JP2020030136A - Detection method of target substance - Google Patents

Abstract

To provide a detection method of target substances capable of detecting plural kinds of target substances in detection of target substances from a specimen utilizing magnetic force.SOLUTION: A magnetic particle 14 and labeling particles 16a, 16b are combined with target substances 12a, 12b and coupled bodies 20a, 20b are moved by magnetic force, thus using plural kinds of labeling particles combined with different target substances in detection of target substances, and solving a task by allowing plural kinds of labeling particles to satisfy at least one of a first condition in which particle sizes differ mutually and a second condition in which signal light is generated by irradiation with light and the signal light differs mutually.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for detecting a target substance used for detecting a target substance such as bacteria from a subject.

  In the detection of small organisms such as proteins, viruses and bacteria, it is preferable that the detection can be performed with high sensitivity even if the target substance is in a small amount. In particular, it is important that a virus, such as influenza virus and norovirus, for which an infectious disease is likely to spread, can be reliably detected even if the amount in the sample is very small.

As a highly sensitive detection method capable of detecting a small amount of a virus or the like, a polymerase chain reaction (PCR) is known.
In the polymerase chain reaction method, by amplifying only the base sequence to be tested by the polymerase chain reaction, the virus or the like to be tested can be amplified about 1,000,000 times in 20 cycles. Therefore, highly sensitive detection of viruses and the like is possible.
On the other hand, the polymerase chain reaction method has problems such as a large influence of contaminants, complicated pretreatment, and necessity of refrigeration or frozen storage such as a reagent test.

On the other hand, as a method for easily detecting a small amount of virus or the like, a near-field is formed on a detection plate described in Patent Documents 1 and 2, and a target substance (target substance) on the detection plate surface is formed. An optical detection method for detecting a conjugate containing as an optical signal is known.
The optical detection methods described in Patent Literature 1 and Patent Literature 2 use, for example, a conjugate obtained by binding magnetic particles and labeled particles (fluorescent particles or light scattering particles) to a target substance using an antigen-antibody reaction. Generate. On the other hand, a near field is generated on the front surface of the detection plate by light irradiated from the back surface side under the condition of total reflection. In this state, the generated combined body is attracted to the near field (detection plate) by the magnetic force and is moved in parallel with the detection plate. In the optical detection methods described in Patent Literature 1 and Patent Literature 2, a target substance is detected by measuring a change in light amount and a movement of light (bright point) caused by the movement of the subject.

JP 2017-219512 A WO 2017/187744

In an optical detection method in which only labeled particles such as fluorescent particles are bound to a target substance, the label particles adsorbed to a substance other than the target substance due to non-specific adsorption become noise, and the detection sensitivity is reduced. There is a problem that the substance cannot be detected.
In contrast, in the methods described in Patent Documents 1 and 2, non-specifically adsorbed labeled particles other than the target substance do not move due to the magnetic force, and the magnetic particles, the target substance and the labeled particle Only move by magnetic force while generating fluorescence or scattered light. Therefore, according to the method described in Patent Document 1, by detecting the movement of light (bright point), noise due to nonspecifically adsorbed labeled particles can be removed, and the target substance can be detected with high sensitivity. .

  However, in the detection methods described in Patent Literature 1 and Patent Literature 2, one kind of target substance can be suitably detected, but a plurality of kinds of target substances cannot be detected.

  An object of the present invention is to solve such problems of the prior art, and a method for detecting a target substance by binding magnetic particles and labeled particles to the target substance and moving the combined substance by magnetic force. The object of the present invention is to provide a target substance detection method capable of detecting a plurality of types of target substances.

In order to solve this problem, the present invention has the following configuration.
[1] A detection method for detecting a target substance by binding a magnetic particle and a label particle to the target substance and moving a combination of the target substance, the magnetic particle, and the label particle by a magnetic force,
Using multiple types of labeled particles that bind to different target substances,
The plurality of types of labeling particles generate signal light by irradiating light with a first condition in which the particle sizes are different from each other, and the signal light satisfies at least one of the second conditions in which the signal light is different from each other. The method for detecting a target substance.
[2] The description of [1], wherein when the plurality of types of labeled particles satisfy the first condition, the size of the large labeled particle is twice or more the size of the small labeled particle among the labeled particles having the closest sizes. Method of detecting target substance.
[3] When a plurality of types of labeled particles satisfy the second condition, the labeled particles are particles that emit light when irradiated with light, and the difference in emission wavelength between labeled particles having the closest emission wavelengths is 15 nm or more. , [1] or the method for detecting a target substance according to [2].
[4] The method for detecting a target substance according to any one of [1] to [3], wherein the plurality of types of labeled particles satisfy both the first condition and the second condition.
[5] The method for detecting a target substance according to any one of [1] to [4], wherein the detection of the target substance is performed by irradiating the labeled particles with light that generates signal light.
[6] The object according to any one of [1] to [4], wherein the detection of the object substance is performed using an observation light for observing a detection position of the object substance by enlarging a detection field of the object substance. How to detect substances.
[7] The method for detecting a target substance according to any one of [1] to [6], wherein the detection of the target substance is performed while moving the binder by magnetic force.
[8] The method for detecting a target substance according to any one of [1] to [6], wherein the detection of the target substance is performed after the movement of the conjugate by magnetic force.

  ADVANTAGE OF THE INVENTION According to the present invention, a plurality of types of target substances can be detected in a detection method of detecting a target substance by binding magnetic particles and label particles to the target substance and moving the binder by magnetic force.

It is a conceptual diagram for explaining an example of the detection method of the target substance of the present invention. FIG. 2 is a conceptual diagram illustrating a method for detecting a target substance shown in FIG. 1. It is a conceptual diagram for explaining an example of the detection method of the target substance of the present invention. It is a conceptual diagram for explaining another example of the detection method of the target substance of the present invention. It is a conceptual diagram for explaining another example of the detection method of the target substance of the present invention. It is a conceptual diagram for explaining another example of the detection method of the target substance of the present invention.

  Hereinafter, the method for detecting a target substance of the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings.

  In this specification, a numerical range represented by using “to” means a range including numerical values described before and after “to” as a lower limit and an upper limit.

  The method for detecting a target substance of the present invention comprises forming a conjugate in which magnetic particles and label particles are bound to the target substance, and moving the conjugate by magnetic force to form the conjugate, ie, the labeled particles bound to the target substance. By detecting, the target substance is detected.

Here, in the detection method of the present invention, a plurality of types of labeled particles that bind to different target substances are used.
Further, the plurality of types of labeled particles satisfy at least one of the first condition and the second condition. The first condition is that a plurality of labeled particles have different particle sizes. The second condition is that the marker particles generate signal light by light irradiation, and the signal light is different from each other in a plurality of marker particles.
The plurality of types of labeled particles may satisfy only the first condition, may satisfy only the second condition, or may satisfy both the first condition and the second condition. That is, the plurality of types of labeling particles may differ only in size from each other, or may only differ from each other in generated signal light, or may differ in size from each other and generate signal light from each other.

  FIG. 1 conceptually shows a state in which an analyte is dissolved in a detection solution 10 containing magnetic particles and labeled particles. In the present invention, “dissolved in the detection liquid 10” includes not only a state in which the substance is dissolved in the detection liquid 10 but also a state in which the substance is dispersed in the detection liquid 10.

The detection liquid 10 is contained in a transparent cell 24. In the detection liquid 10, the magnetic particles 14, and the first labeled particles 16a and the second labeled particles 16b are dissolved.
By dissolving the analyte in the detection liquid 10, for example, the first target substance 12a and the second target substance 12b are supplied to the detection liquid 10.
After the analyte is dissolved in the detection liquid 10, the sample may be left standing for a predetermined time, stirring may be performed, the stirring may be performed after standing for a predetermined time, and the stirring may be performed for a predetermined time after the stirring. You may leave still.

The magnetic particles 14 specifically bind to the first target substance 12a and the second target substance 12b. The first labeled particles 16a specifically bind to the first target substance 12a. The second labeling particles 16b specifically bind to the second target substance 12b.
Therefore, by dissolving the analyte in the detection solution 10, the conjugate 20a in which the first target substance 12a, the magnetic particles 14, and the first labeling particles 16a are bonded, and the second target substance 12b, and the magnetic particles 14a A conjugate 20b, in which the second labeling particles 16b are bonded to the second labeling particles 16b, is formed.
Although not shown in the drawings, the detection liquid 10 includes a conjugate of the first target substance 12a and the magnetic particles 14, a conjugate of the second target substance 12b and the magnetic particles 14, and a first target substance. There are also a conjugate of the second target substance 12b with the second labeled particle 16b, and the like, and the like.

Here, as described above, the first labeling particles 16a and the second labeling particles 16b are particles that generate signal light by the first condition having different sizes from each other and signal light generated by light irradiation, and the signal light generated is different from each other. At least one of the second conditions is satisfied.
The first labeling particles 16a and the second labeling particles 16b in the illustrated example are fluorescent particles that have the same size and emit fluorescence when irradiated with excitation light, and have different emission wavelengths. As an example, the first labeling particles 16a emit red fluorescence when irradiated with the excitation light, and the second labeling particles 16b emit blue fluorescence when irradiated with the excitation light. That is, in the illustrated example, the first marker particles 16a and the second marker particles 16b satisfy only the second condition.
The target substance detection method of the present invention enables detection of a plurality of types of target substances by having such a configuration.
This will be described in detail later.

In the present invention, the target substance (target substance) to be detected is not limited. Examples include viruses, bacteria, exosomes, DNA (deoxyribonucleic acid), RNA (ribonucleic acid), proteins, polysaccharides, contaminants, and the like. Among them, viruses, proteins and polysaccharides are suitable as target substances.
In addition, there is no limitation on the analyte (sample to be collected) from which the target substance is to be detected, and various substances that are considered to contain the target substance can be used. Examples of the subject include, for example, body fluids such as blood and lymph, cells, endothelium (epithelial cells), saliva, sweat, runny nose, tears, vomit, urine, feces, drugs, environmental water, clean water, sewage, and A wiping liquid or the like is exemplified.
These subjects may be collected by a known method according to the subject. For example, a method of wiping a door knob, a table, or the like at a site where food poisoning or the like has occurred with a swab (a cotton swab) or the like to collect a subject is exemplified. As another method, a method in which a swab is brought into contact with a vomit, urine, or the like to collect a subject is exemplified. In addition, cells, endothelium, and the like may be collected using an endoscope or the like.

  There is no limitation on the magnetic particles (magnetic particles, magnetic substances, and magnetic substances), and various types of known magnetic particles used for detecting a target substance using magnetism can be used. As an example, magnetic beads, magnetic powder, and the like are exemplified. These can also use a commercial item.

There is no limitation on the labeling particles, and the type can be identified by a method using signal light generated by light irradiation, observation of a captured image or further image analysis, and magnification observation using a microscope and a magnification optical system. Various known particles can be used.
Suitable examples of the labeling particles include fluorescent particles that emit fluorescence upon irradiation with excitation light and light scattering particles that scatter the irradiated light to generate scattered light.

There is no limitation on the fluorescent particles, and various known fluorescent particles used as labeling particles in the detection of a target substance as described in Patent Documents 1 and 2 described above can be used. Examples of the fluorescent particles include polymer particles containing a fluorescent dye, a fluorescent pigment and a rare earth, silica particles, silicon particles, and the like. Furthermore, quantum dots, biological fluorescent molecules, and the like can also be used. These can also use a commercial item. Further, the labeling particles may be phosphorescent particles.
When fluorescent particles are used as the labeling particles, the plurality of labeling particles may have the same size and satisfy only the second condition in which the wavelength of the generated signal light, that is, the emission wavelength (fluorescence wavelength) is different from each other, Alternatively, they may have the same emission wavelength and different sizes and satisfy only the first condition, or may satisfy both the first condition and the second condition.

  There is no limitation on the light scattering particles, and similarly, various types of known light scattering particles used as labeling particles in the detection of a target substance as described in Patent Documents 1 and 2 described above can be used. is there. Examples of the light scattering particles include metal particles such as polystyrene beads and gold particles, and silicon particles. These can also use a commercial item.

Note that, depending on the type of the target substance, the target substance may emit fluorescence by irradiation with the excitation light. In this case, the target substance may also serve as the label particles.
Also, depending on the type of the labeling particles, the labeling particles may be magnetic. In this case, the labeling particles may also serve as the magnetic particles (the magnetic particles may also serve as the labeling particles).

  The binding method between the target substance and the magnetic particles, and furthermore, the binding method between the target substance and the label particles is not limited, and the target substance and the magnetic particles, and a known method depending on the type of the target substance and the label particles. Is available. Examples include physical adsorption, antigen-antibody reaction, binding of a receptor to a ligand, DNA hybridization, chelate binding, amino binding, and the like.

Physical adsorption is a method of bonding a target substance and magnetic particles using an electrostatic bonding force such as a hydrogen bond. Physical adsorption is easy to carry out because it does not require treatment of magnetic particles. On the other hand, in the physical adsorption, the selectivity is low because the magnetic particles and the labeled particles do not specifically adsorb to the target substance. That is, in the physical adsorption, the magnetic particles and the labeled particles may bind to substances other than the target substance contained in the subject.
On the other hand, since the antigen-antibody reaction and the binding between the receptor and the ligand utilize specific binding to the target substance, the magnetic particles and labeled particles can be selectively bound to the target substance. There is an advantage.

When utilizing the antigen-antibody reaction, if the target substance is an antigen such as a bacterium, a virus, or an exosome, at least one of the magnetic particles and the labeled particles is modified in advance with an antibody against the target substance. It is necessary to prepare antibody-modified magnetic particles and antibody-modified labeled particles. When the target substance is a bacterium, a virus, an exosome, or the like, it is necessary to bind an antibody against an antigen present on the surface or the like of the target substance to at least one of the magnetic particles and the labeled particles in advance. There is.
When utilizing the binding between a receptor and a ligand, it is necessary to bind a receptor or a ligand that specifically binds to a target substance to at least one of the magnetic particles and the labeled particles in advance. Further, when the target substance is a bacterium, a virus, an exosome, or the like itself, a ligand for a receptor existing on the surface of the target substance or a receptor for a ligand existing on the surface of the target substance or the like in advance Must be bound to at least one of the magnetic particles and the labeled particles.

When both the magnetic particles and the labeled particles are bound to the target substance, at least one of the bindings is carried out by an antigen-antibody reaction and a specific binding to the target substance such as a binding between a receptor and a ligand. It is preferred that
When both the magnetic particles and the labeled particles are bound to the target substance, if both of the bindings are non-specific bonds, both the magnetic particles and the labeled particles bind to foreign substances other than the target substance. In this case, there is a disadvantage that the target substance and the foreign substance cannot be distinguished.

As described above, in the present invention, magnetic particles (magnetically labeled particles) that generate signal light upon irradiation with light, such as magnetic particles that generate fluorescence upon irradiation with excitation light, can also be used. .
In this case, it is preferable that the binding between the magnetic particle that emits fluorescence upon irradiation with the excitation light and the target substance and the particle is a specific bond with the target substance such as an antigen-antibody reaction. When the magnetic particles that emit fluorescence when irradiated with excitation light and the target substance are non-specifically bonded, the magnetic particles that emit fluorescence when irradiated with excitation light bind to foreign substances other than the target substance. In this case, there is a disadvantage that the target substance and the foreign substance cannot be distinguished from each other.

  In the present invention, as the detection liquid 10, various liquids capable of dissolving the analyte, the magnetic particles, and the target substance can be used. Specifically, the detection solution 10 includes a phosphate buffer, a Tris buffer, an acetate buffer, a citrate buffer, a tartrate buffer, PBS (phosphate buffered saline), water, and an aqueous liquid. Etc. are exemplified. As the water, it is preferable to use any of pure water, ion-exchanged water, and distilled water.

  Hereinafter, the method for detecting a target substance of the present invention will be described in more detail with reference to FIGS.

As described above, in the example shown in FIG. 1, the magnetic particles 14 and the first labeling particles 16 a and the second labeling particles 16 b are dissolved in the detection liquid 10 contained in the transparent cell 24. ing. The first labeling particles 16a and the second labeling particles 16b are both fluorescent particles, and the first labeling particles 16a emit red fluorescent light and the second labeling particles 16b emit blue light when irradiated with excitation light. Is emitted as described above.
By dissolving the analyte in the detection liquid 10, the first target substance 12a and the second target substance 12b are supplied to the detection liquid 10.

In the method for detecting an object of the present invention, the analyte may be dissolved in the detection solution first, and then the magnetic particles and the labeled particles may be dissolved (dispersed) in the detection solution simultaneously or sequentially. .
That is, in the method for detecting a target substance of the present invention, there is no limitation on the order of dissolution (supply order) of the analyte (target substance), magnetic particles, and label particles in the detection liquid.

By dissolving the analyte in the detection liquid 10 and supplying the first target substance 12a and the second target substance 12b, the first target substance 12a, the magnetic particles 14, and the first labeled particles 16a are bound. The combined body 20a and the combined body 20b in which the second target substance 12b, the magnetic particles 14, and the second labeled particles 16b are combined are formed.
Although illustration is omitted, in the detection liquid 10, a conjugate between the magnetic particles and the target substance and a conjugate between the label particles and the target substance are also formed as described above.

  As an example, a magnet 28 is arranged on the right side of the cell 24 in the drawing as conceptually shown in FIG. The magnet 28 is a known magnet.

Further, an excitation light irradiating unit 34 having a light source 30 and a condensing optical system 32 is provided so as to irradiate the inside of the cell 24 with excitation light from the bottom surface of the cell 24. Further, an image pickup means 40 having an image pickup element 36 and a condensing optical system 38 is provided so as to photograph an irradiation area of the excitation light by the excitation light irradiation means 34 inside the cell 24 from the bottom surface of the cell 24.
In addition, it is preferable that the imaging position (focal point of imaging) by the imaging unit 40 is not inside the wall surface (near field) of the cell 24 but inside the cell 24.

As the light source 30, various types of light sources that can excite the first labeling particles 16a and the second labeling particles 16b and emit light that emits fluorescence can be used. Examples include a light bulb such as a mercury lamp, a fluorescent lamp, a halogen lamp, an LED (Light Emitting Diode), and a laser such as a semiconductor laser.
As the image sensor 36, various types of image sensors capable of measuring the fluorescence emitted by the first marker particles 16a and the second marker particles 16b are available. As an example, a CMOS (Complementary Metal Oxide Semiconductor) image sensor (CMOS camera) and a CCD (Charge-Coupled Device) image sensor (CCD camera) are exemplified.
Both the condensing optical systems are publicly known condensing optical systems.
In addition, the light source 30 may be provided with an optical filter such as a band-pass filter as necessary. Further, the image sensor 36 and / or the condensing optical system 38 may be provided with an optical filter such as a sharp cut filter as needed.

Note that the method for detecting a target substance of the present invention is not limited to a configuration using an optical system for irradiating such excitation light and an optical system for imaging, and fluorescence is generated in labeled particles in the cell 24. Various methods can be used as long as the inside of the cell 24 can be observed.
For example, when the inside of the cell 24 can be observed with a fluorescence microscope, the target substance may be detected by observing the inside of the cell 24 with a fluorescence microscope. At this time, a microscope image by a fluorescence microscope may be taken by a CCD image sensor or the like as necessary.
Further, in the method for detecting a target substance of the present invention, the configuration for imaging the inside of the cell 24 using the imaging means 40 is not limited, and movement of a conjugate (labeled particle) described later is observed by visual observation. The target substance may be detected.

The excitation light irradiating means 34 irradiates the inside of the cell 24 with excitation light, and the imaging means 40 images the excitation light irradiation area inside the cell 24, and causes the magnet 28 to act.
By the irradiation of the excitation light, the first marker particles 16a emit red fluorescence, and the second marker particles 16b emit blue fluorescence.
In addition, the magnetic force of the magnet 28 causes the magnetic particles 14, the first target substance 12a, the combined body 20a of the magnetic particles 14 and the first labeling particles 16a to be bonded, and the second target substance 12b and the magnetic particles 14 The combined body 20b combined with the second labeling particles 16b moves toward the magnet 28 by magnetic force.

Here, the first labeling particles 16a and the second labeling particles 16b (further, a conjugate of the target substance and the labeling particles) emit fluorescence by irradiation with the excitation light, but do not have magnetic particles. Does not move due to magnetic force.
On the other hand, the magnetic particles 14 (further, a combination of the target substance and the magnetic particles) move by magnetic force, but do not emit fluorescent light because they have no labeled particles.
That is, only when at least one of the first target substance 12a and the second target substance 12b is present in the detection liquid 10, a conjugate of the target substance, the magnetic particles, and the label particles is formed, and the excitation light The fluorescent light (light-emitting body) moves toward the magnet 28 by the irradiation and the magnetic force of the magnet 28.

Specifically, when only the first target substance 12a is contained in the detection liquid 10, that is, the specimen, the first target substance 12a, the magnetic particles 14, and the first labeled particles 16a are bonded. Only the conjugate 20a formed is formed, and the conjugate 20b in which the second target substance 12b, the magnetic particles 14, and the second labeling particles 16b are bonded is not formed.
Therefore, in this case, although the red light and the blue light are generated by the irradiation of the excitation light, only the red light moves toward the magnet 28 by the magnetic force of the magnet 28.

When only the second target substance 12b is contained in the detection liquid 10, that is, the subject, only the conjugate 20b in which the second target substance 12b, the magnetic particles 14, and the second labeling particles 16b are bonded is provided. Are formed, and the conjugate 20a in which the first target substance 12a, the magnetic particles 14, and the first labeling particles 16a are bonded is not formed.
Therefore, in this case, although the excitation light irradiates the red fluorescence and the blue fluorescence, only the blue fluorescence moves toward the magnet 28 due to the magnetic force of the magnet 28.

When both the first target substance 12a and the second target substance 12b are contained in the detection liquid 10, that is, the specimen, the first target substance 12a, the magnetic particles 14, and the first labeling particles 16a Is formed, and a conjugate 20b is formed in which the second target substance 12b, the magnetic particles 14, and the second labeling particles 16b are bonded.
Therefore, in this case, the irradiation of the excitation light generates red fluorescence and blue fluorescence, and both the red fluorescence and the blue fluorescence move toward the magnet 28 by the magnetic force of the magnet 28.

  Furthermore, when neither the first target substance 12a nor the second target substance 12b is contained in the subject, the conjugate 20a and the conjugate 20b are not formed. Accordingly, in this case, red and blue fluorescent light are generated by the irradiation of the excitation light, but no fluorescent light moves due to the magnetic force of the magnet 28.

  Therefore, by analyzing the image captured by the imaging element 36 and detecting or further counting the movement of the red fluorescence and the movement of the blue fluorescence in the image, the first Whether or not the target substance 12a and the second target substance 12b exist can be detected.

  That is, according to the present invention using a plurality of types of labeled particles that bind to different target substances and emit different fluorescences, a plurality of target substances can be detected.

The above example is an example in which the excitation light emitted from the light source 30 excites both the first labeling particles 16a and the second labeling particles 16b to emit fluorescence. However, in some cases, one type of excitation light cannot excite both the first labeled particles 16a and the second labeled particles 16b.
In this case, first excitation light irradiating means for exciting the first labeling particles 16a to emit fluorescence and second excitation light irradiating means for exciting the second labeling particles 16b to emit fluorescence are provided. For example, first, the conjugate 20a having the first labeled particles 16a is detected by using the first excitation light irradiating means, and thereafter, the second excitation light is irradiated by using the second excitation light irradiating means. The conjugate 20b having the labeled particles 16b may be detected or further counted.
Alternatively, a light source such as a halogen lamp capable of irradiating broadband light, a first filter that transmits light that excites the first label particles 16a to emit fluorescence, and a second filter that excites the second label particles 16b are excited. For example, first, the first filter is inserted into the optical path to detect the conjugate 20a having the first labeled particles 16a, and then the second filter is used. May be inserted into the optical path to detect the conjugate 20b having the second labeled particles 16b, or to perform further counting.
Furthermore, a light source capable of irradiating broadband light such as a halogen lamp or a plurality of light sources capable of exciting each labeling particle, and a plurality of light receiving elements for receiving various fluorescent wavelengths are provided, corresponding to the respective combined bodies. The fluorescence may be detected by a corresponding light receiving element, or may be further counted.

In the method for detecting a target substance of the present invention, various methods can be used for moving and detecting the combined body 20a and the combined body 20b by magnetic force.
For example, as conceptually shown in FIG. 3, one magnet 28 is arranged, the inside of the cell 24 is irradiated with excitation light, and imaging is performed in the irradiation area of the excitation light, while the magnet 28 By moving the conjugate 20a and the conjugate 20b in one direction and detecting or further counting the fluorescence moving toward the magnet 28 in the captured image, the first target substance 12a and the second target substance are detected. 12b may be detected. Note that this method can be used even in a configuration having a plurality of magnets.
Also, as conceptually shown in FIG. 4, two magnets 28 are arranged so as to sandwich the cell 24, and the inside of the cell 24 is irradiated with excitation light, and imaging is performed in the irradiation area of the excitation light. The two target magnets alternately act to reciprocate the combined body 20a and the combined body 20b, and detect or further count the reciprocating fluorescence in the captured image, thereby obtaining the first target substance 12a. Alternatively, the detection of the second target substance 12b may be performed. Alternatively, magnets are also arranged above and below the cell 24 in the drawing, and the inside of the cell 24 is irradiated with excitation light, and while imaging is performed in the irradiation area of the excitation light, each magnet is caused to act sequentially, The first target substance 12a and the second target substance 12b may be detected by moving the combined body 20a and the combined body 20b two-dimensionally (rectangular) by magnetic force.
Further, as conceptually shown in FIG. 5, one magnet 28 is arranged, and first, the magnet 28 is actuated to collect the combined body 20a and the combined body 20b on the magnet 28 side in the cell 24 by magnetic force. . Thereafter, the magnet 28 is not operated as required, and the region where the combined bodies 20a and the combined bodies 20b are collected in the cell 24 is irradiated with excitation light, and the irradiation area of the excitation light is imaged. In the obtained image, the first target substance 12a and the second target substance 12b may be detected by detecting or further counting the fluorescence located on the magnet 28 side. Note that this method can also be used in a configuration having a plurality of magnets.

In the method for detecting a target substance in the illustrated example, the first labeled particles 16a and the second labeled particles 16b are different from each other in signal light generated by light irradiation. Specifically, the first labeling particles 16a and the second labeling particles 16b emit fluorescence when irradiated with excitation light, the first labeling particles 16a emit red fluorescence, and the second labeling particles 16a emit red fluorescence. 16b emits blue fluorescence and has different emission wavelengths.
As described above, when a plurality of types of labeled particles are particles that emit light by irradiation with light such as excitation light and satisfy the second condition in the present invention, the difference in emission wavelength between labeled particles having the closest emission wavelengths is obtained. Is preferably 15 nm or more, more preferably 25 nm or more, and even more preferably 50 nm or more.
By making the difference between the emission wavelengths 15 nm or more, it is possible to more appropriately identify a plurality of types of labeled particles that move by binding to the target substance, and the detection of a plurality of types of target substances can be improved. Accuracy can be easily achieved.

The examples shown in FIGS. 1 and 2 both detect two kinds of target substances, but the present invention is not limited thereto, and the third target substance, the fourth target substance, .., It is also possible to detect three or more target substances such as the n-th target substance.
In this case, the third labeled particles, the fourth labeled particles,..., And the n-th labeled particles, which generate different signal lights and specifically bind to a target substance different from the others, are similarly used. The target substance may be detected first.

In the examples shown in FIGS. 1 and 2, the same magnetic particles 14 specifically bind to both the first target substance 12a and the second target substance, but the present invention is not limited thereto.
That is, in the present invention, as will be described later in the examples, the magnetic particles include a magnetic particle that specifically binds to the first target substance 12a and a magnetic particle that specifically binds to the second target substance 12b. May be used. In this case, the magnetic particles that specifically bind to the first target substance 12a and the magnetic particles that specifically bind to the second target substance are different in size, magnetic force, forming material, and the like. Types of magnetic particles may be used.
In the case of detecting three or more target substances, magnetic particles that specifically bind to one target substance and magnetic particles that specifically bind to a plurality of target substances are mixed. Is also good.

  Regarding the above points, a case where a plurality of types of labeled particles satisfy the first condition that the particles are different in size from each other, and a case where the plurality of labeled particles satisfy both the first condition and the second condition, which will be described later. But it is the same.

FIG. 6 conceptually shows an example of the detection method of the present invention when a plurality of types of labeled particles satisfy the first condition having different sizes.
In the example shown in FIG. 6, the same reference numerals are given to the same substances as those in FIG. 1 and the like described above, and the description will mainly be made of different substances.

In the example shown in FIG. 6, the first labeling particles 46a and the second labeling particles 46b are different in size from each other. In the illustrated example, the first marker particles 46a are larger than the second marker particles 46b. In the following description, the light irradiated to detect the size of the label particles is also referred to as “detection light” for convenience.
When a plurality of types of labeled particles satisfy only the first condition having different sizes, fluorescent particles having the same emission wavelength may be used as the labeled particles, as described above.

Further, the first labeling particles 46a specifically bind to the first target substance 12a, similarly to the above-described first labeling particles 16a. On the other hand, the second labeling particles 46b specifically bind to the second target substance 12b, similarly to the above-described second labeling particles 16b.
Therefore, the analyte is dissolved in the detection liquid 10 containing the magnetic particles 14, the first labeling particles 46a and the second labeling particles 46b, and the first target substance 12a and the second target substance 12b are supplied. And a conjugate 50a in which the first target substance 12a, the magnetic particles 14, and the first labeling particles 46a are bonded, and a conjugate, in which the second target substance 12b, the magnetic particles 14, and the second labeling particles 46b are bonded. The combined body 50b is formed.

As described above, in the first labeling particles 46a and the second labeling particles 46b, the first labeling particles 46a are larger than the second labeling particles 46b. Therefore, when the detection liquid 10 containing the first label particles 46a and the second label particles 46b is irradiated with the detection light, two types of label particles having different sizes are observed, and the first label particles 46a Is larger than the second labeling particle 46b. When the labeling particles are fluorescent particles, two types of fluorescence having different sizes are observed.
When a plurality of types of labeling particles satisfy the first condition that the sizes are different from each other, the labeling particles that move by the same operation as the case of using a plurality of types of labeling particles having different emission wavelengths described above, The first target substance 12a and the second target substance 12b can be detected depending on the difference in particle size.
When a plurality of types of labeled particles satisfy the first condition having different sizes, the difference in the size of the labeled particles can be detected as scattered light.
In this case, when the detection liquid 10 containing the first labeling particles 46a and the second labeling particles 46b is irradiated with the detection light, two types of scattering light having the same signal light but different sizes are obtained. Light is observed and the first labeling particles 46a produce greater scattered light than the second labeling particles 46b. When the labeling particles are fluorescent particles, two types of fluorescence having different sizes are observed.
When a plurality of types of labeling particles satisfy the first condition that the sizes are different from each other, and scattered light is generated by irradiation of detection light, the same operation as when the above-described plurality of types of labeling particles having different emission wavelengths is used. Thus, the first target substance 12a and the second target substance 12b can be detected based on the scattered light that moves and the size of the scattered light.

Specifically, when only the first target substance 12a is contained in the detection liquid 10, that is, the subject, the first target substance 12a, the magnetic particles 14, and the first labeled particles 46a are bonded. Only the conjugate 50a formed is formed, and the conjugate 50b in which the second target substance 12b, the magnetic particles 14, and the second labeling particles 46b are bonded is not formed.
Therefore, in this case, the large labeled particles and the small labeled particles are detected by the irradiation of the detection light, but the magnetic force of the magnet 28 causes only the conjugate 50a to which the first labeled particles 46a, which are the large labeled particles, are bound. Move toward the magnet 28.

When only the second target substance 12b is contained in the detection liquid 10, that is, the specimen, only the conjugate 50b in which the second target substance 12b, the magnetic particles 14, and the second labeling particles 46b are bonded. Is formed, and the conjugate 50a in which the first target substance 12a, the magnetic particles 14, and the first labeling particles 56a are bonded is not formed.
Therefore, in this case, the large labeled particles and the small labeled particles are detected by the irradiation of the detection light, but the magnetic force of the magnet 28 causes only the conjugate 50b to which the second labeled particles 46b, which are the small labeled particles, are bound. Move toward the magnet 28.

When both the first target substance 12a and the second target substance 12b are contained in the detection liquid 10, that is, the subject, the first target substance 12a, the magnetic particles 14, and the first label particles 46a Is formed, and a conjugate 50b is formed in which the second target substance 12b, the magnetic particles 14, and the second labeling particles 46b are bonded.
Therefore, in this case, the large labeled particles and the small labeled particles are detected by the irradiation of the detection light, and the conjugate 50a in which the first labeled particles 46a, which are the large labeled particles, are bound by the magnetic force of the magnet 28, and The conjugate 50b to which the second labeling particles 46b, which are small labeling particles, are bound together moves toward the magnet 28.

Furthermore, when neither the first target substance 12a nor the second target substance 12b is contained in the subject, the conjugate 50a and the conjugate 50b are not formed.
Therefore, in this case, the large label particles and the small label particles are detected by the irradiation of the detection light, but even if the magnet 28 is actuated, no moving label particles exist.

  Therefore, the image captured by the image sensor 36 is observed or further image-analyzed, and in the image, the movement of the conjugate 50a to which the first labeling particle 46a that is a large labeling particle is bound, and the second labeling particle that is a small labeling particle The movement of the conjugate 50b to which the two labeled particles 46b are bound is detected or further counted to determine whether the first target substance 12a and the second target substance 12b are present in the subject. Can be detected.

  That is, according to the present invention using a plurality of types of labeled particles that bind to different target substances and have different sizes, a plurality of target substances can be detected.

In the present example, the light source 30 is a light source that emits detection light that enables the first marker particles 46a and the second marker particles 46 to be observed, and the imaging element 36 detects the light emitted by the light source 30. An image sensor capable of measuring light is used.
As the light source 30 and the imaging device 36, various known devices described above can be used as long as they satisfy the above conditions.

The image captured by the image sensor 36 may be subjected to image processing. For example, a particle size filter corresponding to the size of each labeled particle may be provided, and the size may be identified by the particle size filter to detect each labeled particle (fluorescence). Alternatively, a threshold may be set for the light intensity, and light exceeding the threshold may be counted.
Furthermore, if necessary, the intensity of the detection light (excitation light) may be changed to detect or further count the labeled particles.
The above points can be used even when the above-described signal light such as fluorescence generated by a plurality of types of labeled particles satisfies different second conditions.

When a plurality of types of labeled particles satisfy the first condition that the sizes are different from each other, the plurality of types of labeled particles have a size of a large labeled particle and a size of a small labeled particle of the closest labeled particles. It is preferably at least two times, more preferably at least 2.5 times, even more preferably at least three times.
By making the difference between the sizes of the labeled particles closest to each other two times or more, it is possible to more appropriately identify multiple types of labeled particles that move by binding to the target substance. It becomes possible to detect species target substances with higher accuracy and more easily.
In the present invention, the size of the labeled particles is the maximum length of the labeled particles, that is, the diameter of the smallest sphere that includes and inscribes the labeled particles. When commercially available particles are used as the labeled particles, the average particle size described in a catalog or the like may be used as the size of the labeled particles. In the present invention, the difference in the size of the labeled particles may be such that at least one of the maximum length and the catalog value is twice or more between the labeled particles having the closest size.

In the method for detecting a target substance according to the present invention, even when a plurality of types of labeled particles satisfy the first condition that the sizes are different from each other, the detection of the target substance is performed as shown in FIG. The combined body may be moved only in one direction while performing the irradiation and the imaging by the imaging element 36, or the irradiation with the light from the light source 30 and the imaging by the imaging element 36 may be performed as shown in FIG. Alternatively, the combination may be reciprocated, or, as shown in FIG. 5, after the combination is moved to the magnet 28 side, irradiation of the detection light from the light source 30 to the position where the combination has moved and The imaging by the imaging element 36 may be performed.
This point is the same even when a plurality of types of labeled particles satisfy both the first condition and the second condition.

In the above example, the target substance is detected by causing the labeled particles to emit fluorescence by irradiating the excitation light, or by detecting the labeled particles having different sizes by irradiating the detection light. Is not limited to this.
That is, the method for detecting a target substance of the present invention includes irradiating excitation light or the like for emitting labeled particles, and irradiating detection light for detecting the labeled particles with light for generating signal light on the labeled particles. The method of detecting by irradiation is not limited.
For example, when the inside of the cell 24 can be observed by a fluorescence microscope or the like, or when the imaging magnification of the condensing optical system 38 of the imaging unit 40 can be increased, light irradiation for generating signal light is not performed. In addition, by the observation light for observing the inside of the cell 24, if it is possible to directly visualize or image the label particles, without irradiating the label particles with light for generating signal light, The target substance may be detected by observation using observation light or further by imaging.
Note that, in the present invention, when the target substance is detected using only the observation light, the observation light also includes environmental light in the environment where the cell 24 exists.

The target substance detection method of the present invention is not limited to a configuration in which a plurality of types of labeled particles satisfy only the first condition having different sizes or a configuration in which generated signal light satisfies only the second condition different from each other. That is, in the method for detecting a target substance of the present invention, a plurality of types of labeled particles may satisfy both the first condition in which the sizes are different from each other and the second condition in which the generated signal light is different from each other.
When a plurality of types of labeling particles satisfy both the first condition and the second condition, for example, detection of a plurality of types of target substances by detecting movement of fluorescence having different emission wavelengths (colors) and sizes. Is possible, so that the target substance can be detected with higher accuracy and sensitivity, which is more preferable.

  As described above, the method for detecting the target substance of the present invention has been described in detail, but the present invention is not limited to the above-described example, and various improvements and changes may be made without departing from the gist of the present invention. Is, of course.

  Hereinafter, the features of the present invention will be described more specifically with reference to examples. The materials, reagents, amounts used, amounts of substances, ratios, processing details, processing procedures, and the like shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples described below.

<Preparation of anti-influenza A antibody-modified magnetic particles>
Mouse monoclonal anti-influenza A (nucleoprotein) antibody was biotin-modified using Biotin Labeling Kit-NH ₂ (manufactured by Dojindo Laboratories) according to the protocol.
Next, using the obtained biotinylated modified antibody and the spread-avidin-bound magnetic particles Dynabeads MyOne Streptavidin C1 (manufactured by Thermo Fisher Scientific Inc., average particle size 1 μm), the anti-influenza A antibody-modified magnetic particles were modified according to the protocol. Was prepared.

<Preparation of anti-influenza B antibody-modified magnetic particles>
Anti-influenza B antibody-modified magnetic particles were prepared in the same manner except that a mouse monoclonal anti-influenza B (nucleoprotein) antibody was used instead of a mouse monoclonal anti-influenza A (nucleoprotein) antibody.

<Production of anti-influenza A antibody-modified labeled particles-A1>
A mouse monoclonal anti-influenza A (nucleoprotein) antibody (an antibody different from the mouse monoclonal anti-influenza A (nucleoprotein) antibody used in the preparation of the anti-influenza A antibody-modified magnetic particles) was used. Biotin modification was performed using NH ₂ (manufactured by Dojindo Laboratories) according to the protocol.
Next, using the obtained biotinylated modified antibody and streptavidin-conjugated fluorescent particles Streptavidin Fluoresbrite YG Microspheres, 6.0 μm (Polysciences Inc., average particle size 6 μm), an anti-influenza A antibody according to the protocol Modified labeled particles-A1 were prepared.
The labeled particles are fluorescent particles having an average particle size of 6 μm, an excitation wavelength peak of 441 nm, and an emission wavelength peak of 486 nm.

<Preparation of anti-influenza B antibody-modified labeled particles-B1>
A mouse monoclonal anti-influenza B (nucleoprotein) antibody (an antibody different from the mouse monoclonal anti-influenza B (nucleoprotein) antibody used in the preparation of the anti-influenza B antibody-modified magnetic particles) was used for the Biotin Labeling Kit- Biotin modification was performed using NH ₂ (manufactured by Dojindo Laboratories) according to the protocol.
Next, using the obtained biotinylated modified antibody and streptavidin-conjugated fluorescent particles Streptavidin Fluoresbrite YG Microspheres, 1.0 μm (Polysciences Inc., average particle diameter 1 μm), an anti-influenza B antibody according to the protocol. Modified labeled particles-B1 were produced.
The labeled particles are fluorescent particles having an average particle size of 1 μm, an excitation wavelength peak of 441 nm, and an emission wavelength peak of 486 nm.

<Preparation of detection solution-1>
Made,
Anti-influenza A antibody-modified magnetic particles,
Anti-influenza B antibody-modified magnetic particles,
Anti-influenza A antibody-modified labeled particles-A1 (average particle diameter 6 μm, excitation wavelength peak 441 nm, emission wavelength peak 486 nm), and
Anti-influenza B antibody-modified labeled particles-B1 (average particle diameter 1 μm, excitation wavelength peak 441 nm, emission wavelength peak 486 nm)
Was dispersed in 1 mL (liter) of PBS to prepare a detection solution-1. The addition amount of each particle was all 1 × 10 ⁷ .
The detection of the target substance using this detection liquid-1 is to detect an influenza A antibody and an influenza B antibody as the target substances.

[Example 1]
<Detection of influenza A nuclear protein antigen using labeled particles of different sizes (first condition)>
A mixed solution was prepared by adding the same amount of a PBS solution of influenza A nuclear protein antigen adjusted to a concentration of 1 × 10 ³ PFU (Plaque Forming Unit) to 100 μL of the prepared detection solution-1 (100 μL). .
Next, the obtained mixture was placed in a cell (Photon Silde Ultra-low Fluorescence Counting Slides, manufactured by Logos Biosystems, Inc.) and allowed to stand for 3 minutes.
Next, the permanent magnet is brought close to the cell, and while the permanent magnet is manually moved, the inside of the cell is irradiated with excitation light by a blue LED light source (peak wavelength 440 nm) as conceptually shown in FIG. The excitation light irradiation area was imaged by an imaging means including a general color CCD image sensor, a sharp cut filter (a transmission limit wavelength of 520 nm), and a condensing optical system.

As a result of analyzing the captured image, it was confirmed that 35 green fluorescent lights having a size of about 6 μm were moved in synchronization with the movement of the permanent magnet. In addition, green fluorescence having a size of about 1 μm was also observed in the detection solution, but this fluorescence did not move even when the magnet was moved.
Thereby, in the mixed solution, a conjugate of influenza A nuclear protein antigen, anti-influenza A antibody-modified magnetic particles, and anti-influenza A antibody-modified labeled particles -A1 having a size of 6 μm is formed, and A conjugate of the influenza B nuclear protein antigen, the anti-influenza B antibody-modified magnetic particles, and the anti-influenza B antibody-modified labeled particles-B1 having a size of 1 μm is not formed, and the influenza A nucleus is contained in the mixture. It was confirmed that the protein antigen was present and the influenza B nuclear protein antigen was not present.

[Example 2]
<Detection of influenza B nuclear protein antigen using labeled particles of different sizes (first condition)>
A mixture was prepared using the same concentration and the same amount of the influenza B nuclear protein antigen PBS solution instead of the influenza A nuclear protein antigen PBS solution.
A target substance was detected in the same manner as in Example 1 except that this mixture was used.
As a result, it was confirmed that 28 green fluorescent lights having a size of about 1 μm were moved in synchronization with the movement of the permanent magnet. In the detection solution, green fluorescence having a size of about 6 μm was also observed, but this fluorescence did not move even when the magnet was moved.
Thereby, in the mixed solution, a conjugate of influenza B nuclear protein antigen, anti-influenza B antibody-modified magnetic particles, and anti-influenza B antibody-modified labeled particles-B1 having a size of 1 μm is formed, and A conjugate of the influenza A nuclear protein antigen, the anti-influenza A antibody-modified magnetic particles, and the anti-influenza A antibody-modified labeled particle-A1 having a size of 6 μm is not formed, and the influenza B nucleus is contained in the mixture. It was confirmed that the protein antigen was present and the influenza A nuclear protein antigen was not present.

[Example 3]
<Detection of influenza A nuclear protein antigen and influenza B nuclear protein antigen using labeled particles having different sizes (first condition)>
To 100 μL of the prepared detection solution-1, the same amount of a PBS solution of influenza A nuclear protein antigen and a PBS solution of influenza B nuclear protein antigen adjusted to a concentration of 1 × 10 ³ PFU were added, A mixture was prepared.
A target substance was detected in the same manner as in Example 1 except that this mixture was used.
As a result, 24 green fluorescent lights having a size of about 6 μm move in synchronization with the movement of the permanent magnet, and 31 green fluorescent lights having a size of about 1 μm move in the movement of the permanent magnet. It was confirmed that they were moving synchronously.
Thereby, in the mixed solution, a conjugate of influenza A nuclear protein antigen, anti-influenza A antibody-modified magnetic particles, anti-influenza A antibody-modified labeled particles-A1 having a size of 6 μm, and influenza B A conjugate of a nuclear protein antigen, an anti-influenza B antibody-modified magnetic particle, and a 1 μm-sized anti-influenza B antibody-modified labeled particle-B1 is formed, and the influenza A nuclear protein antigen, and The presence of the influenza B nuclear protein antigen was confirmed.

  Further, Examples 1 to 3 have revealed that according to the present invention, influenza A nuclear protein antigen and influenza B nuclear protein antigen can be detected with high sensitivity.

<Preparation of anti-influenza A antibody-modified labeled particles-A2>
A mouse monoclonal anti-influenza A (nucleoprotein) antibody (an antibody different from the mouse monoclonal anti-influenza A (nucleoprotein) antibody used in the preparation of the anti-influenza A antibody-modified magnetic particles) was used. Biotin modification was performed using NH ₂ (manufactured by Dojindo Laboratories) according to the protocol.
Next, anti-influenza A antibody-modified labeled particles-A2 were prepared according to the protocol using the obtained biotinylated modified antibody and the FP Cyanine3 Streptavidin beads (supplied by Tamagawa Seiki Co., Ltd.).
The labeled particles are fluorescent particles having an average particle size of 0.4 μm, an excitation wavelength peak of 550 nm, and an emission wavelength peak of 576 nm.

<Preparation of anti-influenza B antibody-modified labeled particles-B2>
A mouse monoclonal anti-influenza B (nucleoprotein) antibody (an antibody different from the mouse monoclonal anti-influenza B (nucleoprotein) antibody used in the preparation of the anti-influenza B antibody-modified magnetic particles) was used for the Biotin Labeling Kit- Biotin modification was performed using NH ₂ (manufactured by Dojindo Laboratories) according to the protocol.
Next, anti-influenza B type antibody-modified labeled particles-B2 were prepared using the obtained biotinylated modified antibody and FS Eu Streptavidin beads (manufactured by Tamagawa Seiki Co., Ltd.) according to the protocol.
The labeled particles are fluorescent particles having an average particle size of 0.4 μm, an excitation wavelength peak of 340 nm, and an emission wavelength peak of 616 nm.

<Preparation of detection solution-2>
Made,
Anti-influenza A antibody-modified magnetic particles,
Anti-influenza B antibody-modified magnetic particles,
Anti-influenza A antibody-modified labeled particles-A2 (average particle size 0.4 μm, excitation wavelength peak 550 nm, emission wavelength peak 576 nm), and
Anti-influenza B antibody-modified labeled particles-B2 (average particle size 0.4 μm, excitation wavelength peak 340 nm, emission wavelength peak 616 nm)
Was dispersed in 1 mL of PBS to prepare a detection solution-2. The addition amount of each particle was all 1 × 10 ⁷ .
The detection of the target substance using this detection solution-2 is to detect an influenza A antibody and an influenza B antibody as the target substances.

[Example 4]
<Detection of influenza A nuclear protein antigen by labeled particles having different emission wavelengths (second condition)>
To 100 μL of the prepared detection solution-2, an equal amount of a PBS solution of influenza A nuclear protein antigen adjusted to a concentration of 1 × 10 ³ PFU was added.
Next, the obtained mixture was placed in a cell (Photon Silde Ultra-low Fluorescence Counting Slides, manufactured by Logos Biosystems, Inc.) and allowed to stand for 3 minutes.
Then, while the permanent magnet is brought close to the cell and the permanent magnet is manually moved, the excitation light having a peak wavelength of 510 nm is peaked by the green LED light source and the ultraviolet LED light source is peaked inside the cell as conceptually shown in FIG. Excitation light having a wavelength of 340 nm was sequentially irradiated, and the excitation light irradiation area inside the cell was imaged by an image pickup means including a general color CCD image sensor, a sharp cut filter (transmission limit wavelength: 560 nm), and a condensing optical system. .

As a result of analyzing the captured image, it was confirmed that 21 orange fluorescent lights were moved in synchronization with the movement of the permanent magnet when the excitation light having the peak wavelength of 510 nm was irradiated. On the other hand, in the state where the excitation light having the peak wavelength of 340 nm was irradiated, red fluorescence was confirmed, but this fluorescence did not move even when the magnet was moved.
Thereby, in the mixed solution, a conjugate of the influenza A nuclear protein antigen, the anti-influenza A antibody-modified magnetic particles, and the anti-influenza A antibody-modified labeled particles -A2 exhibiting orange fluorescence is formed, and No conjugate of influenza B nuclear protein antigen, anti-influenza B antibody-modified magnetic particles, and anti-influenza A antibody-modified labeled particles-B2 exhibiting red fluorescence is formed, and influenza is contained in the mixture. It was confirmed that the type A nuclear protein antigen was present and the influenza type B nuclear protein antigen was not present.

[Example 5]
<Detection of influenza B nuclear protein antigen by labeled particles having different emission wavelengths (second condition)>
A mixture was prepared using the same concentration and the same amount of the influenza B nuclear protein antigen PBS solution instead of the influenza A nuclear protein antigen PBS solution.
A target substance was detected in the same manner as in Example 4 except that this mixed solution was used.
As a result, in a state where the excitation light having a peak wavelength of 510 nm was irradiated, orange fluorescence was confirmed, but the fluorescence did not move even when the permanent magnet was moved. On the other hand, in the state where the excitation light having the peak wavelength of 340 nm was irradiated, it was confirmed that 19 pieces of red fluorescence moved in synchronization with the movement of the permanent magnet.
Thereby, in the mixed solution, a conjugate of the influenza B nuclear protein antigen, the anti-influenza B antibody-modified magnetic particles, and the anti-influenza B antibody-modified labeled particles-B2 exhibiting red fluorescence is formed, and No conjugate of influenza A nuclear protein antigen, anti-influenza A antibody-modified magnetic particles, and anti-influenza B antibody-modified labeled particles-A2 exhibiting orange fluorescence was formed, and influenza B was contained in the mixture. It was confirmed that there was a type nucleoprotein antigen and no influenza A type nucleoprotein antigen was present.

[Example 6]
<Detection of influenza A nuclear protein antigen and influenza B nuclear protein antigen by labeled particles having different emission wavelengths (second condition)>
To 100 μL of the prepared detection solution-2, the same amount of a PBS solution of influenza A nuclear protein antigen and a PBS solution of influenza B nuclear protein antigen adjusted to a concentration of 1 × 10 ³ PFU were added, A mixture was prepared.
A target substance was detected in the same manner as in Example 4 except that this mixed solution was used.
As a result, it was confirmed that 23 pieces of orange fluorescence moved in synchronization with the movement of the permanent magnet when the excitation light having a peak wavelength of 510 nm was irradiated, and further, the excitation light having a peak wavelength of 340 nm was confirmed. It was confirmed that 24 pieces of red fluorescence were moved in synchronization with the movement of the permanent magnets in the state of irradiation of.
Thereby, in the mixed solution, a conjugate of the influenza A nuclear protein antigen, the anti-influenza A antibody-modified magnetic particles, the anti-influenza A antibody-modified labeled particles -A2 exhibiting orange fluorescence, and the influenza B A conjugate of a type nucleoprotein antigen, an anti-influenza B antibody-modified magnetic particle, and an anti-influenza B antibody-modified labeled particle-2 exhibiting red fluorescence is formed, and the influenza A nuclear protein antigen is contained in the mixture. , And the presence of influenza B nuclear protein antigen.

  Further, Examples 4 to 6 revealed that the present invention can detect influenza A nuclear protein antigen and influenza B nuclear protein antigen with high sensitivity.

<Production of anti-influenza A antibody-modified labeled particles-A3>
A mouse monoclonal anti-influenza A (nucleoprotein) antibody (an antibody different from the mouse monoclonal anti-influenza A (nucleoprotein) antibody used in the preparation of the anti-influenza A antibody-modified magnetic particles) was used. Biotin modification was performed using NH ₂ (manufactured by Dojindo Laboratories) according to the protocol.
Next, using the obtained biotinylated modified antibody and streptavidin-conjugated fluorescent particles Streptavidin Fluoresbrite YG Microspheres, 1.0 μm (Polysciences Inc., average particle diameter 1 μm), an anti-influenza A antibody according to the protocol. Modified labeled particles-A3 were produced.
The labeled particles are fluorescent particles having an average particle size of 1 μm, an excitation wavelength peak of 441 nm, and an emission wavelength peak of 486 nm.

<Preparation of anti-influenza B antibody-modified labeled particles-B3>
A mouse monoclonal anti-influenza B (nucleoprotein) antibody (an antibody different from the mouse monoclonal anti-influenza B (nucleoprotein) antibody used in the preparation of the anti-influenza B antibody-modified magnetic particles) was used for the Biotin Labeling Kit- Biotin modification was performed using NH ₂ (manufactured by Dojindo Laboratories) according to the protocol.
Next, anti-influenza B antibody-modified labeled particles-B3 were produced using the obtained biotinylated modified antibody and the fluorescent particles FS Eu Streptavidin conjugated with streptavidin (manufactured by Tamagawa Seiki Co., Ltd.) according to the protocol.
The labeled particles are fluorescent particles having an average particle size of 0.4 μm, an excitation wavelength peak of 340 nm, and an emission wavelength peak of 616 nm.

<Preparation of detection solution-3>
Made,
Anti-influenza A antibody-modified magnetic particles,
Anti-influenza B antibody-modified magnetic particles,
Anti-influenza A antibody-modified labeled particles-A3 (average particle size 1 μm, excitation wavelength peak 441 nm, emission wavelength peak 486 nm), and
Anti-influenza B antibody-modified labeled particles-B3 (average particle size 0.4 μm, excitation wavelength peak 340 nm, emission wavelength peak 616 nm)
Was dispersed in 1 mL of PBS to prepare a detection solution-3. The addition amount of each particle was all 1 × 10 ⁷ .
The detection of the target substance using the detection solution-3 is to detect an influenza A antibody and an influenza B antibody as the target substances.

[Example 7]
<Detection of influenza A nuclear protein antigen by labeled particles having different sizes (first conditions) and emission wavelengths (second conditions)>
To 100 μL of the prepared detection solution- ³ , an equal amount of a PBS solution of influenza A nuclear protein antigen adjusted to a concentration of 1 × 10 ³ PFU was added.
Next, the obtained mixture was placed in a cell (Photon Silde Ultra-low Fluorescence Counting Slides, manufactured by Logos Biosystems, Inc.) and allowed to stand for 3 minutes.
Then, while the permanent magnet is brought close to the cell and the permanent magnet is manually moved, the excitation light having a peak wavelength of 440 nm is emitted from the green LED light source and the ultraviolet LED light source is peaked inside the cell as conceptually shown in FIG. Excitation light having a wavelength of 340 nm was sequentially irradiated, and an excitation light irradiation area inside the cell was imaged by an image pickup means including a general color CCD image sensor, a sharp cut filter (a transmission limit wavelength of 520 nm), and a condensing optical system. .

As a result of analyzing the captured image, it was confirmed that 21 pieces of green fluorescent light having a size of about 1 μm moved in synchronization with the movement of the permanent magnet when the excitation light having a peak wavelength of 440 nm was irradiated. Was done. On the other hand, in the state where the excitation light having the peak wavelength of 340 nm was irradiated, red fluorescence having a size of about 0.4 μm was confirmed, but this fluorescence did not move even when the magnet was moved.
Thereby, in the mixed solution, the conjugate of the influenza A nuclear protein antigen, the anti-influenza A antibody-modified magnetic particles, and the anti-influenza A antibody-modified labeled particles -A3 having a size of 1 μm and exhibiting green fluorescence is formed. A conjugate formed with the formed influenza B nuclear protein antigen, the anti-influenza B antibody-modified magnetic particles, and the anti-influenza B antibody-modified labeled particles -B3 having a size of 0.4 μm and exhibiting red fluorescence is formed. However, it was confirmed that the influenza A nuclear protein antigen was present and the influenza B nuclear protein antigen was not present in the mixture.

Example 8
<Detection of influenza B nuclear protein antigen by labeled particles having different sizes (first condition) and emission wavelengths (second condition)>
A mixture was prepared using the same concentration and the same amount of the influenza B nuclear protein antigen PBS solution instead of the influenza A nuclear protein antigen PBS solution.
A target substance was detected in the same manner as in Example 7, except that this mixture was used.
As a result, in the state where the excitation light having the peak wavelength of 440 nm was irradiated, green fluorescence having a size of about 1 μm was confirmed, but the fluorescence did not move even when the permanent magnet was moved. On the other hand, in the state where the excitation light having the peak wavelength of 340 nm was irradiated, it was confirmed that 19 pieces of red fluorescence having a size of about 0.4 μm were moved in synchronization with the movement of the permanent magnet. .
Thereby, in the mixed solution, the binding of the influenza B nuclear protein antigen, the anti-influenza B antibody-modified magnetic particles, and the anti-influenza B antibody-modified labeled particles -B3 having a size of 0.4 μm and exhibiting red fluorescence. A body is formed, and a conjugate of influenza A nuclear protein antigen, anti-influenza A antibody-modified magnetic particles, and anti-influenza A antibody-modified labeled particles -A3 having a size of 1 μm and exhibiting green fluorescence is formed. However, it was confirmed that the influenza B nuclear protein antigen was present and the influenza A nuclear protein antigen was not present in the mixture.

[Example 9]
<Detection of influenza A nuclear protein antigen and influenza B nuclear protein antigen by labeled particles having different sizes (first conditions) and emission wavelengths (second conditions)>
To 100 μL of the prepared detection solution-2, the same amount of a PBS solution of influenza A nuclear protein antigen and a PBS solution of influenza B nuclear protein antigen adjusted to a concentration of 1 × 10 ³ PFU were added, A mixture was prepared.
A target substance was detected in the same manner as in Example 7, except that this mixture was used.
As a result, in a state where the excitation light having the peak wavelength of 440 nm was irradiated, it was confirmed that 23 pieces of green fluorescence having a size of about 1 μm were moved in synchronization with the movement of the permanent magnet. In a state where the excitation light having the wavelength of 340 nm was irradiated, it was confirmed that 24 pieces of red fluorescence having a size of about 0.4 μm were moved in synchronization with the movement of the permanent magnet.
Thereby, in the mixed solution, a conjugate of the influenza A nuclear protein antigen, the anti-influenza A antibody-modified magnetic particles, and the anti-influenza A antibody-modified labeled particles -A3 having a size of 1 μm and exhibiting green fluorescence, A conjugate of an influenza B nuclear protein antigen, anti-influenza B antibody-modified magnetic particles, and anti-influenza B antibody-modified labeled particles -B3 having a size of 0.4 μm and exhibiting red fluorescence is formed and mixed. It was confirmed that influenza A nuclear protein antigen and influenza B nuclear protein antigen were present in the solution.

Further, according to Examples 7 to 9, according to the present invention, by satisfying the first condition and the second condition, the influenza can be more suitably determined by the combination of the size of the labeled particles and the emission wavelength (color of emission). It has been revealed that the A-type nuclear protein antigen and the influenza B-type nuclear protein antigen can be detected with high sensitivity.
From the above results, the effect of the present invention is clear.

  It can be suitably used for detection of viruses and the like in medical care and research.

DESCRIPTION OF SYMBOLS 10 Detection liquid 12a 1st target substance 12b 2nd target substance 14 Magnetic particle 16a, 46a 1st label particle 16b, 46b 2nd label particle 20a, 20b, 50a, 50b Combined body 24 Cell 28 Magnet 30 Light source 32 38, condensing optical system 34 excitation light irradiation means 36 imaging device 40 imaging means

Claims (8)

A detection method for detecting the target substance by binding the magnetic particles and the label particles to the target substance, and moving the conjugate of the target substance, the magnetic particles and the label particles by magnetic force,
Using a plurality of types of the labeled particles that bind to the target substance different from each other,
The plurality of types of labeled particles have a first condition in which particle sizes are different from each other and a signal light is generated by light irradiation, and the signal light satisfies at least one of the second conditions different from each other. A method for detecting a target substance, the method comprising:   2. When the plurality of types of labeling particles satisfy the first condition, the size of the large labeling particle is twice or more the size of the small labeling particle among the labeling particles having the closest size. 2. The method for detecting a target substance according to 1.   When the plurality of types of labeling particles satisfy the second condition, the labeling particles are particles that emit light when irradiated with light, and the labeling particles having the closest emission wavelength have a difference in emission wavelength of 15 nm or more. 3. The method for detecting a target substance according to claim 1 or 2.   The method for detecting a target substance according to any one of claims 1 to 3, wherein the plurality of types of labeled particles satisfy both the first condition and the second condition.   The method for detecting a target substance according to any one of claims 1 to 4, wherein the detection of the target substance is performed by irradiating the labeling particles with light that generates signal light.   The object according to any one of claims 1 to 4, wherein the detection of the target substance is performed by using an observation light for observing a detection position of the target substance by enlarging a detection visual field of the target substance. How to detect substances.   The method for detecting a target substance according to any one of claims 1 to 6, wherein the detection of the target substance is performed while moving the combination by the magnetic force.   The method for detecting a target substance according to any one of claims 1 to 6, wherein the detection of the target substance is performed after the combined body is moved by the magnetic force.