JP6684545B2 - Detection or quantification method, resonant additive, method of using resonant structure and container - Google Patents

Description

  The present invention relates to a detection or quantification method, a resonant additive, a method of using a resonant structure and a container.

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

  According to this latex agglutination method, an antigen added as a sample cross-links a plurality of latex-bound antibodies to promote latex agglutination. However, when the amount of the antigen is very small, the cross-linking is unlikely to occur, so that the latex does not sufficiently aggregate, and even if it aggregates, the detection sensitivity becomes lower than the detection sensitivity and cannot be detected. Therefore, it has been difficult to detect a small amount of antigen rapidly.

  Therefore, methods utilizing the enzyme substrate reaction such as the ELISA method and the CLEIA method have been 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 substrate of the enzyme is added and the degree of the reaction catalyzed by the enzyme is measured to detect or quantify the antigen. According to these methods, for example, when a coloring reagent or a luminescent reagent is used as the substrate, the detection sensitivity of the coloring or the luminescence after adding the substrate is high.

  In order to shorten the stirring time for reacting the sample and the reagent, a stirring device has been proposed which radiates a sound wave in a resonance frequency band to a liquid sample containing the sample and the reagent to generate an acoustic flow ( See, for example, Patent Document 2). However, this sound wave is generated from the sounding section attached to the wall surface of the reaction container, and requires a high frequency of about several MHz to several hundred MHz to stir the entire liquid sample in the container. Therefore, a measurement target such as a protein may be thermally denatured. In order to minimize damage to the liquid sample, such as heat denaturation of the protein, it is necessary to suppress the sound wave generation time instantly, which may result in insufficient stirring.

  Further, in the ELISA method, it has been proposed to utilize resonance in order to rapidly make contact between the capture antibody immobilized on the inner wall of the tip cone and the antigen in the sample (for example, see Patent Document 3). . However, this resonance also occurs in the entire solution. In addition, it is necessary to perform a suction / discharge operation of a reagent or the like in the well facing the tip cone at a frequency that causes resonance, which requires a special device.

JP-B-58-ll575 JP, 2010-91306, A Japanese Patent Publication No. 2013-532834

  The present invention has been made in view of the above circumstances, and an object of the present invention is to promote binding, aggregation and the like in an antigen-antibody reaction and the like without causing damage to a sample such as heat denaturation of a protein.

  The present inventors can increase the chance of contact of a reactant by vibration by causing a structure that is a dispersoid rather than a dispersion medium or a solvent to resonate in a reaction field where binding, aggregation, etc. are performed, and as a result, The inventors have found that they can promote bonding, aggregation, etc., 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 sample, comprising:
A first affinity substance for the detection target, a resonant structure, and the sample are mixed,
Resonating the resonating structure by applying a wave of a natural frequency to the resonating structure,
A method comprising the step of making a determination regarding a complex including the first affinity substance and the detection target.
[2] A mixture of the first affinity substance, the resonant structure, the sample, and a second affinity substance for the detection target,
The method according to [1], wherein the first affinity substance and the second affinity substance can simultaneously bind to the detection target at different sites of the detection target.
[3] The first affinity substance or the second affinity substance is bound to a labeling enzyme,
The method according to [2], wherein the method further comprises reacting the labeling enzyme present in the complex with a substrate thereof before or during the determination.
[4] The resonant structure is a group consisting of solid particles made of a single substance, hollow particles that may have a structure in a part of the hollow portion, and composite particles made of a metal and an organic polymer. The method according to any one of [1] to [3], which is at least one kind of particles selected from the following.
[5] The method according to any one of [1] to [4], wherein the resonant structure has an average particle size of 0.001 to 500 μm.
[6] A method of allowing a substance A and an affinity substance for the substance A to interact with each other,
Mixing the substance A, the affinity substance, and the resonant structure,
A method comprising resonating the resonant structure by imparting a wave of a natural frequency to the resonant structure.
[7] A resonant additive for adding to a test solution for detecting and / or quantifying a detection target,
The test solution contains a first affinity substance for the detection target,
The resonant additive has a resonant structure that resonates when a wave having a specific frequency is applied.
[8] A method of using a resonant structure, which comprises adding a test liquid for detecting and / or quantifying an object to be detected, and causing resonance by applying a wave of a natural frequency,
The method for use, wherein the test liquid contains a first affinity substance for the detection target.
[9] A container for detecting and / or quantifying a detection target,
The container includes a resonator that resonates when a wave having a specific frequency is applied.
[10] The container according to [9], wherein the resonator is located at a portion that does not block transmitted light for detection and / or quantification.
[11] A method of allowing a substance A and an affinity substance for the substance A to interact with each other,
The substance A and the affinity substance are present in the fluid held in the container according to [9] or [10],
A method, comprising the step of causing a resonator contained in the container to resonate by applying a wave having a frequency unique to the resonator.

  In the present specification, the “first affinity substance” and the “second affinity substance” may be collectively referred to as “affinity substance”. In addition, the “first detection or quantification method of the present invention” and “second detection or quantification method of the present invention” described below may be collectively referred to as “the detection or quantification method of the present invention”.

  According to the present invention, in a reaction field between a detection target and a substance having an affinity for the detection target, vibration is transmitted to the reaction field by causing a resonant structure to exist and resonate, and thus exists in the reaction field. Vibration is also transmitted to the affinity substance. If the detection target exists in the reaction field, the vibration is 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 such binding, a complex containing 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 imparted to resonate the resonant structure requires low energy. Therefore, no damage to the sample, such as thermal denaturation of the protein, occurs. Further, since it is sufficient to apply a wave with low energy, the wave can be applied for a relatively long time as compared with the conventional stirring method by resonance, and thus vibration sufficient for promoting bonding, aggregation, etc. can be applied.

Therefore, according to the present invention, it is possible to improve the detection accuracy even if the detection target is very small. Further, it is possible to reduce the time required for binding, aggregation and the like. Furthermore, since binding, aggregation, etc. can be promoted even in a very narrow reaction field, not only in conventional cells, wells, etc., but also in reactions in microchemical processes, microchannels, microreactors, etc. Can also be used.
INDUSTRIAL APPLICABILITY The present invention is suitable for detecting and quantifying a detection target in a sample such as a biological sample that utilizes an antigen-antibody reaction.

It is a schematic diagram showing one embodiment of the 1st detection or quantification method which makes an antigen a detection object. It is a schematic diagram showing one embodiment of the 1st detection or quantification method which makes an antibody a detection object. It is a schematic diagram showing one embodiment of the 2nd detection or quantification method which makes an antigen a detection object. It is a schematic diagram showing one embodiment of the 2nd detection or quantification method which makes an antibody a detection object. 1 is a schematic perspective view of an embodiment of a container for detecting or quantifying. 1 is a schematic plan view of an embodiment of a reaction container in a discrete-type clinical biochemical automatic analyzer.

  Hereinafter, embodiments of the present invention will be described.

<Method of detecting or quantifying>
The detection or quantification method of the present invention comprises mixing an analyte, at least a first affinity substance and a resonant structure, and generating a natural frequency (herein, “natural frequency”) of the resonant structure. The process of causing the resonance structure to resonate by applying the wave and performing a determination on the complex including the first affinity substance and the detection target.
In the present invention, the resonant structure is separated from the first affinity substance.

  In the present specification, the terms “bind”, “bonded” and the like, in principle, include directly or indirectly bound or the state thereof, unless otherwise specified. In the present specification, for example, the terms “directly bond”, “directly bond” and the like with respect to A and B mean that A and B are bonded without any intervening substance or the state thereof. means. Further, in the present specification, for example, regarding A and B, terms such as “indirectly bound”, “indirectly bound” and the like mean that A and B have other molecules or the like interposed therebetween. And the state thereof.

(Resonant structure)
In the present invention, the resonant structure is not particularly limited as long as it resonates by imparting a wave of a frequency unique to the resonant structure, and may be, for example, solid particles of a single substance, As used herein, a "solid particle" is a solid particle. The solid particles preferably do not have a hollow portion in the hollow particles described below. The resonant structure may be hollow particles composed of a shell portion and a hollow portion surrounded by the shell portion, composite particles composed of a metal and an organic polymer, and the like. Examples of the former hollow particles include those made of an organic polymer such as polystyrene, and the structure may have, for example, a rod-shaped or mesh-shaped structure in a part of the hollow portion. Examples of the latter composite particles include, for example, those composed of a metal such as iron and an organic polymer such as polystyrene, and the structure thereof includes, for example, a structure in which particles made of a metal are coated with an organic polymer, and a metal. A core-shell structure consisting of a core part consisting of and a shell part surrounding the core part and consisting of an organic polymer, other, a structure corresponding to a structure in which a part of particles of the organic polymer is replaced with a metal, and the like, and The former may be a hollow particle made of an organic polymer and a composite particle having a rod-shaped or mesh-shaped structure existing in a part of the hollow portion made of a metal.

In the present invention, the resonant structure does not necessarily have a spherical shape or a shape similar to a sphere, and may have various shapes.
Since the resonant structure is, for example, a structure as described above, it is possible to resonate by imparting a wave of a specific frequency to the resonant structure and continuously transmit the vibration due to the resonance to the reaction field. You can

The lower limit of the average particle diameter of the resonant structure is preferably 0.001 μm, more preferably 0.01 μm, even more preferably 0.05 μm, even more preferably 20 μm, and the upper limit is preferably 500 μm, more preferably It is 400 μm, more preferably 300 μm, and even more preferably 280 μm.
The resonant structure in the present invention has an average particle diameter within the above numerical range, and since the average particle diameter of the affinity substance is usually large enough to be neglected, the vibration of the frequency peculiar to the resonant structure is generated. It has high energy efficiency for application, and resonance can be caused by application of relatively low energy wave.

  Since the resonant structure is relatively large as described above, it is preferable that the resonant structure has a small specific gravity in order to prevent it from settling and being unable to transmit vibration to the reaction field. From the viewpoint of reducing the specific gravity, the resonant structure is preferably a structure having a hollow portion or a composite structure having a metal portion but also an organic polymer portion, as described above, and reacts with vibration due to resonance. In the case of the former hollow structure, it is more preferable that the shell portion is thin, and in the case of the latter composite structure, it is more preferable that the organic polymer portion is larger than the metal portion as long as it can be transmitted to the field.

  In the present invention, the resonant structure is not bound to the first affinity substance, and preferably exists independently in the reaction field without being anchored to anything. Since the resonant structures are independently scattered in the reaction field, it is easy to widely transmit vibration to the reaction field by resonance, and it is possible to increase the chance of contact between the detection target and the affinity substance in the reaction field. .

  As the resonant structure, only one kind may be used, or two or more kinds may be used. As described above, the opportunity of contact between the detection target and the first affinity substance, and when the second affinity substance described below is further included, the detection target, the first affinity substance, and the second affinity substance The chances of contact with the substance are increased and the binding between them can be promoted.

(Affinity substance)
The first affinity substance is preferably bound to the solid phase. The solid phase is not particularly limited and may be in various forms, for example, latex particles, magnetic particles, beads, membranes, wells such as microtiter wells, slide materials, plates, microfabricated chips, pellets, disks, Capillary tubes, hollow fibers, needles, solid fibers and the like can be mentioned. The beads include, for example, cellulose beads, porous glass beads, silica gel, low- and high-crosslinked polystyrene beads optionally crosslinked with divinylbenzene, grafted co-poly beads, poly- Examples include acrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N, N-bis-acryloyl ethylenediamine, and glass particles coated with a hydrophobic polymer. It may contain various compositions including alkanethiolate-derived gold, polyamide, acrylic copolymer, nylon, dextran, polyacrolein, etc., polystyrene beads such as low cross-linking degree and high cross-linking degree polystyrene beads, poly -Prefer acrylamide beads Ku, polystyrene beads is more preferable.

In the present specification, a bound substance in which the first affinity substance and at least the solid phase are bound may be referred to as a first bound substance.
In the present specification, the detection or quantification method of the present invention that does not use the second affinity substance described later may be referred to as the first detection or quantification method of the present invention.
The first detection or quantification method of the present invention is suitable for the latex agglutination method.

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 simultaneously bind to the detection target at different sites of the detection target.
In the present specification, the detection or quantification method of the present invention using the second affinity substance may be referred to as the second detection or quantification method of the present invention.

The first affinity substance and the second affinity substance may be, for example, antibodies when the detection target is an antigen.
The antibody used here may be any type of immunoglobulin molecule, or 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 antigenic determinants having different antigens.
In the second detection or quantification method of the present invention, the first affinity substance and the second affinity substance are two types of monoclonal antibodies having different antigen recognition sites for one type 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 this antibody. In the second detection or quantification method of the present invention, the second affinity substance is usually the second antibody capable of binding 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 a method utilizing an enzyme substrate reaction such as the ELISA method and the CLEIA method, and specific examples thereof include alkaline phosphatase and horseradish peroxidase.
The labeling enzyme may directly bind to the first affinity substance or the second affinity substance, and when the first affinity substance is bound to the solid phase, it preferably binds to the second affinity substance. It is directly linked.

(Magnetic substance)
In the second detection or quantification method of the present invention, the first affinity substance or the second affinity substance may be further 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 applying a magnetic force, the aggregated magnetic substance can be separated, and the detection accuracy can be improved.

  In the present specification, a conjugate in which the second affinity substance and at least the labeling enzyme and / or the magnetic substance are bound may be referred to as a second bound substance.

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

(Method for producing the above-mentioned components used in the detection or quantification method of the present invention)
The first bound substance is produced by binding the first affinity substance and the solid phase, for example. When the second affinity substance constitutes the second bound substance, it is produced by binding the second affinity substance and other constituents of the second bound substance.

For example, when the affinity substance is an antibody or an antigen and is directly bound to the solid phase, a conventional method such as a physical adsorption method or a chemical binding method can be used. Further, for example, in the case of directly binding the affinity substance and the labeling enzyme, a conventional method can be used. Further, when the affinity substance and the magnetic substance are bound to each other, for example, a chemical bonding method for a special functional group can be used, and specifically, both the affinity substance and the magnetic substance can be used. It is also possible to bind substances having affinity to each other (for example, avidin and biotin, glutathione and glutathione S transferase), and indirectly bind the affinity substance and the magnetic substance 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)
The detection method according to the present invention comprises mixing a specimen, at least a first affinity substance and a resonant structure, and causing the resonant structure to resonate by imparting a wave of a frequency unique to the resonant structure, The step of determining the presence / absence of a complex containing one affinity substance and a detection target is included.

(mixture)
First, the first affinity substance, the resonant structure, and the sample are mixed in the container. In this case, the resonant structure and the sample may be added to a kit or a test solution containing the first affinity substance but not the resonant structure such as a commercially available kit. The first affinity substance may form a first bound substance.
Further, a second affinity substance may be further mixed. The second affinity substance may form a second bound substance. For the mixing, for example, the sample can be put into a container holding a test solution containing the first affinity substance, the second affinity substance, and the resonant structure. In this case, the resonant structure and the sample may be added to a kit or a test solution containing the first affinity substance and the second affinity substance but not the resonant structure such as a commercially available kit. .

(Aggregation)
A wave having a frequency unique to the resonant structure is applied to the obtained mixture to cause the resonant structure to resonate. When two or more kinds of resonant structures having different natural frequencies from each other are present in the mixture, a wave having a unique frequency may be applied to each of the resonant structures.

  Resonance of the resonating structure is caused by the application of a wave having a frequency unique to the resonating structure, and the vibration is transmitted to the field (reaction field) surrounding the resonating structure in the mixture. Since the mixture and the reaction field are liquids, the vibration is also transmitted to the first affinity substance existing in the reaction field. If a detection target exists in the reaction field, vibration is further transmitted to the detection target. When there is a detection target in the sample, these vibrations increase the chances 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. It By this binding, a complex containing the first affinity substance and the detection target is formed and aggregated.

  When the second affinity substance is present in the mixture, the vibration transmitted to the reaction field based on the resonance of the resonant structure also transmits the vibration to the second affinity substance present in the reaction field. When the detection target is present, 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 substance are increased. The binding of the substance and the second affinity substance is promoted. The complex containing the first affinity substance and the detection target further contains the second affinity substance and aggregates.

  The above phenomenon will be described with reference to the drawings. It should be noted that the embodiment of the present invention shown in each drawing is merely one embodiment and is not limited to these.

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 bound substance 10 is a latex particle as a solid phase 11. And the antibody 12 as the first affinity substance for the antigen 51 are bound.
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 the antibody 52, the first bound substance 10 is the solid phase 11. The latex particles are bound to the antigen 13 as the first affinity substance for the antibody 52.
In any of the above embodiments, the resonant structures 40 are interspersed with the mixture.

  In FIG. 1 and FIG. 2, when a sample is put into each of the embodiments of the first detection or quantification method of the present invention and mixed, and then a wave with a unique frequency is applied to the resonant structure, resonance occurs. The elastic structure 40 resonates. The vibration due to resonance is also transmitted to the reaction field surrounding the resonant structure 40. Then, it is also transmitted to the antibody 12 and the antigen 13 which are the first affinity substance existing in the reaction field. When the antigen 51 or the antibody 52 which 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. 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 containing at least the antigen 51 and the antibody 12 and a complex containing at least the antibody 52 and the antigen 13 are formed and aggregated.

  As an 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 bound substance 10 is a solid phase 11 and an antigen. The antibody 12 is bound as the first affinity substance for 51. The detection or quantification method of this one embodiment further includes an antibody 22 as a second affinity substance for the antigen 51. The antibody 22 may optionally bind the labeling enzyme 30 to form the second bound product 20. The solid phase 11 may be a magnetic substance. 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 the antibody 52, the first bound substance 10 is the solid phase 11 and , The antigen 13 is bound as the first affinity substance for the antibody 52. The detection or quantification method according to this embodiment further includes the antibody 23 as the second affinity substance for the antibody 52. The antibody 23 may optionally bind the labeling enzyme 30 to form the second bound product 20. The solid phase 11 may be a magnetic substance. The antigen 13 and the antibody 23 can simultaneously bind to the antibody 52 at different sites (Fab and Fc) of the antibody 52.

In FIG. 3 and FIG. 4, after the sample is put into each of the embodiments of the second detection or quantification method of the present invention and mixed, and a wave with a specific frequency is applied to the resonant structure, Upon formation of the complex in one embodiment of the first detection or quantification method of the invention, the antibody 22 or the antigen 23, which is the second affinity substance, is added. At that time, the vibration of the reaction field due to the resonance of the resonant structure 40 also transmits the vibration to the antibody 22 and the antigen 23.
Specifically, since the number of contact opportunities increases due to the resonance of the resonant structure 40, 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 respectively promoted. It By this binding, a complex containing at least the antibody 12, the solid phase 11, the antigen 51 and the antibody 22 and a complex containing at least the antigen 13, the solid phase 11, the antibody 52 and the antibody 23 are formed and aggregated.

In the present invention, the wave to be imparted is not particularly limited as long as it causes resonance in the resonating structure, for example, within the range that does not cause damage to the sample such as thermal denaturation of protein, Stokes-Einstein formula below. In (1), it is preferable to impart a wave that maximizes the diffusion coefficient D.
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 in which the resonant structure exists, and η ₀ is the viscosity of the reaction field in which 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 a molecule, and can be obtained by, for example, measuring a diffraction scattering pattern by a laser diffraction method. In the present invention, for example, the resonant structure in the measurement cell is measured. It can be determined by the cumulant method and the histogram method analysis after the laser light which has been irradiated with the laser beam and reflected by the laser beam and which has passed through the pinhole provided outside the measurement cell is multiplied by 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 low frequency to ultrasonic wave to medium wave.
The conditions such as frequency, time, and output of the applied wave can be selected according to the natural frequency, material, size, etc. of the resonant structure used.

  The frequency of the applied wave is the natural frequency of the resonant structure, which is the object of resonance. As the frequency of the wave to be imparted, a frequency having a large vibration width is preferable, a frequency having a wavelength having a higher vibration width with lower energy is more preferable, and the resonant structure has a particle size distribution. In some cases, it is preferable to select one having a particle size with a large distribution.

The lower limit of the frequency of the applied wave is preferably 10 Hz, more preferably 15 Hz, and the upper limit is preferably 3 MHz, more preferably 2 MHz.
In the present invention, what is specifically used as the resonant structure may be selected according to the frequency used, such as when the frequency of the wave generated by the device used is fixed.

The lower limit of the output of the applied wave 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, as compared with the conventional stirring method by resonance, so that the wave can be imparted for a relatively long time, and it is sufficient to promote coupling, aggregation and the like. Vibration can be given. Further, since the temperature rise in the reaction field can be suppressed to a small level, damage to the sample, such as thermal denaturation of protein, does not occur.

Examples of a method of imparting a wave of a specific frequency to the resonant structure include a piezo element, a crystal oscillator, a dynamic speaker, and the like, and the piezo element is preferable.
When a piezo element is used, 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.

(Process of making a determination)
The determination step is specifically a step of determining the presence or absence of a complex containing a first affinity substance and a detection target in the case of a detection method, and in the case of a quantification method, the above-mentioned complex Is a step of calculating the amount of the detection target in the sample based on the correlation formula between the amount of the detection target and the 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. The addition of substrate can generate a detectable signal and can replace the turbidity measurement described above. The substrate can be added before the determination or at the time of the determination.

(Judgment)
The presence or absence of the complex containing the detection target can be determined, for example, visually or by measuring the turbidity. The turbidity can be calculated from the light transmittance of a light scattering device, and if the turbidity is high, the complex is aggregated, which suggests the presence of a detection substance. Here, the wavelength of the light 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) from the viewpoint that conventional general-purpose devices can be used.

  Visual observation or turbidity measurement may be performed intermittently at a certain time point or continuously over time. Further, the determination may be performed 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 or quantification method of the present invention includes not only directly measuring the turbidity but also measuring a parameter that reflects the turbidity. Examples of such parameters include differences in turbidity measurement values at a plurality of time points, the amount of separated aggregates, the turbidity of non-aggregates after separation, and the like. Here, one of the plurality of time points is preferably near the time point at which the turbidity reaches the maximum value when a magnetic force is applied to the negative control in which the detection target is absent. As a result, 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.

(Quantification method)
The quantification method according to the present invention comprises mixing a specimen, at least a first affinity substance and a resonant structure, and causing the resonant structure to resonate by applying a wave of a frequency unique to the resonant structure, The turbidity based on the complex containing the affinity substance of 1 and the detection target, that is, the turbidity of the mixture obtained by resonance is measured, and the turbidity of the sample in the sample is determined based on the correlation formula between the amount of the detection target and the turbidity. Calculate the amount to be detected. Since the procedure of the first half is similar to the above-mentioned detection method, the description is omitted.

(Correlation formula)
A correlation formula between the amount of the detection target and the turbidity is created in advance. Regarding the measurement of the amount of the detection target and the turbidity forming the correlation equation, the more the data is, the more reliable the correlation equation is obtained. Therefore, the data only needs to 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 the detection target and the turbidity is not only an equation showing the direct correlation between the amount of the detection target and the turbidity, but also the correlation between the amount of the detection target and the parameter reflecting the turbidity. It may be an expression.

(Calculation)
By substituting the measured turbidity 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 included, it is preferable that the detection or quantification method of the present invention further includes separating the aggregated magnetic substance by applying magnetic force. As a result, the aggregated magnetic substance is separated from the impurities containing the non-aggregated magnetic substance. Therefore, the measured values of the amount of the separated magnetic substance, the light transmittance when dispersed in the solvent, and the like exclude the influence of impurities and more faithfully reflect the existence of the detection substance.

  The magnetic force can be applied by bringing the magnet close to the magnetic substance. The magnetic force of this magnet depends on the magnitude of the magnetic force of the magnetic substance used. Examples of magnets include Neodymium magnets manufactured by Magna.

  Further, the magnetic force may be applied before the judgment or the measurement of the turbidity, or at the same time as the judgment or the measurement of the turbidity, but the simultaneous parallel is preferable in that the time spent in the process can be shortened. It should be noted that when magnetic force is applied, the aggregated magnetic substance is separated by entraining the contaminants, so it is presumed that the turbidity of the mixture after the separation is rather smaller when the contaminants are present. .

(Detection target)
Examples of the detection target in the sample include substances used for clinical diagnosis, and 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 virus 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.

<Resonant additive>
A resonant additive for adding to a test liquid for detecting and / or quantifying a detection target, the test liquid containing a first affinity substance for the detection target, and imparting a wave of a specific frequency A resonant additive having a resonant structure that resonates with is also one aspect of the present invention.
Examples of the resonant additive include those described above as the resonant structure. Examples of the detection target include immunological detection targets such as antigens and antibodies. Examples of the affinity substance include antibodies and antigens.

<How to use the resonant structure>
A method of using the resonating structure, which is added to the test solution and is caused to resonate by applying a wave having a specific frequency, is also one aspect of the present invention.

<Method of interacting using resonant structure>
A method of interacting a substance A with a substance having an affinity for the substance A, which comprises mixing the substance A, the affinity substance, and a resonant structure, and imparting a wave of a frequency unique to the resonant structure. A method including the step of resonating the resonant structure is also one aspect of the present invention.
The substance A is not particularly limited and may be the one described above as a detection target, but it is not limited to the reaction substance in the antigen-antibody reaction and can be widely selected. The affinity substance for the substance A may be the above-mentioned substance such as an antigen or an antibody, but is not limited to the antigen-antibody reaction and may be a substance capable of interacting with the substance A. The interaction may be binding, coordination, inclusion or the like between the substance A and the affinity substance, and may 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, low-molecular or high-molecular compounds. The above-mentioned thing can be used as a resonant structure and the 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 additive of the present invention, the method of using the resonant structure of the present invention, and the method of interacting of the present invention include causing the resonant additive or the resonant structure to function as a stirrer at a molecular level by resonance. You can Then, the interaction between the affinity substance existing in the reaction field surrounding the resonant structure and the detection target or the substance A is enabled in the minute reaction field while suppressing the damage to the reaction field and its surroundings. . Therefore, the resonant structure of the present invention and the method for interacting with the present invention are widely used not only in conventional cells and wells but also in reactions in microchemical processes, microchannels, microreactors, and the like. be able to.

<Container>
A container for detecting and / or quantifying an object to be detected, which container includes a resonator that resonates when a wave having a specific frequency is applied, is also one aspect of the present invention.
INDUSTRIAL APPLICABILITY The container of the present invention can transfer the vibration due to resonance to a fluid such as a liquid held inside the container by imparting a vibration of a specific frequency to the resonator to cause the resonance of the resonator. It is possible to increase the chances of bringing the reactants existing in the fluid into contact with each other. As a result, interactions such as binding and coordination of the reactants can be promoted. Therefore, according to the present invention, the efficiency of interaction can be enhanced even if the amount of the reaction substance is very small. In addition, the time required for interaction can be shortened.

As used herein, the term "reactive substance" with respect to the container of the present invention means a substance, such as two or more compounds, present in a fluid held inside the container and capable of interacting with each other. The term “reactive substance” is a concept that includes not only starting materials in a chemical reaction but also reaction intermediates, catalysts, and the like, and also detection targets (antigens) that are involved in detection and quantification of detection targets in specimens such as biological samples. , Antibodies, etc.) and affinity substances (antibodies, antigens, etc.) for the detection target.
In the present specification, the “interaction” with respect to the container of the present invention is a concept including binding, coordination, and inclusion of reactants, and is, for example, binding between a detection target and an affinity substance for the detection target. May be. The “interaction” may be one that has advanced to a reaction or the like.

  The container of the present invention is not particularly limited, and examples thereof include cells used for measurement of turbidity, absorbance and the like. According to the present invention, like a cell, a stirrer cannot normally be placed inside, and even if it is difficult to stir by vibrating the whole container from the outside, etc., it will resonate with the container itself. By causing the resonator, including the body, to resonate, the liquid and the like held inside the container can be easily stirred.

The resonator is preferably located at a portion that does not block transmitted light for detection and / or quantification, and more preferably at such a portion on the wall surface and / or bottom surface of the container.
The resonator is preferably provided at a location where vibration due to resonance is easily transmitted to the fluid held inside the container, and can be provided, for example, on a side surface and / or a bottom surface of the inner wall of the container.

The resonator is a solid that can resonate when a wave having a specific frequency is applied, and is not particularly limited as long as it is suitable for inclusion in a container, and examples thereof include glass and metal. As the resonator, glass is preferable from the viewpoint of minimizing the interaction with the fluid held inside the container and maintaining.
As the resonator, one that resonates by a wave having a lower output than that of the container body is preferable, and a structure that is discontinuous with the container body is preferable.
The shape of the resonator is not particularly limited.

The size of the resonator is not particularly limited, but the lower limit of the ratio of the total surface area of the resonator to the total surface area of the inner wall of the entire container is preferably 0.1%, and the upper limit is preferably 90%. is there.
Since the container of the present invention includes the resonator in a part of the container, deterioration of the container can be prevented as compared with the case where the entire container is the resonator. Further, since the container of the present invention preferably contains the resonator in a proportion of the total surface area within the above range, deterioration of the container can be prevented and vibration can be sufficiently transmitted to the fluid in the container based on the resonance.

In the container of the present invention, the wave to be applied is not particularly limited as long as it causes resonance in the resonator, and specifically, it may be a wave having a frequency unique to the resonance property, for example, an ultrasonic wave. , Microwaves, etc., and low frequencies are preferable.
The conditions such as the frequency of the wave to be applied, the time, and the output can be selected according to the material, size, and natural frequency of the resonator used.

  The frequency of the applied wave is the natural frequency of the resonator. The lower limit of the frequency of the applied wave is preferably 10 Hz, more preferably 15 Hz, and the upper limit is preferably 10 MHz, more preferably 2 MHz.

The lower limit of the output of the applied wave 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, as compared with the conventional stirring method by resonance, so that the wave can be imparted for a relatively long time and sufficient vibration is provided to promote the reaction. be able to. In addition, the sample is not damaged by heat denaturation.

Examples of the method of imparting a wave include a piezo element and a piezoelectric electron, and the piezo element is preferable.
When the container of the present invention is, for example, a cell used for measuring turbidity and the like, it is preferable that the wave is applied substantially simultaneously with the measurement of turbidity and the like in a measuring device for turbidity and the like.

The container of the present invention may be provided in a wave generating device that imparts a wave, or may be provided with a wave generating device.
One embodiment of the container of the present invention is, for example, as shown in FIG. 5, a cell 2 for measuring turbidity, and a wave generator 1 for imparting a wave to the contents of the cell. It may be provided. In FIG. 5, the direction in which the wave motion represented by the arrow in the figure is applied to the cell 2 from the wave generator 1 is the turbidity represented by the arrow passing through the cell 2 in the figure from the other side to the front side. Although it is orthogonal to the irradiation direction of the light for measuring the degree, it is not limited to this, and both directions may form any angle, for example, they may be parallel, but if they are parallel, they are given. It is preferable that the generated waves and the light for measuring the turbidity do not overlap so as not to affect their generators.

  As another embodiment of the container of the present invention, for example, as shown in FIG. 6, clinical biochemical automatic analysis mainly for automated quantitative analysis by colorimetric analysis of components such as blood, urine and other body fluids. Among the devices, the reaction container 3 in the discrete system may be provided with a wave generator. The wave generator 1 can be provided in the space between the respective mechanisms such as the reagent dispensing parts 61, 62 and 63, the stirring parts 64 and 65, the photometric part 66, and the cleaning area 67. Therefore, the reaction container 3 of the present invention can be used, for example, as a substitute for the reaction container in the existing discrete type clinical biochemical automatic analyzer, and the wave generator 1 can be provided in the existing device. The wave generation device 1 may be provided with a plurality as shown in FIG. 6, or may be one.

[Method for producing container of the present invention]
A resonator is provided at a predetermined portion of the container. For example, it can be manufactured by previously manufacturing a portion other than the resonator of the container, and fitting the resonator into a predetermined portion to fix it. When the resonator is made of glass, it is possible to place the resonator at a predetermined position in the process of manufacturing the container and mold it together with other parts of the container into a final shape.

<Method of interacting using the above container>
A method of causing a substance A and an affinity substance for the substance A to interact with each other, wherein the substance A and the affinity substance are present in the fluid held in the container of the present invention, and the resonator contained in the container is A method including the step of causing the resonator to resonate by applying a wave having a frequency unique to the resonator is also one aspect of the present invention.
The substance A is not particularly limited and may be the one described above as a detection target, but it is not limited to the reaction substance in the antigen-antibody reaction and can be widely selected. The affinity substance for the substance A may be the above-mentioned substance such as an antigen or an antibody, but is not limited to the antigen-antibody reaction and may be a substance capable of interacting with the substance A. The interaction may be binding, coordination, inclusion or the like between the substance A and the affinity substance, and may 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, low-molecular or high-molecular compounds. The fluid containing the substance A and the affinity substance may be, for example, a test liquid containing the affinity substance. The above-mentioned thing can be used as a wave to give.
The interaction method of the present invention is capable of transmitting vibration to a fluid held in a container by causing the resonator included in the container of the present invention to resonate by giving a wave of a natural frequency to the resonator. Therefore, the interaction between the substance A existing in the fluid and the affinity substance can be promoted.

DESCRIPTION OF SYMBOLS 1 Wave generator 10 1st binding substance 11 Solid phase 12 Antibody as 1st affinity substance 13 Antigen as 1st affinity substance 20 2nd binding substance 22, 23 As 2nd affinity substance Antibody 30 Labeling enzyme 40 Resonant structure 50 Detection target 51 Antigen as detection target 52 Antibody as detection target

Claims (7)

A method for detecting or quantifying a detection target in a sample,
Mixing the first affinity substance and the second affinity substance for the detection target, the resonant structure, and the analyte,
Resonating the resonating structure by applying a wave of a natural frequency to the resonating structure,
Comprising a step of determining a complex containing the first affinity substance and the detection target,
The resonant structure has an average particle size of 0.001 to 500 μm, and is composed of solid particles made of a single substance, hollow particles made of an organic polymer, and composite particles made of a metal and an organic polymer. At least one or more types of particles selected,
The method, wherein the first affinity substance and the second affinity substance can simultaneously bind to the detection target at different sites of the detection target. The first affinity substance or the second affinity substance is bound to a labeling enzyme,
The method according to claim 1, wherein the method further comprises reacting the labeling enzyme present in the complex with a substrate thereof before or during the determination.   The method according to claim 1, wherein the resonant structure is at least one kind of particles selected from the group consisting of hollow particles made of polystyrene and composite particles made of iron and polystyrene.   The method according to claim 1, wherein the frequency of the wave is 10 Hz or more and 3 MHz or less.   The method according to claim 1, wherein the application of the wave is performed by a piezo element, a crystal oscillator, or a dynamic speaker. A method of interacting a substance A with a first affinity substance and a second affinity substance for the substance A, comprising:
Mixing the substance A, the first affinity substance, the second affinity substance, and the resonant structure,
A step of causing the resonant structure to resonate by applying a wave of a frequency unique to the resonant structure,
The resonant structure has an average particle size of 0.001 to 500 μm and is selected from the group consisting of solid particles made of a single substance, hollow particles made of an organic polymer, and composite particles made of a metal and an organic polymer. At least one or more types of particles,
The method, wherein the first affinity substance and the second affinity substance can simultaneously bind to the substance A at different sites of the substance A. A method of using a resonating structure, which comprises adding to a test solution for detecting and / or quantifying a detection target, and causing resonance by applying a wave of a natural frequency,
The test liquid contains a first affinity substance and a second affinity substance for the detection target,
The resonant structure has an average particle size of 0.001 to 500 μm and is selected from the group consisting of solid particles made of a single substance, hollow particles made of an organic polymer, and composite particles made of a metal and an organic polymer. At least one or more types of particles,
The use method, 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.

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