JP2000131320A - Method for measuring substance to be inspected using particles carrying immobilized specific binding material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly reliable and highly sensible method for measuring substance to be inspected by which the presence/-absence of a substance to be inspected in a sample can be discriminated in a short time even when the quantity of the substance exists is extremely small at the time of measuring the substance visually or by using an instrument by utilizing a specific binding reaction, such as the antigen-antibody reaction, etc. SOLUTION: A method for measuring substance to be inspected in sample includes 1) an operation for bringing particles carrying an immobilized specific binding substance for a substance to be inspected contained in a sample into contact with the sample, 2) an operation for binding the substance to be inspected into contact with the specific binding substance immobilized on the particles in the mixed solution of the particles and sample, and 3) an operation for bringing the particles in the mixed solution into contact with the discriminating section of a carrier having the discriminating section in which a specific binding substance for the substance to be inspected that is equal to or different from that immobilized on the particles is immobilized and which is covered with the specific binding substance or the portion of the carrier connected to the discriminating section. The method also includes 4) an operation for moving the particles along the discriminating section and 5) an operation for discriminating the presence/absence of the substance to be inspected from the distributed state of the particles in the discriminating section.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring a test substance in a sample using particles having a specific binding substance to the test substance immobilized thereon, and particularly to a method for measuring a very small amount of the test substance contained in the sample. The present invention relates to a method for measuring a test substance capable of performing measurement in a short time and easily without being affected by interfering substances. INDUSTRIAL APPLICABILITY The present invention is useful in the field of analytical chemistry, the field of life science, particularly the field of clinical examination.

[0002]

2. Description of the Related Art A trace amount of a sample contained in a sample utilizing a reaction between a substance having a specific affinity such as an antigen and an antibody, a sugar and a lectin, a nucleotide chain and its complementary nucleotide chain, and a ligand and a receptor. Various types of measuring methods or measuring instruments for analytes are known.

[0003] Among them, immunological measurement methods utilizing an antigen-antibody reaction (immune reaction) between an antigen and an antibody are widely practiced. Among these immunological measurement methods, the indirect agglutination measurement method is widely used because it is a simple and inexpensive method.

[0004] In this indirect agglutination assay, particles (such as latex particles or gelatin particles) to which an antibody to a test substance (an antigen or an antibody to this antibody can be used if the test substance is an antibody) can be used. Polymer particles or erythrocytes) and a sample presumed to contain the test substance were mixed and reacted in a measurement container having a U-shaped or V-shaped bottom surface such as a microtiter plate. The presence or absence of the test substance in the sample is determined by observing the aggregation image between the particles via the test substance.

FIG. 7 shows an agglutination image when a test substance in a sample is measured by this indirect agglutination reaction measurement method. If the test substance does not exist in the sample (negative), the particles bound with the antibody (or antigen) do not cause aggregation, so they settle down by gravity, and fall on the inner wall surface of the U-shaped or V-shaped measurement container. It rolls down (slids down) and gathers in the center of the bottom of the measuring container. Therefore, in the negative case, when viewed from above the measurement container, an image in which the particles are converged at the center of the bottom surface of the measurement container is observed (A in FIG. 7).

On the other hand, when the test substance is present (positive) in the sample, the particles to which the antibody (or antigen) is bound undergo three-dimensional aggregation via the test substance to form an aggregate.
This agglomerate is less likely to roll on the inner wall surface of the measurement container (harder to slide down) and has a lower rolling speed (sliding down speed) than particles that do not cause aggregation. Stop. Therefore, in the case of positive, when viewed from above the measurement container, particles are spread on the bottom surface of the measurement container, that is, a “button” -like aggregation image can be observed (FIG. 7B), and the test substance is present in the sample. Can be confirmed.

However, in this indirect agglutination measurement method, the presence or absence of a test substance in a sample, that is, whether it is positive or negative, is determined by the size of a circular image formed by particles on the bottom surface of a measurement container. Therefore, in the case of a sample in which the concentration of the test substance is low, both the particles aggregated via the test substance and the particles not bonded to the test substance are mixed in the center of the bottom surface of the measurement container, and Since the circle of the agglutination image was small, it was difficult to discriminate it from the negative case. And, because of the difficulty of the determination in the case of a sample having a low concentration of the test substance, it is not possible to measure the low-concentration test substance, and it is necessary to take a long time to sufficiently form an agglutination image and then make a determination. Therefore, the time required for the measurement was long, and the measurement time was usually one hour or more.

In the indirect agglutination measurement method, particles bound to an antibody (or antigen) aggregate or particles bound to an antibody (or antigen) via components in a sample other than the test substance. In some cases, an agglutination image was formed as if the test substance were present (positive), despite the absence of the test substance in the sample (negative), when bound to the inner wall surface of the measurement container (non-specific) Agglutination reaction). The possibility that a negative sample may be erroneously determined to be positive has been a serious problem in diagnosing a disease by a clinical test.

On the other hand, JP-A-64-69954 discloses that a sample sample is added to a measurement container in which an antibody or antigen corresponding to the antigen or antibody which is the test substance in the sample sample is fixed on the inner wall. Simultaneously or subsequently, the same antibody or antigen as that immobilized in the measurement container without washing the sample sample is added to the measurement container with the same insoluble carrier particles immobilized thereon, and the presence or absence of an agglutination reaction that develops Discloses an immunological measurement method for determining the presence or absence of an antigen or antibody as a test substance in a specimen sample. In this measurement method, when the state in the measurement container when the antigen (or antibody) as the test substance is not present (negative) in the sample sample is viewed from above, an image similar to the agglutination image shown in FIG. Is observed.
In addition, when an antigen (or antibody) as a test substance is present (positive) in the sample sample, when the state in the measurement container is viewed from above, an image similar to the aggregation image shown in FIG. 7B is observed. Is done.

The measuring method described in JP-A-64-69954 can measure a low concentration of a test substance, suppress a zone phenomenon, and obtain a clear aggregated image in a short time. However, since the presence or absence of the test substance in the sample is determined by the size of the circular image due to the particles on the bottom of the measurement container, if the sample has a very low concentration of the test substance, The problem that it is difficult to judge is inevitable.

Japanese Patent Application Laid-Open No. 2-124644 discloses a magnetic material in which a substance (antibody or antigen, etc.) that specifically binds to or competes with a test substance (test substance) is immobilized on magnetic particles. Mix the marker particles and the sample solution (sample),
The reaction solution is acted on by a magnet disposed outside a predetermined wall region of the measurement container, whereby the substance to be measured in the sample is measured based on the distribution state of the magnetic marker particles collected on the predetermined wall region. An immunoassay has been disclosed. In this measurement method, when the substance to be measured does not exist in the sample (negative), when the state in the measurement container is viewed from above, an image similar to the agglutination image shown in FIG. When the state inside the measurement container is viewed from above when the measurement substance is present (positive), an image similar to the aggregation image shown in FIG. 7B is observed.

According to the measuring method described in Japanese Patent Application Laid-Open No. Hei 2-124466, the sedimentation speed of the magnetic particles is increased by attracting the magnetic particles to the bottom surface of the measuring container by using a magnet.
The formation of the aggregation image on the bottom surface of the measurement container can be performed in a short time, and the time required for the determination can be reduced. However, since the presence or absence of the test substance in the sample is also determined based on the size of the circular image formed by the particles on the bottom of the measurement container, the case of a sample having a low test substance concentration is considered to be negative. There was no change in that it was difficult to determine.

[0013]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a method for measuring a test substance in a sample visually or using an instrument by utilizing a specific binding reaction such as an antigen-antibody reaction. Provides a highly sensitive measurement method that can determine the presence or absence of a test substance in a short time even if the substance is contained in a very small amount in the sample, and that is highly reliable in its determination It is to be.

[0014]

Means for Solving the Problems The present invention provides: 1) an operation of bringing a sample into contact with particles on which a specific binding substance to a test substance in a sample is fixed; 2) an operation of mixing said particles and a sample in a liquid mixture. An operation of binding the test substance to the specific binding substance fixed to the particles; 3) a specific binding substance to the test substance that is the same or different from the specific binding substance fixed to the particles; An operation of bringing the particles in the mixed solution into contact with the determination portion or a portion connected to the determination portion of the carrier having the determination portion fixed and coated with the specific binding substance; 4) determining the particles by the determination And 5) a method of measuring a test substance in a sample, including an operation of determining the presence or absence of a test substance from the distribution state of particles in the determination section. (First Measurement Method of the Present Invention) In the first measurement method of the present invention, a specific binding substance which is the same as or different from the specific binding substance immobilized on the particles is immobilized, and Particles in the mixed liquid to be brought into contact with this judgment part or a part connected to this judgment part of the carrier having the coated judgment part, particles settled or settled at the lower part of the mixture, or particle concentration in the mixture liquid are reduced. Preferably, it is a high part. In the first measurement method of the present invention, it is preferable that the determination unit is partially covered with the specific binding substance.

[0015] The present invention also provides: 1) a particle holding section for holding particles to which a specific binding substance to a test substance in a sample is fixed, and a specific binding substance to the test substance, which is fixed to the particles. In the particle holding portion of the carrier having a determination portion, the same or different specific binding substance as the fixed specific binding substance is fixed and coated with the specific binding substance,
An operation of bringing a sample into contact with the sample; 2) an operation of binding a test substance and a specific binding substance immobilized on the particles in a mixture of the particles and the sample in the particle holding unit; 4) an operation of moving the particles to the determination unit and bringing the particles into contact with the determination unit; 4) an operation of moving the particles along the determination unit; and 5) an analyte from the distribution state of the particles in the determination unit. This is a method for measuring a test substance in a sample, which includes an operation for determining the presence or absence of a sample. (Second measurement method of the present invention) In the second measurement method of the present invention, the particles in the mixed solution that was moved to the determination section and contacted settled or settled at the lower part of the mixed solution. It is preferable that the particle or the portion where the particle concentration in the mixed solution is high. Then, in the second measurement method of the present invention, it is preferable that the determination unit is partially covered with the specific binding substance.

Furthermore, the present invention provides: 1) a mixing section for bringing a sample into contact with particles to which a specific binding substance to a test substance in a sample is fixed, and a specific binding substance to the test substance, An operation of bringing the particles and the sample into contact with each other in the mixed portion of the carrier having a determination portion on which a specific binding substance the same as or different from the immobilized specific binding substance is immobilized and coated with the specific binding substance; A) an operation of binding a test substance and a specific binding substance immobilized on the particles in a mixture of the particles and the sample in the mixing section; 3) transferring the particles in the mixture to the determination section. 4) an operation of moving the particles along the determination unit; and 5) an operation of determining the presence or absence of a test substance from the distribution state of the particles in the determination unit. This is a method for measuring a test substance in a sample. (Third Measurement Method of the Present Invention) In the third measurement method of the present invention, the particles in the mixed solution that was moved to the determination section and contacted settled or settled at the lower part of the mixed solution. It is preferable that the particle or the portion where the particle concentration in the mixed solution is high. In the third measurement method of the present invention, it is preferable that the determination unit is partially covered with the specific binding substance. Further, in the third measuring method of the present invention, the mixing part of the carrier and the determination part are in the same part, and the operation of bringing the particles into contact with the sample may be performed in the part that is the mixing part and the determination part. It is suitable.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION "Measurement operation of the first measurement method of the present invention" The first measurement method of the present invention may be performed by the following operation.

Contacting Particles with Sample First, particles in which a specific binding substance to a test substance in a sample is immobilized are brought into contact with a sample suspected of containing the test substance. The contact between the particles and the sample may be performed by adding each of the particles and the sample to a container such as a test tube.

The optimum ratio of the amount of the sample to the amount of the particles at the time of contact is determined by the concentration of the particles in the dispersion, the concentration of the test substance in the sample, and the specificity of the test substance in the sample fixed to the particles. Binding substances (hereinafter simply referred to as specific binding substances)
Depends on the amount of the specific binding substance and the affinity between the specific binding substance and the test substance, and cannot be unconditionally determined. Therefore, it may be appropriately selected according to the conditions at that time.

The particles may be dispersed in an appropriate dispersion medium as described below and brought into contact with a sample.

Further, the sample may be diluted with an appropriate diluent as described below and brought into contact with the particles.

Next, the test substance is bound to the specific binding substance immobilized on the particles in the mixed solution. Next, in the mixed solution of the particles and the sample obtained by contacting the particles with the sample, The test substance is bound to the immobilized specific binding substance. This binding can be performed by bringing the sample into contact with the particles. By bringing the sample into contact with the particles in a container such as a test tube or the like, the test substance in the sample can be bound to the specific binding substance immobilized on the particles.

When the test substance is present in the sample, the test substance binds to the specific binding substance fixed on the particles.
Conversely, when the test substance is not present in the sample, the specific binding substance fixed to the particles does not bind to the test substance and thus remains unbound.

In the present invention, it is preferable that more test substances can be bound to the specific binding substance immobilized on the particles. That is, if the test substance in the sample is larger and can bind to the specific binding substance fixed to the particles, it can be determined that the test substance is present even if the test substance in the sample is very small. Because it can be.

In order to allow more test substances to bind to the specific binding substance immobilized on the particles, the following method may be used.

A) The contact time between the particles and the sample in the mixture of the particles and the sample is increased. Note that the contacting time may be any time as long as it is longer than a certain time at which the specific binding substance fixed to the particles and the test substance in the sample can be sufficiently bound. This period of time is determined by the concentration of the test substance in the sample, the concentration of the particles, the amount of the specific binding substance fixed to the particles, the amount of the sample to be mixed, the amount of the particles or the particle dispersion to be mixed, or the specific binding. Since it depends on the affinity of the substance and the test substance, etc., it cannot be said unconditionally.
It is particularly preferable that the time is 0 minutes or more.

B) Use a larger amount of sample to contact the particles. Since an image is not formed at the time of measurement unless a certain number of test substances in the sample to be brought into contact with the particles are present, the amount of the sample brought into contact with the particles also needs to be a certain amount or more. The predetermined amount may be the concentration of the test substance in the sample, the concentration of the particles, the amount of the specific binding substance immobilized on the particles, the amount of the sample to be mixed, the amount of the particles or the particle dispersion to be mixed, or Since it differs depending on the affinity between the target binding substance and the test substance, it cannot be said unconditionally.

C) The temperature at which the particles are brought into contact with the sample is increased. The temperature at which the sample is brought into contact with the particles may be room temperature (1 to 30 ° C.) or a temperature equal to or higher than room temperature such as 37 ° C. However, if the temperature is too high, for example, at 70 ° C. or higher, the test substance or the specific binding substance is denatured or decomposed, which is not preferable.

D) The mixture is sufficiently stirred. It is preferable to sufficiently stir the mixture so that the sample and the particles in the mixture are well mixed. This stirring may be performed with a stirrer, a mixer, or the like. Further, it is preferable to use a container having a shape with good mixing and stirring efficiency.

Then, the particles are brought into contact with the determination part of the carrier or the like. Thereafter, a specific binding substance to the test substance, which is the same as or different from the specific binding substance fixed to the particles, is immobilized. The particles in the mixed solution are brought into contact with the determination portion of the carrier having the determination portion coated with the target binding substance or a portion connected to the determination portion.

[0031] The above-mentioned judging part is the above-mentioned carrier,
The site (surface) on which the specific binding substance is fixed and coated.

Further, the portion connected to the determination section is
In the carrier, the particles in the contacted liquid mixture are naturally moved by the magnet or by tilting the carrier, and the like, are moved to the determination unit and can be brought into contact with the determination unit. .

For example, a part on the same plane as the determination unit,
Alternatively, a portion connected to the determination portion by a groove or the like can be given.

The amount of the particles to be brought into contact with the judgment part of the carrier or the part connected to the judgment part may be appropriately selected,
It is preferable that the amount does not overflow from the carrier.

The contact of the particles in the mixed solution with the determination portion of the carrier or the portion connected to the determination portion is performed by adding a part or the entirety of the mixture directly to the determination portion of the carrier or the portion connected to the determination portion. Alternatively, particles settled or settled at the lower part of the mixed solution, or a portion having a high particle concentration in the mixed solution may be added.

The sedimentation of the particles at the bottom of the mixture
It may be carried out by leaving the mixture to settle naturally by gravity, or by centrifuging it with a centrifuge to settle. In addition,
In the case where the particles are magnetic particles described later, a magnet may be arranged in the lower part of the mixed solution, and a magnetic force may be applied to magnetically settle the magnetic particles.

Further, particles settled or settled at the lower part of the mixed solution, or a portion where the particle concentration in the mixed solution is high,
To be added to the determination part of the carrier or a part connected to this determination part, the particles or particles in the container after the supernatant is transferred out of the container by tilting the container containing the mixed solution and performing decantation or the like. It may be carried out by adding a liquid containing the particles, or by adding particles absorbed by a pipette or the like or a portion having a high particle concentration. And, when the particles are magnetic particles, after the magnets are arranged on the wall surface of the container to collect the magnetic particles on the wall surface, the container is moved out of the container by tilting the container and performing decantation, etc. Particles or a liquid containing particles may be added.

The particles settled or settled at the lower part of the above-mentioned liquid mixture, or the portion where the particle concentration in the liquid mixture is high, is mixed with a small amount of liquid and then dispersed in a high concentration liquid. Alternatively, the carrier may be brought into contact with a determination part of the carrier or a part connected to the determination part.

When a sample is blood (a whole blood sample), when a test substance in the blood sample is measured, the distribution of particles in the determination section is usually interrupted by red blood cells contained in the sample. The state cannot be confirmed, and it is difficult to determine whether it is positive or negative. However, when measuring a test substance in a blood sample in the first measurement method of the present invention,
The particles settled or settled at the lower part of the mixed solution, or a portion having a high particle concentration in the mixed solution, are added to the determination portion of the carrier or a portion connected to the determination portion and brought into contact with the carrier to suspend the mixture. The turbid red blood cells can be removed and the particles can be brought into contact with the determination section, and the distribution state of the particles can be confirmed without interference of the red blood cells in the determination section, and positive / negative determination can be accurately performed.

Moving the particles along the determination section As described above, after bringing the particles in the mixed solution into contact with the determination section of the carrier or a portion connected to the determination section, the particles are moved along the determination section. The movement of the particles along the determination unit is movement on the surface of the carrier.

When the particles are brought into contact with a portion of the carrier which is connected to the determination portion, the particles are moved to the determination portion naturally by the action of a magnet or by tilting the carrier, and then the determination is made. Move the particles along the part.

The movement of the particles along the determination section can be performed by operating a magnet, or by tilting the carrier.

A) Movement by Acting on a Magnet When particles are moved by acting on a magnet, the following magnetic particles are used. Then, the magnet is operated so that the magnetic particles move along the determination portion which is the surface of the carrier coated with the specific binding substance.

As long as the determination of positive or negative can be made based on the distribution state of the magnetic particles on the surface of the carrier, the magnet is actuated before or during the contact of the magnetic particles with the determination portion of the carrier. May be.

When the entire surface of the carrier is covered with the specific binding substance, that is, when the entire surface of the carrier is the determination portion, the magnetic particles can be moved along the surface. The magnet may be arranged anywhere, for example, when the surface is a plane, the magnet may be arranged on the same plane.

When the surface of the carrier is partially covered with the specific binding substance, that is, when the determination part is a part of the surface of the carrier, the magnetic particles may be moved in a desired direction. The magnet may be placed wherever possible, for example, in the direction in which it is to be moved or in the vicinity thereof.

More specifically, for example, when a carrier whose one half of the surface is coated with a specific binding substance is used, a portion of the surface which is not coated with the specific binding substance is coated (a determination section). ) (In the direction of the arrow in FIG. 1), the magnets are preferably arranged so that the magnetic particles move.

When a carrier having a specific binding substance coated in a strip shape is used, a portion of the surface of the carrier which is not coated with the specific binding substance passes through the strip-shaped coated portion (determination portion) to form a specific binding substance. It is preferable to dispose the magnet so that the magnetic particles move in the direction of the portion not covered by the target binding material (the direction of the arrow in FIGS. 2 to 6 and 8).

Any magnet may be used as long as it generates a magnetic field to magnetize the magnetic particles, and a permanent magnet, an electromagnet, or the like may be used.

The magnetic flux density depends on the interaction between the magnetic particles to be used and the surface of the carrier, but is usually 5 to 100 Gauss (G).

In the region where the specific binding substance is not fixed on the surface of the carrier, the movement of the magnetic particles is promptly moved in the direction of the magnet depending on the interaction between the particles and the surface of the carrier and the strength of the magnetic field. I do.

On the other hand, in the region of the surface of the carrier where the specific binding substance is immobilized (determination section), the specific binding substance immobilized on the magnetic particles is bound to the case where the test substance is bound. When there is no magnetic particle, a large difference occurs in the moving speed of the magnetic particles.

When a test substance is present in a sample, the test substance is bound to the specific binding substance immobilized on the particles. When a magnet acts on the magnetic particles to which the test substance is bound, The magnetic particles are attracted to the magnet and move in the direction of the magnet along the surface of the carrier. In the process, when a specific binding substance fixed on the surface (determination section) is encountered, the magnetic particles are coated. The movement is stopped by binding to the specific binding substance immobilized on the surface (determination section) via the test substance, or the movement is significantly slowed down. Therefore, when the test substance is present in the sample, the magnetic particles are collected on the portion of the surface of the carrier that is coated with the specific binding substance (determination section).

When the test substance does not exist in the sample, the magnetic particles do not bind to the test substance, so that the magnetic particles do not show an affinity with the specific binding substance fixed on the surface (determination section).
It moves in the direction of the magnet as quickly as the region where the specific binding substance is not fixed. Therefore, when the test substance does not exist in the sample, the magnetic particles gather at a position close to the magnet, that is, at the end of the surface of the carrier.

When a container is used as the carrier, the magnetic particles mixed with the sample settle below the concave portion of the container due to the specific gravity.

At this time, the sedimentation of the magnetic particles may be promoted by applying a magnet from the top of the container to the bottom.

When a magnet is applied to the magnetic particles so as to move along the surface (determination portion) covered with the specific binding substance of the carrier, the magnetic particles to which the test substance is not bound and the surface (determination portion) ) Is weak, and its moving speed depends almost on the strength of the magnetic field. Therefore, when a high moving speed is required, that is, when it is desired to obtain a measurement result in a short time, a magnet that generates a strong magnetic field may be used. The measurement may be performed by adjusting the strength of the magnetic field using an electromagnet.

B) Movement by tilting the carrier When the particles are moved by tilting the carrier, the carrier is tilted so that the particles move along the determination portion which is the surface of the carrier coated with the specific binding substance.

The angle at which the carrier is inclined may be any angle at which the particles move along the surface (judgment) covered by the specific binding substance of the carrier by gravity, and an angle of 90 ° or less is appropriately set. An angle between 25 ° and 90 ° is preferred, and an angle between 45 ° and 65 ° is particularly preferred.

The carrier may be tilted before or while the particles are in contact with the determination portion of the carrier, as long as the determination of positive or negative can be made based on the distribution state of the particles on the surface of the carrier. .

When the entire surface of the carrier is covered with the specific binding substance, that is, when the entire surface of the carrier is the determination part, the direction of tilting the carrier may be any direction.

When the surface of the carrier is partially covered with the specific binding substance, that is, when the judgment part is a part of the surface of the carrier, the particles are covered with the specific binding substance of the carrier. The particles may be moved by gravity by inclining the carrier in such a direction as to move on the part (determination unit) where it is located. For example, the carrier may be arranged with the direction to be moved downward.

More specifically, for example, when a carrier whose one half of the surface is coated with a specific binding substance is used, a portion of the surface that is not coated with the specific binding substance is covered by a portion (determination unit). ) (In the direction of the arrow in FIG. 1), the carrier may be inclined so as to move the particles (in this case, the carrier may be inclined in the direction of the arrow in FIG. 1).

When a carrier whose surface is coated in a band with a specific binding substance is used, a portion of the surface not covered with the specific binding substance passes through the band-coated portion (judgment portion) and the specific surface. The carrier may be tilted so that the particles move in the direction of the part not covered by the target binding substance (the direction of the arrow in FIGS. 2 to 6 and 8) (in this case, FIGS.
6 and 8).

In the area of the surface where the specific binding substance is not fixed, the movement of the particles depends on the interaction between the particles and the surface and the angle at which the carrier is tilted, and the particles move quickly in the downward direction of the tilt.

On the other hand, in the area of the surface where the specific binding substance is fixed (determination section), the case where the test substance is bound to the specific binding substance fixed to the particles and the case where the test substance is not bound are as follows. Then, a large difference occurs in the moving speed of the particles.

When the test substance is present in the sample, the test substance is bound to the specific binding substance immobilized on the particles,
When the carrier is tilted so that the particles move along the surface (judgment section) covered with the specific binding substance of the carrier, the particles bound to the test substance are tilted downward along the surface of the carrier due to gravity. The particles move, and in the process, if they encounter a specific binding substance immobilized on the determination unit, the particles bind to the carrier via the test substance and stop moving, or the movement becomes extremely slow. Therefore, when the test substance is present in the sample, the particles gather at the determination part of the carrier.

When the test substance is not present in the sample, the particles do not bind to the test substance, so that the particles do not show an affinity with the specific binding substance immobilized on the judgment part, and the specific binding substance is immobilized. It moves quickly downward in the same inclination as in the area where the movement is not performed. Thus, if no analyte is present in the sample, the particles will collect at the lower end of the carrier.

When the particles are moved along the determination part of the carrier by tilting the carrier, the interaction between the particles to which the test substance is not bound and the surface is weak, and the moving speed is almost dependent on the angle of tilting the carrier. . Therefore, when a large moving speed is required, that is, when it is desired to obtain a measurement result in a short time, the angle of tilting the carrier may be increased. Further, the angle at which the carrier is inclined may be changed over time.

Further, when a plate-shaped carrier is used, by bending the plate-shaped body and changing the angle of the plate-shaped body,
The speed at which the particles move may be changed.

Determining the Presence or Absence of the Test Substance As described above, after the particles are moved along the determination part of the carrier, the presence or absence of the test substance is determined from the distribution state of the particles in this determination part.

The state of distribution of particles in the determination unit, that is, the image, can be easily confirmed by the naked eye or by an optical reading device such as a microplate reader based on absorbance measurement or pattern recognition.

FIG. 1C is a diagram showing a well viewed from above when a sample containing a test substance (positive sample) is measured using a flat bottom microplate.
D is a diagram of a well viewed from above when a sample containing no test substance (negative sample) was measured using a flat-bottom microplate. If positive, particles that have migrated from the part of the flat bottom of the well that is not coated with the specific binding substance are coated with the specific binding substance (determination part)
Trapped in

FIG. 2C shows a sample in which a test substance is present (positive sample) using a rectangular parallelepiped transparent container having a rectangular bottom surface.
FIG. 2D shows a sample in which no test substance is present (negative sample) using a rectangular parallelepiped transparent container having a rectangular bottom surface.
FIG. 5 is a view of the container as viewed from above when the measurement is performed for. In the case of positive, particles that have migrated from the portion of the bottom surface that is not coated with the specific binding substance are trapped in the band-shaped coated portion (determination portion).

FIG. 3C shows a plate obtained by measuring a sample containing two kinds of test substances (a sample in which each test substance is positive) using a rectangular plate. FIG. 3D shows the plate-like body when the measurement was performed on a sample (a negative sample) in which no test substance was present using a rectangular plate-like body, as viewed from above. FIG. In the case of a positive result, the particles that have migrated from the portion of the surface that is not coated with the specific binding substance are trapped in the strip-shaped coating portions (determination sections) corresponding to the respective test substances.

FIG. 4C shows a measurement in which a sample containing two types of test substances (a sample positive for each test substance) was measured using a rectangular plate-shaped body having grooves on the surface. FIG. 4D is a diagram of the plate-like body viewed from above, and FIG. 4D shows the plate-like body when a sample containing no test substance (negative sample) is measured using the plate-like body. It is the figure which looked at the body from the upper part. In the case of positive, particles that have migrated from the portion of the bottom of the groove that is not coated with the specific binding substance are trapped in the strip-shaped coating portions (determination sections) corresponding to the respective test substances.

FIG. 8C shows the measurement of a sample containing two types of test substances (a sample positive for each test substance) using a rectangular plate having a groove on the surface. FIG. 8D is a view of the plate-like body as viewed from above. FIG. 8D shows the plate-like body when a sample containing no test substance (negative sample) is measured using the plate-like body. It is the figure which looked at the body from the upper part. In the case of positive, particles that have migrated from the portion of the bottom of the groove that is not coated with the specific binding substance are trapped in the strip-shaped coating portions (determination sections) corresponding to the respective test substances.

When particles to which a test substance or an analog thereof is immobilized are used in place of the specific binding substance, a result opposite to the distribution of the positive or negative particles obtained by the above-described measurement method is obtained. .

2. "Measurement operation of the second measurement method of the present invention" The second measurement method of the present invention may be performed by the following operation.

First, the sample is brought into contact with the particle holding portion of the carrier. First, the particle holding portion holding the particles on which the specific binding substance to the test substance in the sample is fixed, and the specific binding substance to the test substance, The presence of the test substance is present in the particle holding portion of the carrier having the determination portion in which the same or different specific binding material as the specific binding material fixed to the particles is fixed and coated with the specific binding material. Contact the suspected sample. This contact may be performed by adding the sample to the particle holding portion of the carrier.

When the sample is brought into contact with the particle holding portion, the sample may be brought into direct contact with the particle holding portion, or the sample may be brought into contact with a portion of the carrier which is connected to the particle holding portion. May be moved by tilting the carrier or the like to bring the carrier into contact with the particle holding unit.

The amount of the sample to be brought into contact with the particle holding unit depends on the concentration of the test substance in the sample, the amount of the particles in the particle holding unit, the amount of the specific binding substance immobilized on the particles, or the specific binding. The optimum amount differs depending on the affinity between the substance and the test substance, and cannot be unambiguously determined, so that it may be appropriately selected according to the conditions at that time.

The sample may be diluted with an appropriate diluent as described below and brought into contact with the particle holding unit.

As described above, when the sample is brought into contact with the particle holding portion of the carrier, the particles held in the particle holding portion are separated from the particle holding portion by the liquid sample, and the mixed liquid of the particles and the sample is removed. To form

Next, the test substance is bound to the specific binding substance immobilized on the particles in the mixed solution. Next, in the mixed solution of the particles and the sample obtained by bringing the particles into contact with the sample, The test substance is bound to the immobilized specific binding substance. This binding can be performed by bringing the particles into contact with the sample at the particle holding portion of the carrier.

When the test substance is present in the sample, the test substance binds to the specific binding substance fixed on the particles.
Conversely, when the test substance is not present in the sample, the specific binding substance fixed to the particles does not bind to the test substance and thus remains unbound.

In the present invention, it is preferred that more test substances can be bound to the specific binding substance immobilized on the particles. That is, if the test substance in the sample is larger and can bind to the specific binding substance fixed to the particles, it can be determined that the test substance is present even if the test substance in the sample is very small. Because it can be.

To enable more test substances to bind to the specific binding substance immobilized on the particles, the following method may be used.

A) The contact time between the particles and the sample is longer in the mixed solution of the particles and the sample. Note that the contacting time may be any time as long as it is longer than a certain time at which the specific binding substance fixed to the particles and the test substance in the sample can be sufficiently bound. This period of time includes the concentration of the test substance in the sample, the concentration of the particles, the amount of the specific binding substance immobilized on the particles, the amount of the sample to be mixed, the amount of the particles to be mixed, or the specific binding substance and the test substance. Since it differs depending on the affinity of the substance and the like, it cannot be said unconditionally. In general, it should be several seconds or more, and is preferably one minute or more, and particularly preferably 10 minutes or more.

B) Use a larger amount of sample to contact the particles. The amount of the sample to be brought into contact may be a certain amount or more for the reason described above. In addition, this fixed amount is the concentration of the test substance in the sample, the concentration of the particles, the amount of the specific binding substance fixed to the particles, the amount of the sample to be mixed, the amount of the particles to be mixed, or the amount of the specific binding substance. Since it differs depending on the affinity of the test substance and the like, it cannot be said unconditionally.

C) The temperature at which the particles are brought into contact with the sample is increased. The temperature at which the sample is brought into contact with the particles may be room temperature (1 to 30 ° C.) or a temperature equal to or higher than room temperature such as 37 ° C.

However, if the temperature is too high, for example, if the temperature is 70 ° C. or more, the test substance or the specific binding substance is undesirably denatured or decomposed.

D) The mixture is sufficiently stirred. It is preferable to sufficiently stir the mixture so that the sample and the particles in the mixture are well mixed. This stirring may be performed with a stirrer, a mixer, or the like. Further, it is preferable to use a container having a shape with good mixing and stirring efficiency.

The particles are moved to the determination part of the carrier and brought into contact therewith. Thereafter, the particles in the mixed solution are moved to the determination part and then brought into contact with the determination part. In addition, the said determination part is a site | part (surface) in which the said specific binding substance was fixed and covered in the said carrier.
The movement of the particles along the determination unit is movement on the surface of the carrier.

Further, a part or the whole of the mixed solution subjected to the operation of binding the test substance to the specific binding substance fixed to the particles in the above-mentioned particle holding part is moved to the determination part as it is and brought into contact therewith. Although good, particles settled or settled at the lower part of the particle holding part, or a part of the particle holding part where the particle concentration in the mixed solution is high may be moved and contacted.

The movement of the particles or the mixed liquid containing the particles in the mixed liquid in the particle holding section to the determination section can be performed by tilting the carrier, applying magnetism with a magnet, sucking and adding with a pipette or the like, or What is necessary is just to perform by using a capillary phenomenon.

In particular, moving the settled or settled particles or the portion having a high particle concentration in the mixed solution to the determination section is performed by transferring the solvent (supernatant) portion of the mixed solution in the particle holding section to a pipette or the like. Alternatively, it may be carried out by absorbing with a water-absorbing substance, or by tilting the carrier and removing the carrier by flowing out of the carrier, and then tilting the carrier to move the particles to the determination unit.

Alternatively, particles in the mixed solution or a portion having a high particle concentration may be added to a suction determination unit using a pipette or the like.

Further, when the particles are magnetic particles, magnetism may be applied to the particles by a magnet, and the particles may be moved from the particle holding unit to the determination unit by a magnetic force.

The particles settled or settled at the lower part of the above-mentioned liquid mixture, or the portion where the particle concentration in the liquid mixture is high, are mixed with a small amount of liquid and then dispersed in a high concentration liquid. Alternatively, the carrier may be moved and brought into contact with the determination section of the carrier.

When a sample is blood (a whole blood sample), when a test substance in the blood sample is measured, the distribution of particles in the determination section is usually interrupted by red blood cells contained in the sample. The state cannot be confirmed, and it is difficult to determine whether it is positive or negative. However, when measuring a test substance in a blood sample in the second measurement method of the present invention,
The particles settled or settled at the lower part of the mixed solution, or a portion having a high particle concentration in the mixed solution, are added to the determination portion of the carrier or a portion connected to the determination portion and brought into contact with the carrier to suspend the mixture. The turbid erythrocytes can be eliminated and the particles can be brought into contact with the determination section, and the distribution state of the particles can be confirmed in the determination section without being affected by the erythrocytes, and positive / negative determination can be accurately performed.

Moving the particles along the determination section As described above, the particles in the liquid mixture are moved to and brought into contact with the determination section of the carrier, and then the particles are moved along the determination section.

The operation of moving the particles along the judging section may be performed in the same manner as in the above-described first measuring method of the present invention.

Determining the Presence or Absence of the Test Substance After the particles are moved along the determination section of the carrier as described above, the presence or absence of the test substance is determined from the distribution state of the particles in this determination section.

The operation of judging the presence or absence of a test substance from the distribution state of particles in this judgment unit may be performed in the same manner as in the case of the first measurement method of the present invention.

In FIG. 5C, a sample (positive sample) in which a test substance is present was measured using a rectangular plate having a groove on the surface and having a particle holding portion (a). FIG. 6 is a view of the plate-like body as viewed from above, and FIG.
FIG. 3 is a diagram of the plate-like body viewed from above when a sample containing no test substance (negative sample) is measured using the plate-like body. In the case of positive, particles that have migrated from the portion of the bottom of the groove that is not covered with the specific binding substance are trapped in the band-shaped covering portion (determination section) corresponding to the test substance.

3. "Measurement operation of the third measurement method of the present invention" The third measurement method of the present invention may be performed by the following operation.

Contacting Particles and Sample in Mixing Section of Carrier First, a mixing section for bringing a sample into contact with particles in which a specific binding substance to a test substance in a sample is immobilized, and a specific binding substance to a test substance. And a specific binding substance that is the same as or different from the specific binding substance fixed to the particles, and a determination unit coated with the specific binding substance. Contact a sample suspected of having an analyte. This contact may be performed by adding the particles and the sample to the mixing portion of the carrier.

The amount of the sample brought into contact with the particles in the mixing section depends on the concentration of the test substance in the sample, the amount of the particles, the amount of the specific binding substance immobilized on the particles, or the amount of the specific binding substance and the test substance. Since the optimum amount varies depending on the affinity and the like and cannot be determined unconditionally, it may be appropriately selected according to the conditions at that time.

The amount of the particles brought into contact with the sample in the mixing section also depends on the concentration of the test substance in the sample, the amount of the sample, the amount of the specific binding substance immobilized on the particles, or the specific binding substance and the test substance. The optimum amount varies depending on the affinity of the substance and the like, and cannot be determined unconditionally. Therefore, it may be appropriately selected according to the conditions at that time.

The sample may be diluted with an appropriate diluent as described below and brought into contact with the particles in the mixing section.

In the carrier in the third measuring method of the present invention, the mixing section and the judging section may be in the same portion of the carrier. In this case, an operation of bringing the particles into contact with the sample is performed in a portion (determination portion) of the carrier that is covered with the specific binding substance. That is, this part of the carrier is the mixing part and the determination part.

The test substance is bound to the specific binding substance immobilized on the particles in the liquid mixture in the mixing section. Next, the liquid mixture of the particles and the sample obtained by bringing the particles into contact with the sample in the mixing section Inside, the test substance is bound to the specific binding substance immobilized on the particles. This binding can be performed by bringing the particles into contact with the sample in the mixing section of the carrier.

When the test substance is present in the sample, the test substance binds to the specific binding substance fixed on the particles.
Conversely, when the test substance is not present in the sample, the specific binding substance fixed to the particles does not bind to the test substance and thus remains unbound.

In the present invention, it is preferable that more test substances can be bound to the specific binding substance immobilized on the particles. That is, if the test substance in the sample is larger and can bind to the specific binding substance fixed to the particles, it can be determined that the test substance is present even if the test substance in the sample is very small. Because it can be.

In order to allow more test substances to bind to the specific binding substance immobilized on the particles, the following method may be used.

A) The particles and the sample are brought into contact in the mixture of the particles and the sample for a longer time. Note that the contacting time may be any time as long as it is longer than a certain time at which the specific binding substance fixed to the particles and the test substance in the sample can be sufficiently bound. This period of time is determined by the concentration of the test substance in the sample, the concentration of the particles, the amount of the specific binding substance fixed to the particles, the amount of the sample to be mixed, the amount of the particles or the particle dispersion to be mixed, or the specific binding. Since it depends on the affinity of the substance and the test substance, etc., it cannot be said unconditionally.
It is particularly preferable that the time is 0 minutes or more.

B) Use a larger amount of sample to contact the particles. The amount of the sample to be brought into contact may be a certain amount or more for the reason described above. The predetermined amount may be the concentration of the test substance in the sample, the concentration of the particles, the amount of the specific binding substance immobilized on the particles, the amount of the sample to be mixed, the amount of the particles or the particle dispersion to be mixed, or It cannot be said unconditionally because it differs depending on the affinity of the target binding substance and the test substance.

C) The temperature at which the particles are brought into contact with the sample is increased. The temperature at which the sample is brought into contact with the particles may be room temperature (1 to 30 ° C.) or a temperature equal to or higher than room temperature such as 37 ° C. However, if the temperature is too high, for example, at 70 ° C. or higher, the test substance or the specific binding substance is denatured or decomposed, which is not preferable.

D) The mixture is sufficiently stirred. It is preferable to sufficiently stir the mixture so that the sample and the particles in the mixture are well mixed. This stirring may be performed with a stirrer, a mixer, or the like. Further, it is preferable to use a container having a shape with good mixing and stirring efficiency.

The particles are moved to the determination part of the carrier and brought into contact therewith. Thereafter, the particles in the mixed liquid in the mixing part are moved to the determination part and then brought into contact with the determination part. In addition, the said determination part is a site | part (surface) in which the said specific binding substance was fixed and covered in the said carrier.

Further, a part or all of the mixed solution subjected to the operation of binding the test substance to the specific binding substance fixed to the particles in the mixing section is moved to the determination section as it is,
The particles may be brought into contact with each other, or particles that have settled or settled at the lower part of the mixing part, or a part of the mixing part where the particle concentration in the mixed solution is high may be moved and brought into contact.

The movement of the particles or the mixed liquid containing the particles in the mixed liquid in the mixing section to the determination section can be performed by tilting the carrier, applying magnetism with a magnet, sucking and adding with a pipette, or adding a capillary. This may be performed by utilizing the phenomenon.

In particular, moving the sedimented or sedimented particles or the portion having a high particle concentration in the mixed solution to the judging section is performed by transferring the solvent (supernatant) portion of the mixed solution in the mixing section to a pipette or the like. Alternatively, the removal may be performed by absorbing with a water-absorbing substance, or by tilting the carrier and removing the carrier by flowing it out of the carrier, and then tilting the carrier to move the particles to the determination unit.

Alternatively, particles in the mixed solution or a portion having a high particle concentration may be added to a suction judging section using a pipette or the like.

When the particles are magnetic particles, magnetism may be applied to the particles by a magnet, and the particles may be moved from the mixing section to the determination section by a magnetic force.

The particles settled or settled in the lower part of the above-mentioned liquid mixture, or the portion where the particle concentration in the liquid mixture is high, is mixed with a small amount of liquid and then dispersed in a high concentration liquid. Alternatively, the carrier may be moved and brought into contact with the determination section of the carrier.

When a sample is blood (a whole blood sample), when a test substance in the blood sample is measured, the distribution of particles in the determination section is usually interrupted by red blood cells contained in the sample. The state cannot be confirmed, and it is difficult to determine whether it is positive or negative. However, when measuring a test substance in a blood sample in the third measurement method of the present invention,
The particles settled or settled at the lower part of the mixed solution, or a portion having a high particle concentration in the mixed solution, are added to the determination portion of the carrier or a portion connected to the determination portion and brought into contact with the carrier to suspend the mixture. The turbid red blood cells can be removed and the particles can be brought into contact with the determination section, and the distribution state of the particles can be confirmed without interference of the red blood cells in the determination section, and positive / negative determination can be accurately performed.

Moving the Particles Along the Judgment Section As described above, the particles in the mixed liquid are moved to and brought into contact with the judgment section of the carrier, and then the particles are moved along the judgment section.
The movement of the particles along the determination unit is movement on the surface of the carrier.

The operation of moving the particles along the determination section may be performed in the same manner as in the case of the first measuring method of the present invention.

Determination of the Presence or Absence of the Test Substance After the particles are moved along the determination section of the carrier as described above, the presence or absence of the test substance is determined from the distribution state of the particles in this determination section.

The operation of judging the presence or absence of a test substance from the distribution state of particles in this judging section may be performed in the same manner as in the case of the first measuring method of the present invention.

In FIG. 6C, a groove is provided on the surface,
When a sample (positive sample) containing a test substance is measured using a rectangular plate having a mixing portion (b) partitioned by a partition (c), the plate is removed from above. FIG. 6D is a view of the plate-like body viewed from above when a measurement is performed on a sample (a negative sample) in which no test substance is present using such a plate-like body. . In the case of positive, particles that have migrated from the portion of the bottom of the groove that is not covered with the specific binding substance are trapped in the band-shaped covering portion (determination section) corresponding to the test substance.

3. Sample In the present invention, a sample refers to a liquid that has a possibility that a test substance to be measured is present and that is to be used for confirming the presence or absence or in some cases quantification of the test substance.

For example, human or animal blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, etc .; human or animal brain and other organs, hair, skin, nails, etc.
An extract of muscle or nerve tissue; an extract or suspension of human or animal feces; an extract of cells or cells; an extract of a plant;

The sample may be diluted with a diluent before use. As this diluent, various buffers such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer and phosphate buffered saline, or physiological saline can be used. The pH of this buffer is pH
It is preferably in the range of 4 to 12.

In addition, various dilutions of this sample include various proteins such as bovine serum albumin, human serum albumin, casein, and salts thereof; various salts such as sodium chloride; various sugars; skim milk powder; Animal serum; various preservatives such as sodium azide; additives such as various surfactants such as nonionic surfactants, amphoteric surfactants and anionic surfactants can be appropriately added and used. .

The concentration at which these additives are added is not particularly limited, but is 0.001 to 10% (w / v).
Is preferable, and particularly preferably 0.01 to 5% (w / v).

Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, and polyoxyethylene alkyl ether. A nonionic surfactant such as oxyethylene phytosterol, polyoxyethylene phytostanol, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil, or polyoxyethylene lanolin; an amphoteric surfactant such as betaine acetate; Or an anionic surfactant such as polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether acetate. That.

4. Test Substance The test substance to be measured in the present invention may be any organic substance such as proteins, carbohydrates, lipids, and nucleic acids, or any biological substance such as an inorganic substance.

Specifically, HBs antigens, anti-HBs antibodies,
HBe antigen, anti-HBe antibody, anti-HBc antibody, HCV antigen, anti-HCV antibody, anti-HIV antibody, or anti-ATLV
Virus-related antigens or antibodies such as antibodies; Escherichia coli O157
Bacteria-related antigens or antibodies such as antigens, anti-Treponemal pallidum (TP) antibodies, anti-mycoplasma antibodies, or anti-streptolysin O antibodies (ASO); immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM) or immunoglobulin E (I
gE) and other immunoglobulins; C-reactive protein (CR
P), α1-acid glycoprotein, haptoglobin, complement C3
Markers such as α-fetoprotein, CEA or CA19-9; Hormones such as human placental chorionic gonadotropin (hCG); Allergens or allergen-specific IgE antibodies Allergen-related antigens or antibodies such as anti-thrombin III (ATIII); blood coagulation-related substances such as antithrombin III (ATIII); fibrinolytic substance (FDP) or fibrinolytic related substances such as D-dimer; ABO blood group antibodies or irregular Blood group-related antigens or antibodies such as antibodies; viral DNA or RNA; bacterial DNA or RNA; human or other animal or plant DNA or RNA; lipoprotein (a), substances related to other diseases such as ferritin ;
Alternatively, a drug or the like can be exemplified.

5. Specific binding substance In the present invention, a specific binding substance to a test substance refers to a substance having an affinity for the test substance, and nonspecifically binds to the test substance due to a specific interaction with the test substance. A substance that stably binds covalently.

For example, when the test substance is an antigen, the specific binding substance is an antibody thereof. When the test substance is an antibody, the specific binding substance is an antigen or an antibody against the antibody, and the test substance is a nucleotide chain. Is a complementary nucleotide chain. Here, an antibody refers to not only a complete antibody molecule but also fragments produced from the antibody, for example, Fab and F.
(Ab ') 2 and the like. When the test substance is a ligand, it is its receptor.

The specific binding substance immobilized on the particles and the specific binding substance immobilized on the carrier may be the same or different as long as the specific binding substance can be bound via the test substance.

6. Particles The particles used in the present invention may be particles generally used for an indirect aggregation reaction.

For example, latex particles, gelatin particles,
Organic polymer particles such as polyacrylamide particles, microcapsules, liposomes, and emulsions; inorganic polymer particles such as glass beads, silica beads, and bentonite; other artificial particles; and erythrocytes.

In addition, magnetic particles can be used as the particles. In this case, the magnetic particles may be particles that are magnetized at least while a magnet is being applied from the outside.

As the magnetic particles, particles obtained by molding a ferromagnetic material alone into particles; particles having a ferromagnetic material as a nucleus whose surface is coated with a polymer substance such as polystyrene, silica gel, gelatin, or polyacrylamide; Particles coated with ferromagnetic material using high molecular particles such as polystyrene, silica gel, gelatin, or acrylamide as cores, or particles enclosing ferromagnetic material in a closed bag-like material such as red blood cells, liposomes, or microcapsules Can be mentioned.

As the ferromagnetic material, for example,
A ferromagnetic metal such as iron, cobalt, or nickel; an alloy containing the ferromagnetic metal; a nonmagnetic substance containing the ferromagnetic metal or an alloy containing the ferromagnetic metal; An alloy containing a nonmagnetic substance in an alloy containing a ferromagnetic metal can be used.

It is particularly preferable that the magnetic particles have a property of being magnetized while a magnet is being applied from the outside, and being quickly demagnetized by shutting off the magnet from the outside. Examples of such magnetic particles include Dynabeads M-450 uncoated (trade name) (manufactured by Dinal Corporation), which is a magnetic particle in which iron oxide (III) [Fe2O3], which is a ferromagnetic substance, is dispersed. No.

Further, as the particles, those colored by coating with a dye or dispersing or enclosing the dye in the particles may be used.

The particle size of the particles is preferably 0.01 to 100 μm.
m, particularly preferably 0.5 to 10 μm.

The specific gravity of the particles may be any specific gravity that sediments in the dispersion medium. For example, particles having a specific gravity of 1 to 10 are preferable.

In the present invention, particles having the specific binding substance immobilized thereon are used. To fix the specific binding substance to the particles, the specific binding substance is attached to the surface of the particles by hydrophobic bonding, hydrophilic adsorption, or the like. Can be carried out by a physical adsorption method, a chemical bonding method such as a covalent bond, or a combination of these methods.

In the case where the specific binding substance is fixed to the particles by a physical adsorption method, the specific binding substance and the particles are mixed and contacted in a solution such as a buffer according to a known method. be able to.

For example, the specific binding substance and the particles are brought into contact with each other by mixing and stirring in a solution such as a buffer solution and the like.
After the adsorption reaction is performed at a temperature of about 40 ° C. for about 10 minutes to about 1 day, the obtained particles may be washed with a buffer or the like.

In the case where the specific binding substance is fixed to the particles by a chemical bonding method using a cross-linking reagent, it is recommended that the special edition of “Clinical Pathology Extra Edition, No. 53, Immunoassay for Clinical Examination” edited by the Japanese Society of Clinical Pathology be used. Application, "Clinical Pathology Publishing Association, 1983, The Society of Biochemistry, Japan
Protein IV '', according to a known method described in Tokyo Chemical Dojin, 1991, etc., mixing and contacting the specific binding substance and particles with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, The specific binding substance and the functional group such as amino group, carboxyl group, thiol group, aldehyde group, or hydroxyl group of the particle can be fixed by reacting with a crosslinking reagent.

For example, a divalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide is added to a buffer containing particles or the like, and the mixture is stirred and reacted. Next, a specific binding substance is added thereto, and the mixture is stirred and reacted. In some cases, the reaction is then stopped by removing the crosslinking reagent by a treatment such as dialysis or gel filtration, or by adding a reaction terminator for the crosslinking reaction. Then, the obtained particles may be washed with a buffer solution or the like.

Further, by changing the amount of the specific binding substance fixed to the particles, the sensitivity can be easily changed according to the level of the concentration of the test substance in the sample. For example, when a specific binding substance is immobilized on particles, the use of a high concentration of a specific binding substance increases the amount of immobilization, thereby increasing sensitivity.

In the case of measuring a plurality of types of test substances in a sample, use is made of particles in which a specific binding substance to a plurality of types of test substances is simultaneously immobilized on particles, or
What fixed the specific binding substance with respect to several types of test substances to separate particles respectively may be used.

If necessary, in order to suppress non-specific reactions, various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, or skim milk powder are contacted with particles having a specific binding substance immobilized thereon. The particles may be masked by a known method such as making the particles.

For example, in the masking, particles immobilized with a specific binding substance are added to a buffer containing various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, etc., and allowed to stand. Is coated with various proteins and the like.

In the measurement method of the present invention, particles to which a test substance or an analog thereof is immobilized may be used instead of particles to which a specific binding substance is immobilized.

Here, the analog of the test substance is a part of the test substance, a substance in which another substance is bonded to the test substance, or a substance in which a part of the structure of the test substance is substituted. Having a structure of a portion that binds to a specific binding substance of the test substance,
A substance that can bind to a specific binding substance.

For example, when the test substance is an antigen, a substance containing an antigenic determinant of the antigen can be mentioned as an analog of the test substance.

In the first measurement method and the third measurement method of the present invention, the particles may be dispersed in an appropriate dispersion medium and brought into contact with a sample.
Various buffers such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, or phosphate buffered saline, or physiological saline can be used. The pH of the buffer is preferably in the range of pH 4 to 12.

When the particles are dispersed in a dispersion medium, the concentration is as follows:
Although not particularly limited, 0.001% to 10% (w / v)
Is preferable, and 0.01% to 5% (w / v) is particularly preferable.

The additives described in the section of the diluent for the sample can be appropriately added to the dispersion medium of the particles.

The concentration when these additives are added is not particularly limited, but is 0.001 to 10% (w / v).
Is preferable, and particularly preferably 0.01 to 5% (w / v).

[0170] 7. Carrier Carrier In the first measurement method of the present invention, as the carrier, a determination unit in which a specific binding substance that is the same as or different from the specific binding substance immobilized on the particles is immobilized and coated with the specific binding substance, Use what you have.

In the second measuring method of the present invention,
As the carrier, a particle holding portion holding the particles to which the specific binding substance is fixed, and a specific binding substance which is the same as or different from the specific binding substance fixed to the particles, and is coated with the specific binding substance. That has a determination unit.

In the third measuring method of the present invention, as the carrier, a mixing section for bringing the sample in which the specific binding substance is immobilized into contact with the sample, and the same or different as the specific binding substance immobilized on the particle are used. One having a specific binding substance immobilized thereon and having a determination unit coated with the specific binding substance is used.

The material of the carrier is not particularly limited as long as the specific binding substance can be physically or chemically immobilized. Examples thereof include glass, polystyrene, polyvinyl chloride, and the like.
Non-water-absorbing materials such as polyacrylate, polypropylene, nylon, polyethylene, polycarbonate, and polymethacrylate can be used.

The carrier in the present invention may have any shape and structure as long as a solution such as a sample can be brought into contact, and examples thereof include a container and a plate.

Judgment Unit When a container is used as the carrier, the shape of the concave portion of the container may be any shape such as a hemispherical shape, a cylindrical shape, or a rectangular parallelepiped shape, and all or a part of the inner wall surface of the concave portion is made of a specific binding substance. What is necessary is just to cover and make it a determination part.

When the shape of the concave portion is a shape having a flat bottom surface such as a cylindrical shape or a rectangular parallelepiped shape, it is preferable to cover all or a part of the flat bottom surface with a specific binding substance.

The container may have a plurality of recesses.

The container is preferably a container provided with at least one concave portion having a flat bottom, and the whole or a part of the flat bottom is coated with a specific binding substance. Examples of the container provided with at least one concave portion having a flat bottom include a flat bottom microplate, a tray, and the like.

When a plate is used as a carrier, the surface of the plate may be any shape such as a circle, a rectangle or a square, and the whole or part of the surface is coated with a specific binding substance. Then, the determination unit may be used.

Further, by forming a groove on the surface of the plate-like body and introducing a solution such as a sample into the groove, the solution can be prevented from flowing down from the surface.

In the present invention, when a specific binding substance is immobilized on the surface of a carrier to be used as a determination portion, the determination portion is immobilized so that the specific binding substance is partially covered on the surface of the carrier. Is preferred.

This is because when a specific binding substance is present in the sample, that is, when the sample is positive, it is determined that the sample is positive by comparing the images of the part coated with the specific binding substance (determination part) and the part not coated. Is more clear, and it is easy to distinguish between positive and negative, that is, to determine the presence or absence of the test substance in the sample. Here, "partially" means that the specific binding substance is unevenly distributed and is not covered with the specific binding substance over the entire surface.

The shape and area of the portion (judgment portion) covered with the specific binding substance are not particularly limited as long as the presence or absence of the test substance in the sample can be determined.

Examples of the partial coating include a case where one half of the surface of the carrier is coated, and a case where the surface of the carrier is coated in a strip shape.

In the case where the surface of the carrier is partially covered with the specific binding substance, a plurality of covering portions (judgment portions) may exist. For example, in order to measure a plurality of types of test substances in a sample, portions coated with specific binding substances for each test substance (determination sections) can be provided on the surface of the carrier.

In the present invention, the movement of the particles on which the specific binding substance is fixed along the carrier of the carrier is movement on the surface of the carrier. Therefore, in the carrier determination section of the present invention, the specific binding substance may be fixed and coated on at least the surface of the carrier. That is, there is no need to fix and cover the inside of the carrier. Therefore, in the present invention, it is preferable that the specific binding substance fixed and coated on the determination unit is fixed and coated only on the surface of the carrier.

The immobilization of the specific binding substance on the surface of the carrier is as follows.
It can be performed by a physical adsorption method or a chemical bonding method using a crosslinking reagent.

In the case of the physical adsorption method, it can be carried out by bringing a specific binding substance dissolved in a buffer or the like into contact with the surface of a carrier according to a known method. For example, when the carrier is a container, a specific binding substance dissolved in a buffer solution or the like is brought into contact with the container by allowing it to stand in a concave portion of the container and allowed to stand at about 2 ° C. to about 40 ° C. for about 10 minutes to about 1 day. After the reaction, the liquid in the concave portion may be removed by suction and washed with a buffer solution or the like.

In the case of performing a chemical bonding method using a cross-linking reagent, see the Japanese Society of Clinical Pathology, Special Issue No. 53, Special Issue on Clinical Pathology Extraordinary Issue No. 53, “Immunoassay for Clinical Examinations-Techniques and Applications-”, Clinical Pathology Publishing Association, 1983. 19th edition, The Biochemical Society of Japan, “Chemical Experiment Lecture 1 Protein IV”, Tokyo Chemical Dojin, 19
According to a known method described in, for example, 1991, a specific binding substance for a test substance and a carrier are mixed and contacted with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and the specific binding substance is contacted. And the amino group, the carboxyl group, the thiol group, the aldehyde group, the hydroxyl group or the like of the carrier.

For example, when the carrier is a container, a divalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide is added to the concave portion of the container, and the mixture is left to react. Next, a specific binding substance is added thereto, and the mixture is left to stand for reaction. In some cases, the reaction is then stopped by adding a reaction terminator for the crosslinking reaction thereto. Then, washing is performed with a buffer solution or the like.

In the present invention, as a method of forming the judgment portion by partially covering the surface of the carrier with the specific binding substance, for example, the specific binding substance and / or Alternatively, a method in which a crosslinking reagent is dropped and immobilization is performed by the above-mentioned method, a specific binding substance and / or a crosslinking reagent is dropped in a portion surrounded by a plastic plate, etc. Or immobilizing the immobilization by absorbing a specific binding substance and / or a cross-linking reagent into an absorber such as a sponge, and contacting or applying to a portion to be determined as a determination portion of the surface. And the like.

By changing the amount of the specific binding substance fixed to the determination part of the carrier, the sensitivity can be easily changed according to the concentration of the test substance in the sample. For example, when a specific binding substance is immobilized on the surface of a carrier, the use of a high-concentration specific binding substance increases the amount of immobilization and increases the sensitivity.

Further, if necessary, in order to suppress non-specific reactions, various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, or skim milk powder or the like, or a non-fat dry milk or the like is immobilized on a determination unit on which a specific binding substance is immobilized. The determination unit may be masked by a known method such as contacting.

For example, when the carrier is a container, a buffer solution containing various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and the like are added to the concave portion of the container in which the judging portion is present, and allowed to stand. Then, after coating the surface of the determination part of the concave portion of the container with various proteins and the like,
It can be carried out by, for example, sucking and removing the liquid in the recess.

Particle Holder In the carrier in the second measurement method of the present invention, the particle holder holding the particles to which the specific binding substance is fixed is configured to hold the particles until the particles come into contact with the sample. It is.

This means that the particles can be retained, a liquid mixture with the sample brought into contact with the particles can be formed, and the particles in the liquid mixture can be moved to the judgment section. There are no particular restrictions on the shape, size, and the like.

The particle holding section can be produced, for example, by impregnating a particle dispersion in which the above-mentioned particles are dispersed in an appropriate dispersion medium into a porous water-absorbing material and then drying. Alternatively, the particles may be formed into tablets by a method such as freeze-drying without impregnating the porous dispersion with the particle dispersion. Alternatively, the above-mentioned particle dispersion may be directly applied to a carrier and placed thereon, and then dried by a method such as freeze-drying.

Examples of the porous water-absorbing material include papers such as filter paper, paper towel or tissue paper, glass fiber filters, chemical fibers such as rayon or polyester, cloths, nonwoven fabrics, and liquids such as cotton. Any material may be used as long as the material has the same. The shape and size of the porous water absorbent are not particularly limited.

When simultaneously measuring a plurality of types of test substances in a sample, particles in which specific binding substances for a plurality of types of test substances are simultaneously fixed in the particle holding unit or a plurality of types of test substances are simultaneously measured. What is necessary is just to hold | maintain the particle which fixed the specific binding substance with respect to each separately.

FIG. 2 shows a rectangular parallelepiped transparent container having a rectangular bottom surface, which has a bottom surface having a portion coated in a band shape with a specific binding substance (hereinafter, referred to as a band-shaped coating portion) as a judgment portion. The particle holding portion (a) holding the particles is arranged in a portion of the bottom surface which is not coated with the specific binding substance. FIG. 2A is a perspective view of the container (a shaded portion is a determination portion), and FIG. 2B is a plan view of the container (a hatched portion is a determination portion).

FIG. 5 shows a plate-like body having a rectangular surface shape, in which a groove having a rectangular cross-sectional shape is provided on the surface.
A band-shaped covering portion (determination portion) made of a specific binding substance is formed on the bottom surface of the groove, and a particle holding portion (a) is provided at one end of the surface having the determination portion. FIG. 5A is a perspective view of the plate-like body (the shaded portion is a determination portion covered with a specific binding substance), and FIG. 5B is a plan view of the plate-like body (the hatched portion). Is a judgment part coated with a specific binding substance).

When the plate is covered by placing a plate on the judgment portion side of the groove or the like (see FIG. 5A), the sample dropped into the groove on the uncovered side moves toward the judgment portion by capillary action. It is preferred.

Mixing Part The mixing part of the carrier in the third measurement method of the present invention is such that particles and a sample can be brought into contact on the carrier.

[0204] In this method, a particle and a sample are added, and the particles and the sample are brought into contact with each other on a carrier to form a mixture of the particles and the sample. There is no particular limitation on the shape, size, etc., as long as the particles can be held and the particles in the mixed liquid can be moved to the determination unit.

Examples of the shape of the mixing portion of the carrier include a shape having a concave portion such as a hemispherical shape, a cylindrical shape, or a rectangular parallelepiped shape, and a groove shape. In addition, a plane that can satisfy the above conditions may be a plane.

In order to move the particles in the liquid mixture in the mixing section to the determination section, the mixing section and the determination section are on the same plane or adjacent to each other so that the particles can move from the mixing section to the determination section. It is particularly preferable that they are connected by a groove or the like.

During the operation of binding the test substance and the specific binding substance immobilized on the particles in the mixing section, the mixed liquid is prevented from moving to the determination section, and the mixed liquid is not circulated until after the binding operation is completed. In order to move the particles to the determination unit, it is preferable that a partition is provided between the mixing unit and the determination unit so that the mixed liquid cannot move naturally.

As described above, in the carrier in the third measurement method of the present invention, the mixing section and the determination section may be in the same portion of the carrier. That is, in this case, the mixing unit of the carrier is an equal determination unit.

FIG. 6 shows a plate-like body having a rectangular surface shape, in which a groove having a rectangular cross-sectional shape is provided on the surface.
A band-shaped coating portion (determination portion) made of a specific binding substance is formed on the bottom surface of the groove, and a mixing portion (b) is provided at one end of the surface having the determination portion. A partition (c) whose height is lower than the height of the groove is provided at an end of the mixing unit on the determination unit side.

In this carrier, the particles in the mixture in the mixing section can be moved to the judging section by tilting the carrier so that the end on the mixing section side becomes higher and moving it by gravity.

FIG. 6A is a perspective view of this plate-like body (the shaded portion is a judgment portion covered with a specific binding substance), and FIG. 6B is a plan view of this plate-like body. (The shaded area is the determination area covered with the specific binding substance). Also,
It is preferable that the sample is covered by placing a plate on the determination portion side of the groove or the like (see FIG. 6A), since the sample dropped in the groove on the uncovered side moves toward the determination portion by capillary action.

Absorbing Part The carrier may be provided with an absorbing part that absorbs the liquid containing particles in the mixed solution that has been brought into contact with the carrier.

Examples of the absorbing portion include porous absorbents, for example, papers such as filter paper, paper towel, and tissue paper, glass fiber filters, polymer absorbents, chemical fibers such as rayon and polyester,
A material made of a material having a property of absorbing liquid such as cloth, nonwoven fabric, or cotton can be used. There is no particular limitation on the shape and size of the absorbing section.

[0214] The position at which the absorbing section is arranged on the carrier is determined by moving particles in the mixed solution that has been brought into contact with the judging section or a portion connected to the judging section, or particles in the mixed liquid that has been moved to the judging section along the judging section. As long as it does not hinder the movement, it may be placed anywhere.

For example, when a container having a flat bottom is used as a carrier, the absorbing portion is formed in a strip shape, and the absorption portion is placed on a portion of the flat bottom where the specific binding substance is not fixed along the side of the container. It may be arranged.

When a plate is used as the carrier,
For example, the absorbing section may be arranged at the end of the determination section of the carrier, at a position adjacent to the determination section, or the like.

For example, the liquid containing particles can be absorbed by the capillary phenomenon from the direction in which the particles move on the determination unit, that is, the horizontal direction of the determination unit, so that the particles can move smoothly along the determination unit. It is preferable to dispose the absorbing portion at the position.

The absorbing section may be arranged so that it can be removed after absorbing the liquid containing particles. Furthermore, by making the absorbing section detachable or in the form of a cartridge or the like,
The absorber may be replaced many times.

Carrier when the sample is blood As described above, when the sample is blood (a whole blood sample), the distribution state of particles cannot be confirmed due to being blocked by red blood cells contained in the sample, and a positive or negative Judgment becomes difficult.

On the other hand, when the height of the liquid containing particles on the determination portion, which is a portion coated with the specific binding substance on the surface of the carrier, is reduced, the number of red blood cells on the determination portion is reduced, so that the red blood cells are blocked. The distribution state of the particles can be confirmed without being checked.

When blood is used as the sample, the height of the liquid containing the particles on the determination part of the carrier is preferably 1 mm or less.

In order to reduce the height of the liquid containing particles on the determination part of the carrier, for example, when the container shown in FIGS. 1 and 2 is used as the carrier, the depth can be reduced. When a plate-like body having grooves on the surface as shown in FIGS. 4, 5 and 6 is used as a carrier, the depth of the grooves may be reduced.

8. Confirmation test The measurement method of the present invention can be used for a confirmation test. The confirmation test means that, when the test substance is determined to be present (positive) in the sample in the measurement of the test substance in the sample, whether the determination result is truly due to the presence of the test substance, Or, a test to confirm whether it is due to a non-specific reaction.

[0224] 9. Difference from immunochromatography method Note that Japanese Patent Publication No. Hei 7-18878 and Japanese Patent Publication No. Hei 9-506
No. 434, the so-called immunochromatography method uses a water-absorbing material for the carrier, and allows the antigen-antibody reaction to occur while the liquid sample flows through the fine pores in the carrier by capillary action. This is to determine the presence or absence of the test substance in the sample. On the other hand, in the present invention, a test substance in a sample is measured based on whether or not particles collect on a portion of the carrier coated with a specific binding substance. Therefore, a non-water-absorbing material is used for the carrier, and a magnet acts on the magnetic particles, or a gravity acts on the particles, so that the magnetic particles or the particles are moved along the determination portion on the surface of the carrier. Since the measurement is carried out by moving, the principle is essentially different from that of immunochromatography.

[0225]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1 (Measurement of Escherichia coli O157) (1) Preparation of Anti-Escherichia coli O157 Antibody Immobilized Plate Body Acrylic resin extruded plate (width 25 mm, depth 40 mm, thickness 1 mm) [manufactured by Acrysandy] Two acrylic extruded plates (width 10 mm, depth 40 mm, thickness 0.
5 mm) [manufactured by Acrysandy Co., Ltd.] with a solvent at a distance of 5 mm.
mm, a depth of 40 mm and a height of 0.5 mm).

An anti-Escherichia coli O157: H7 antibody solution [manufactured by Kirkegaard & Perry Laboratories, 5 μg / mL] was placed on the bottom of the groove of the plate-like body at a distance of 5 mm to 10 mm from the left end.
Dissolved in 10 mM phosphate buffer at pH 7]
L was put on and contacted with a pipette, and allowed to stand at 37 ° C. for 3 hours.

Next, the solution was removed by suction with a pipette, and then a Tris buffer solution containing 0.5% (W / V) casein [pH 7.5,
50 mM] (hereinafter referred to as diluent A)
In addition, the mixture was allowed to stand at 4 ° C. overnight, and this was removed by suction with a pipette.

Then, an acrylic resin extruded plate (width 25 mm, depth 25 mm, thickness 1 m) is placed on the left end of the plate-like body.
m) [Acry Sunday] was attached with a solvent to cover the plate as shown in FIG. In this way, a specific binding substance (anti-E. Coli O
157: H7 antibody) to prepare a plate-like body on which a band-shaped covering portion (determination portion) was formed.

(2) Preparation of anti-Escherichia coli O157 antibody-immobilized magnetic particles Magnetic particles (Dynabeads M-450 uncoated, manufactured by Dynal, particle size: 4.5 μm, CV of 5% or less, specific gravity of 1.5,
Concentration 3% (w / v)) 1 mL and anti-Escherichia coli O157: H7 antibody solution [Kirkegaard & Perry Laboratories, pH 7.0
Dissolved in 0 mM phosphate buffer at a concentration of 0.1 mg / mL] 1 mL
Were mixed and reacted at 37 ° C. for 30 minutes. Here, about 20 times the amount of the diluent A was added to perform masking.

Next, the obtained magnetic particles were washed with the diluent A, and the magnetic particles were redispersed in the diluent A so as to have a concentration of about 0.1% (w / v). Thus, the anti-E. Coli O
A 157 antibody-immobilized magnetic particle dispersion was prepared.

(3) Measurement of E. coli O157 E. coli O157: H7 [Kirkegaard & Perry Laborato
ries Co., Ltd.] was added to the diluent A to a concentration of 50 cells / mL, and 10 mL of the prepared sample was added to the test tube.

Next, 100 μL of the anti-Escherichia coli O157 antibody-immobilized magnetic particle dispersion prepared in the above (2) was added to the test tube, and the sample and the particles were contacted and mixed.
Stirred for minutes.

Thereafter, a magnet was brought into contact with the side of the stirred test tube to collect the anti-Escherichia coli O157 antibody-immobilized magnetic particles. After the test tube was tilted and the supernatant was discarded, 100 μL of diluent A was added thereto to redisperse the particles.

The dispersion of the above particles was used
μL was placed using a pipette near the boundary between the covered portion and the uncovered portion of the groove of the anti-Escherichia coli O157 antibody-fixed plate prepared in (1) above.

The dispersion liquid in which the particles were redispersed moved to the judgment part of the plate-like body and came into contact with the judgment part.

In order to move the particles along the judging portion, a magnet is provided in the side direction of the plate-like body where the judging portion is formed, and the anti-Escherichia coli O157 antibody on the bottom surface of the groove of the plate-like body is not fixed. A magnetic field having a magnetic flux density of 40 to 60 gauss was generated in the direction (the direction of the arrow in FIG. 5) from the portion to the portion (determination portion) where the anti-Escherichia coli O157 antibody was fixed.

Within three minutes after the generation of the magnetic field, an image of particles as shown in FIG. When the measurement was performed using the diluent A to which Escherichia coli O157: H7 was not added as a sample, no image due to particles was observed as shown in FIG. 5D.

[0239]

According to the measuring method of the present invention, when a test substance in a sample is measured visually or by using an instrument using a specific binding reaction such as an antigen-antibody reaction, the method is simple, rapid, and easy. ,
Since the measurement can be performed with a simple operation, even a person without a technique can obtain a correct measurement result without mistake.

Further, according to the measurement method of the present invention, when a test substance in a sample is measured visually or by using an instrument using a specific binding reaction, the test substance is contained in a very small amount in the sample. Even if it is included, it is possible to determine the presence or absence of the test substance in a short time, and it is possible to perform highly sensitive measurement with high reliability of the determination.

[Brief description of the drawings]

FIG. 1 is a diagram showing a flat-bottom microplate in which one half of the flat bottom of each well is coated with a specific binding substance.

FIG. 2 is a view showing a rectangular parallelepiped transparent container whose bottom surface is covered with a specific binding substance in a band shape.

FIG. 3 is a view showing a plate-like body whose surface is covered in a band shape with a specific binding substance.

FIG. 4 is a view showing a plate-like body having a groove provided on a surface, and a bottom surface of the groove covered with a plurality of specific binding substances in a band shape.

FIG. 5 is a view showing a plate-like body provided with a groove on a surface, a bottom surface of the groove is covered with a specific binding substance in a band shape, and having a particle holding portion.

FIG. 6 is a view showing a plate-like body having a groove on its surface, the bottom surface of which is covered in a band shape with a specific binding substance, and having a mixing portion.

FIG. 7 is a view showing an agglutination image when a test substance in a sample is measured by an indirect agglutination reaction measurement method.

FIG. 8 is a view showing a plate-like body provided with a groove on the surface, and the bottom surface of the groove is covered with a plurality of specific binding substances in a band shape.

[Explanation of symbols]

 B Particle holding unit B Mixing unit C Partition

Claims (10)

[Claims] 1. An operation of bringing a sample into contact with a particle on which a specific binding substance to a test substance in a sample is immobilized; 2) a specific binding immobilized on the particle in a mixed solution of the particle and the sample. An operation of binding a test substance to a substance; 3) a specific binding substance which is the same as or different from the specific binding substance immobilized on the particles, the specific binding substance being immobilized on the test substance; An operation of bringing the particles in the mixed solution into contact with the determination portion or a portion connected to the determination portion of the carrier having the determination portion coated with: 4) an operation of moving the particles along the determination portion; And 5) a method for measuring a test substance in a sample, the method including an operation of determining the presence or absence of the test substance from the particle distribution state in the determination unit. 2. A specific binding substance which is the same as or different from the specific binding substance immobilized on the particles and is connected to the determination section of the carrier having the determination section coated with the specific binding substance or to the determination section. The particles in the mixture according to claim 1, wherein the particles in the mixture to be brought into contact with the site are particles settled or settled at the lower part of the mixture, or a portion where the particle concentration in the mixture is high. Measuring method. 3. The method for measuring a test substance in a sample according to claim 1, wherein the determination section is partially coated with a specific binding substance. 4. A particle holding section holding particles to which a specific binding substance to a test substance in a sample is fixed, and a specific binding substance to the test substance and fixed to the particles. An operation of bringing a sample into contact with the particle holding portion of a carrier having a determination portion on which a specific binding material that is the same as or different from the binding material is immobilized and coated with the specific binding material; An operation of binding a test substance and a specific binding substance fixed to the particles in a mixture of the particles and the sample; 3) moving the particles in the mixture to the determination unit; 4) an operation of moving the particles along the determination unit; and 5) an operation of determining the presence or absence of an analyte from the distribution state of the particles in the determination unit. How to measure . 5. The method according to claim 4, wherein the particles in the mixed solution that are moved to and contacted with the determination unit are particles settled or settled at a lower portion of the mixed solution, or a portion where the particle concentration in the mixed solution is high. 3. The method for measuring a test substance in a sample according to 1. 6. The method for measuring a test substance in a sample according to claim 4, wherein the determination section is partially coated with a specific binding substance. 7. A mixing section for bringing a sample into contact with particles immobilized with a specific binding substance to a test substance in a sample, and a specific binding substance specific to a test substance and immobilized on the particles. An operation in which the sample is brought into contact with the particles in the mixing section of a carrier having a determination section in which a specific binding substance identical or different from the specific binding substance is immobilized and coated with the specific binding substance; 2) the mixing section An operation of binding a test substance and a specific binding substance fixed to the particles in a mixture of the particles and the sample; 3) moving the particles in the mixture to the determination unit; 4) an operation of moving the particles along the determination unit; and 5) an operation of determining the presence or absence of a test substance from the distribution state of the particles in the determination unit. Inspection Method of measuring quality. 8. The particles in the liquid mixture that is moved to and contacted with the determination unit are particles that have settled or settled at a lower portion of the liquid mixture, or a portion where the particle concentration in the liquid mixture is high. 3. The method for measuring a test substance in a sample according to 1. 9. The method for measuring a test substance in a sample according to claim 7, wherein the determination section is partially covered with a specific binding substance. 10. The method according to claim 7, wherein the mixing part and the determination part of the carrier are in the same part, and the operation of bringing the particles into contact with the sample is performed in the part that is the mixing part and the determination part. 13. The method for measuring a test substance in a sample according to the item.