed, a wave having a specific frequency is applied to the first resonant structure. Then, the first resonant structure 11 resonates. The vibration due to the resonance is also transmitted to the antibody 12 and the antigen 13 which are the first affinity substances bonded to the first resonant structure 11. The vibration can also be transmitted to the reaction field surrounding the antibody 12 or the antigen 13. When the antigen 51 or the antibody 52 that is the detection target 50 exists in the reaction field, the vibration can be further transmitted to the antigen 51 and the antibody 52. These vibrations increase the chances of contact between the antigen 51 and the antibody 12 and the chances of contact between the antibody 52 and the antigen 13, respectively. As a result, the binding between the antigen 51 and the antibody 12 and the binding between the antibody 52 and the antigen 13 are promoted. By this binding, a complex including at least the antigen 51, the antibody 12 and the first resonant structure 11, and a complex including at least the antibody 52, the antigen 13 and the first resonant structure 11 are formed and aggregated. To do.

  As one embodiment of the second detection or quantification method of the present invention, for example, as shown in FIG. 3, when the detection target 50 is an antigen 51, the first conjugate 10 has a first resonance structure. The body 11 is bound to the antibody 12 as a first affinity substance for the antigen 51. The detection or quantification method of this embodiment further includes the antibody 22 as the second affinity substance for the antigen 51. The antibody 22 may optionally constitute the second conjugate 20 by binding the labeling enzyme 30. The antibody 22 and the labeling enzyme 30 may be directly bonded or indirectly bonded via the linker 21. The first resonant structure 11 may be a solid phase or a magnetic substance. The antibody 22 may be coupled to the second resonant structure, and the second resonant structure may be, for example, the linker 21. The antibody 12 and the antibody 22 are monoclonal antibodies that can simultaneously bind to the antigen 51 at different sites (antigenic determinants) of the antigen 51.

  As another embodiment of the second detection or quantification method of the present invention, for example, as shown in FIG. 4, when the detection target 50 is an antibody 52, the first conjugate 10 has the first resonance. And the antigen 13 as a first affinity substance for the antibody 52. The detection or quantification method of this embodiment further includes the antibody 23 as a second affinity substance for the antibody 52. The antibody 23 may optionally constitute the second conjugate 20 by binding the labeling enzyme 30. The antibody 23 and the labeling enzyme 30 may be directly bonded, or may be indirectly bonded via a linker 21. The first resonant structure 11 may be a solid phase or a magnetic substance. The antibody 23 may be coupled to the second resonant structure, and the second resonant structure may be, for example, the linker 21. Antigen 13 and antibody 23 can bind to antibody 52 simultaneously at different sites (Fab and Fc) of antibody 52.

3 and FIG. 4, when the sample is put into each of the embodiments of the second detection or quantification method of the present invention and mixed, and then a wave having a specific frequency is applied to the resonant structure, In the formation of the complex in one embodiment of the first detection or quantification method of the invention, the antibody 22 or the antigen 23 as the second affinity substance is added. At that time, the vibration can be transmitted to the antibody 22 and the antigen 23 by the vibration of the reaction field based on the resonance of the first resonant structure 11.
Specifically, due to the increased contact opportunity based on the resonance of the first resonant structure 11, the binding of the antibody 12, the antigen 51 and the antibody 22, and the binding of the antigen 13, the antibody 52 and the antibody 23 are Each is promoted. By this binding, a complex including at least the antibody 12, the first resonant structure 11, the antigen 51 and the antibody 22, and a complex including at least the antigen 13, the first resonant structure 11, the antibody 52 and the antibody 23. Each body is formed and agglomerated.

In the present invention, the wave to be imparted is not particularly limited as long as it causes resonance in the resonant structure. For example, the following Stokes-Einstein equation within a range that does not cause damage to the specimen, such as heat denaturation of protein. In (1), it is preferable to apply a wave that maximizes the diffusion coefficient D as much as possible.
d = κT / 3πη ₀ D Formula (1)
(In the above equation, d is the particle size of the resonant structure, κ is the Boltzmann constant, T is the absolute temperature of the reaction field where the resonant structure exists, η ₀ is the viscosity of the reaction field where the resonant structure exists) , D represents the diffusion coefficient of the resonant structure.)
The diffusion coefficient D is a coefficient indicating the Brownian motion of the molecule, and can be obtained, for example, by measuring a diffraction scattering pattern by a laser diffraction method. In the present invention, for example, a resonant structure in a measurement cell is used. Can be obtained by cumulant method and histogram method analysis after multiplying the laser beam reflected and irradiated through the pinhole installed outside the measurement cell using a photomultiplier tube.

In the present invention, the type of wave to be applied may be, for example, a wave having a frequency unique to the resonant structure, and examples thereof include a low frequency to an ultrasonic wave to a medium wave.
Conditions such as the frequency, time, and output of the wave to be applied can be selected according to the natural frequency, material, size, etc. of the resonant structure to be used.

The frequency of the wave to be applied is preferably the natural frequency of the resonant structure that is the object of resonance. The lower limit of the frequency of waves to be applied is preferably 10 Hz, more preferably 15 Hz, and the upper limit is preferably 10 MHz, more preferably 2 MHz.
In the present invention, when the frequency of the wave generated by the device to be used is fixed, select what is specifically used as the first resonant structure and the second resonant structure according to the frequency. Also good.

The lower limit of the wave output to be applied is not particularly limited, but is preferably 1 W, more preferably 5 W, and the upper limit is preferably 2200 W, more preferably 2000 W.
In the present invention, the output can be kept relatively low as described above, compared with the conventional resonance stirring method, so that the wave can be applied for a relatively long time, which is sufficient for promoting binding, aggregation and the like. Can give vibration. Moreover, since the temperature rise in the reaction field can be suppressed to a small level, damage to the specimen such as heat denaturation of the protein does not occur.

Examples of a method for imparting a wave having a specific frequency to the resonant structure include a piezo element, a crystal resonator, a dynamic speaker, and the like, and a piezo element is preferable.
When using a piezo element, the lower limit of the frequency of the applied wave is preferably 10 Hz, more preferably 15 Hz, the upper limit is preferably 10 MHz, more preferably 2 MHz, and the lower limit of the output of the applied wave is preferably Is 5 W, more preferably 10 W, and the upper limit is preferably 2200 W, more preferably 2000 W.

(Determining process)
Specifically, the determination step is a step of determining the presence or absence of the complex in the case of the detection method. In the case of the method of quantification, the turbidity based on the complex is measured, and the detection target This is a step of calculating the amount of the detection target in the sample based on the correlation equation between the amount and turbidity.

  When the first affinity substance or the second affinity substance is bound to the labeling enzyme, the labeling enzyme present in the complex is reacted with its substrate. Examples of the substrate include conventionally used chromogenic, fluorescent or luminescent substrates. By adding a substrate, a detectable signal can be generated and replaced by the turbidity measurement. The addition of the substrate can be performed before or when the determination is performed.

(Judgment)
The determination of the presence or absence of the complex including the detection target can be performed, for example, by visual observation or turbidity measurement. The turbidity can be calculated from the light transmittance in the light scattering device. If the turbidity is high, the complex is aggregated, which indicates the presence of the detection substance. Here, the wavelength of the light to be used may be appropriately set so as to obtain a desired detection sensitivity according to the particle size of the magnetic substance or the like. The wavelength of light is preferably within the range of visible light (for example, 550 nm) in that a conventional general-purpose device can be used.

  Visual observation or turbidity measurement may be performed intermittently at a certain point in time or continuously over time. Further, the determination may be made based on the difference between the turbidity measurement value at a certain time point and the turbidity measurement value at another time point.

  The “turbidity measurement” in the detection method or quantification method of the present invention includes not only measuring turbidity directly but also measuring a parameter reflecting turbidity. Such parameters include differences in turbidity measurement values at multiple time points, the amount of separated aggregates, the turbidity of non-aggregates after separation, and the like. Here, it is preferable that one point of the plurality of time points is in the vicinity of the time point at which the turbidity becomes a maximum value when a magnetic force is applied to the negative control in which the detection target is absent. Thereby, the difference from the turbidity measurement value at another time point becomes large, and the amount of the detection target can be quantified more accurately.

(Quantitative method)
According to the quantification method of the present invention, the specimen and at least the first combined substance are mixed, and the first resonant structure is resonated by applying a wave having a specific frequency to the first resonant structure. The turbidity based on the complex containing one binding substance and the detection target, that is, the turbidity of the mixture obtained by resonance is measured, and the detection in the specimen is performed based on the correlation equation between the amount of the detection target and the turbidity Calculate the amount of the target. Since the procedure of the first half is similar to the detection method described above, the description is omitted.

(Correlation formula)
A correlation equation between the amount of detection target and turbidity is prepared in advance. In the measurement of the amount of detection target and the turbidity constituting the correlation formula, the more reliable the correlation formula is obtained as the amount of data increases. Therefore, the data may be related to the amount of two or more detection targets, and is preferably related to the amount of three or more detection targets.
Here, the correlation equation between the amount of detection target and turbidity is not only an equation showing a direct correlation between the amount of detection target and turbidity, but also the correlation between the amount of detection target and a parameter reflecting turbidity. It may be a formula.

(Calculation)
By substituting the turbidity measurement value of the mixture into the created correlation equation, the amount of the detection target in the sample can be calculated.

(Separation)
When a magnetic substance is contained, it is preferable that the detection or quantification method of the present invention further includes separating the aggregated magnetic substance by applying a magnetic force. Thereby, the agglomerated magnetic substance is separated from impurities including the non-aggregated magnetic substance. For this reason, the measurement values such as the amount of the separated magnetic substance and the light transmittance when dispersed in the solvent exclude the influence of foreign substances and more accurately reflect the presence of the detection substance.

  The magnetic force can be applied by bringing a magnet close to the magnetic substance. The magnetic force of this magnet varies depending on the magnitude of the magnetic force of the magnetic substance used. An example of the magnet is a neodymium magnet manufactured by Magna.

  The addition of magnetic force may be performed before determination or turbidity measurement or in parallel with determination or turbidity measurement, but simultaneous parallel is preferable in that the time spent in the process can be shortened. When magnetic force is applied, the agglomerated magnetic substance is separated by inclusion of impurities, so the turbidity of the mixture after separation is presumed to be rather smaller when the impurities are present. .

(Detection target)
Examples of the detection target in the sample include substances used for clinical diagnosis. Specifically, human immunoglobulin G, human immunoglobulin M, human immunoglobulin A contained in body fluid, urine, sputum, feces, etc. Human immunoglobulin E, human albumin, human fibrinogen (fibrin and their degradation products), α-fetoprotein (AFP), C-reactive protein (CRP), myoglobin, carcinoembryonic antigen, hepatitis virus antigen, human chorionic gonadotropin ( hCG), human placental lactogen (HPL), HIV viral antigen, allergen, bacterial toxin, bacterial antigen, enzyme, hormone (eg, human thyroid stimulating hormone (TSH), insulin, etc.), nucleic acid, nucleic acid amplified by PCR, etc. , Cytokines, drugs and the like.

<Detection or quantification device>
An apparatus for detecting or quantifying a detection target in a specimen, the apparatus comprising: a first binding substance in which a first affinity substance for the detection target and a first resonant structure are bound; Means for providing a container having a specimen including a specimen, imparting a wave having a specific frequency to the first resonant structure, and means for determining a complex including the first combined substance and the detection target An apparatus including the above is also one aspect of the present invention.
Examples of the first bound substance in which the first affinity substance and the first resonant structure are bound include those described above for the detection or quantification method. The installation facility of the container is not particularly limited, and may be, for example, a facility for installing a cell or the like for normal turbidity measurement. The means for applying the wave having a specific frequency to the first resonant structure may be, for example, a means provided with the above-described wave applying method. The means for determining the complex may be a means for performing the above-described detection method or quantitative method.

The above-mentioned device of the present invention may be provided with a combination of means for applying a wave having a specific frequency to the first resonant structure and means for making a determination on the composite.
As an embodiment of the apparatus of the present invention, for example, as shown in FIG. 5, at least a cell 2 for turbidity measurement and a wave generator 1 for applying a wave to the contents of the cell are provided. It may be a device provided. In FIG. 5, the direction in which the wave represented by the arrow in the figure from the wave generator 1 to the cell 2 is applied is the turbidity represented by the arrow that passes through the cell 2 in the figure from the far side to the near side. It is perpendicular to the light irradiation direction for measuring the degree, but it is not limited to this, and both directions may form any angle, for example, they may be parallel but given in the case of parallel. It is preferable that the wave to be generated and the light for turbidity measurement do not overlap so as not to affect the generator.

  As another embodiment of the apparatus of the present invention, for example, as shown in FIG. 6, automatic clinical biochemistry analysis in which quantitative analysis mainly by colorimetry is automated for components such as blood, urine and other body fluids. Among the devices, in the discrete reaction device 3, the wave generator 1 is provided in the gaps between the respective mechanisms such as the reagent dispensing units 61, 62 and 63, the stirring units 64 and 65, the photometric unit 66, and the washing area 67. Therefore, for example, it can be provided in an existing discrete type clinical biochemical automatic analyzer. As shown in FIG. 6, the wave generator 1 may be provided in plural or one.

<Combined product and its use>
A combined substance including an affinity substance for a detection target and a resonance structure-containing body, the resonance structure-containing body including a resonance structure in a part thereof, and the resonance structure In the present invention, a combination that resonates by applying a wave having a frequency unique to the resonant structure is also one aspect of the present invention.
Examples of the resonance structure include those described above as the first resonance structure and the second resonance structure. The resonant structure-containing body is not particularly limited as long as it contains a resonant structure in a part thereof. For example, the resonant structure-containing body contains a resonant structure in a part thereof by bonding, coordination, inclusion, or the like. And the like. Examples of detection targets include immunological detection targets such as antigens and antibodies. Examples of the affinity substance include an antibody and an antigen.
A method of using a resonant structure in which a resonant structure bonded to an affinity substance for a detection target is resonated by applying a wave having a frequency unique to the resonant structure is also one aspect of the present invention. is there.

<How to interact>
A method in which a substance A and an affinity substance for the substance A interact with each other, wherein a binding substance in which the affinity substance and the resonance structure are bonded to each other and the substance A are mixed to form a resonance structure. A method comprising the step of resonating the resonant structure by applying a wave having a natural frequency is also one aspect of the present invention.
The substance A is not particularly limited and may be those described above as the detection target, but can be selected from a wide variety of substances in addition to the reaction substance in the antigen-antibody reaction. The affinity substance for the substance A may be the above-described substances such as an antigen and an antibody, but is not limited to the antigen-antibody reaction, and may be a substance that can interact with the substance A. The interaction may be binding, coordination, inclusion, etc. between the substance A and the affinity substance, and may further proceed to a reaction or the like. The substance A and the affinity substance are referred to as “substances” for convenience in this specification, but may be, for example, a low-molecular or high-molecular compound. The above-mentioned thing can be used as a resonance structure and a wave to give.
The interaction method of the present invention can promote the interaction between the substance A and the affinity substance by causing the resonant structure to resonate.

  The resonant structure of the present invention and the interacting method of the present invention can cause the resonant structure to function as a stirrer at the molecular level due to resonance. Then, the interaction between the affinity substance to which the resonant structure is bound and the detection target or substance A is made possible in a minute reaction field without causing damage to the reaction field and its surroundings. Therefore, the resonant structure of the present invention and the interaction method of the present invention are widely used not only for conventional cells and wells, but also for reactions in microchemical processes, microchannels, microreactors, etc. be able to.

DESCRIPTION OF SYMBOLS 1 Wave generator 10 1st conjugate | bonded_body 11 1st resonance structure 12 Antibody as 1st affinity substance 13 Antigen as 1st affinity substance 20 2nd conjugate | bonded_body 21 Linker or 1st Resonant structure 22, 23 Antibody as second affinity substance 30 Enzyme for labeling 50 Detection target 51 Antigen as detection target 52 Antibody as detection target

Claims (10)

A method for detecting or quantifying a detection target in a sample,
Mixing the first binding substance in which the first affinity substance for the detection target and the first resonant structure are bound, and the specimen;
Resonating the first resonant structure by applying a wave having a frequency unique to the first resonant structure;
A method comprising a step of determining a complex including the first bound substance and the detection target. Mixing the first binding substance, the specimen, and a second affinity substance for the detection target;
2. The method according to claim 1, wherein the first affinity substance and the second affinity substance are capable of binding to the detection target simultaneously at different sites of the detection target. The second affinity substance is combined with at least the second resonant structure to form a second combined substance,
The second resonant structure has the same or different natural frequency as the first resonant structure,
In the method, after the second affinity substance is mixed with the specimen and before the determination, the second resonance property is applied by applying a wave having a specific frequency to the second resonance structure. The method of claim 2, further comprising resonating the structure. The first affinity substance or the second affinity substance is bound to a labeling enzyme;
The method according to claim 2 or 3, wherein the method further comprises reacting the labeling enzyme present in the complex with its substrate before or when the determination is performed.   The first resonant structure includes a solid particle made of a single substance, a hollow particle that may have a structure in a part of a hollow portion, and a group consisting of particles made of a metal and an organic polymer. The method according to any one of claims 1 to 4, wherein the particles are selected. The first combination and / or the second combination further includes a magnetic substance,
6. The method according to claim 2, wherein the method further includes separating the agglomerated magnetic substance by applying a magnetic force after the resonance and before or when performing the determination. The method according to item. An apparatus for detecting or quantifying a detection target in a sample,
The apparatus includes a facility for installing a container including a first binding substance in which a first affinity substance for the detection target, a first resonant structure is bound, and the specimen,
Means for imparting a wave having a specific frequency to the first resonant structure; and
An apparatus comprising means for determining a complex including the first bound substance and the detection target. A method of causing a substance A to interact with an affinity substance for the substance A,
Mixing the substance A and the binding substance in which the affinity substance and the resonant structure are bound,
And resonating the resonant structure by applying a wave having a natural frequency to the resonant structure. A binding material including an affinity substance for a detection target and a resonant structure-containing body,
The resonant structure-containing body contains a resonant structure in a part thereof,
The resonance structure resonates by applying a wave having a frequency unique to the resonance structure.   A method for using the resonance structure, wherein a resonance structure bonded to an affinity substance for a detection target is resonated by applying a wave having a frequency unique to the resonance structure.

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