JP2002040028A - Capillary immunoassay device and immunoassay method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a capillary immunoassay device capable of detecting target material highly sensitively on a test body by using a specific bonding reaction between the target material and functional material, and an immunoassay method using the device. SOLUTION: This capillary immunoassay device is composed of a base material for a stationary phase formed by mounting integrally capillaries having inner diameters of 10-500 μm on a sheet substrate, and particles for a mobile phase comprising solid phase particles having an average particle size of 0.05-5 μm. In this immunoassay method, the stationary phase is formed by carrying the functional material to be specifically bonded with the target material on the inner surfaces of the capillaries on the base material for the stationary phase of the device, and the mobile phase is composed of the solid phase particles having the average particle size of 0.05-5 μm having the surfaces on which the functional material to be specifically bonded with the target material is carried, and the solid phase particles brought into contact with the test body are moved in the capillaries, and the solid phase particles trapped on the inner surfaces thereof are detected. The solid phase particles are preferably magnetic solid phase particles.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a capillary immunoassay device and an immunoassay method using the same.

[0002]

2. Description of the Related Art At present, as an immunoassay method, an immunochromatography method using a porous membrane or filter paper as a base material constituting a stationary phase is widely applied. In this method, when the target substance to be detected is, for example, a certain kind of antigen, a stationary phase composed of a sensitized substrate having an antibody corresponding to the antigen immobilized at a predetermined site on the substrate is used. The colored solid-phase particles sensitized with the antibody are developed together with the sample in the stationary phase, and when the sample contains an antigen that is a target substance, a reaction in which an antibody-antigen-antibody sandwich type bond occurs, whereby the substrate It is determined whether or not the specimen contains a target substance by utilizing the fact that a colored line is formed by capturing solid phase particles at the antibody immobilization site.

[0003] However, the substrate used in the conventional immunochromatography method has a nonuniform pore size because it is a porous membrane or a filter paper, and for reasons other than the antigen-antibody reaction, for example, a mismatch in the pore size of the substrate. Due to unevenness of the pores and the continuity of the pores, the particles may be aggregated to form a colored line, which may result in a false determination such as a false positive determination. The solid phase particles used as a mobile phase in the immunochromatography method have a particle size of 0.1 to the pore size of the substrate.
It is required to be about 0.5 times, but for this reason the selection range of those that can be used as solid phase particles is narrow,
In addition, although particles having a small particle size are used for reliable development, the detection sensitivity obtained is low because the solid particles are small in size.

[0004] In the conventional immunochromatography method, the type of a sample to be measured or its concentration may be limited. For example, for samples such as blood or sputum,
It may be necessary to add certain additives or dilute to lower the concentration of the analyte to perform the necessary development. However, in these cases, there is a possibility that an accurate determination result cannot be obtained because measurement cannot be performed at a predetermined sample concentration, or since it is necessary to perform several deployment operations for one sample. However, there is a problem that the whole operation becomes complicated.

[0005]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems in the conventional chromatographic development method for a porous substrate. An object of the present invention is to provide a capillary immunoassay device capable of detecting a target substance with high sensitivity for a specimen by utilizing a reaction between a target substance and a functional substance that specifically binds to the target substance. It is in. Another object of the present invention is to provide an immunoassay method using the above-described capillary immunoassay device.

[0006]

The capillary immunoassay device of the present invention comprises a base material for a stationary phase in which a capillary having an inner diameter of 10 to 500 μm is integrally provided on a sheet-like substrate, and a mean particle size of 0.05 to 5 μm. And mobile phase particles comprising solid phase particles.

[0007] The immunoassay method of the present invention has an inner diameter of 10
A stationary phase substrate in which a 500 μm capillary is provided integrally with a sheet-like substrate and a functional substance that specifically binds to a target substance to be detected is supported on the inner surface of the capillary is used as a stationary phase. Using, as a mobile phase, solid phase particles having an average particle diameter of 0.05 to 5 μm, on the surface of which a functional substance that specifically binds to the target substance is supported,
The mobile phase is developed in the stationary phase by moving the solid phase particles of the mobile phase in contact with the sample through the capillary of the stationary phase, and the solid phase captured on the inner surface of the capillary by the reaction between the functional substance and the target substance. A target substance is detected for the sample by detecting phase particles. Here, the solid particles are preferably magnetic solid particles.

[0008]

According to the capillary immunoassay device described above, since the moving path of the mobile phase is formed by a capillary having an inner diameter of 10 to 500 μm, the moving path of the mobile phase particles is uniform and the continuity is uniform. Average particle size 0.0
The solid phase particles having a size of 5 to 5 μm can be smoothly moved by capillary action, and the required development can be performed reliably and stably. Therefore, accurate and reliable detection results can be obtained. . In addition, since the stationary phase substrate is constituted by a sheet-like substrate integrally provided with a capillary, by using an appropriate size as the sheet-like substrate, handling convenience or operation is improved. It is easy to make it easy, so that an accurate and reliable immunoassay can be easily performed.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. In the capillary immunoassay device of the present invention, the base material for the stationary phase is formed by integrally providing one or more of capillaries having an inner diameter of 10 to 500 μm in a specific range on a sheet-like substrate. Here, the “capillary” defines an elongated space extending like a thread, and basically refers to a substance in which water can move by capillary action.

The sheet-like substrate forming the substrate for the stationary phase is
The material is not particularly limited as long as it is a sheet-like or planar-like material having a two-dimensional spread as a whole, and the material is formed into a capillary, depending on the material and configuration of the actually provided capillary. It is preferable that the material be easy to bond with or easily connect to the capillary. Further, when the material of the sheet-like substrate is transparent or white, it is preferable in that the measurement after the development of the mobile phase can be easily performed by an optical method. Examples of the material of the sheet-like substrate include glass, silica glass, quartz glass,
Inorganic materials such as ceramics, synthetic plastic materials such as polystyrene and polypropylene, and metal materials which may be alloys can be used.

The capillary forming the elongated space extending like a thread may be provided integrally with the base material for the stationary phase, and the specific structure is not limited to a specific form or structure. More specifically, the stationary phase is formed by mechanically connecting an independent capillary material, which is itself a small-diameter tubular body and has a narrow elongated space extending in a thread shape by its internal space, to a sheet-like substrate. Substrate can be formed. In the case of using such a capillary material, as a method of achieving integration with a sheet-like substrate, for example, a method of bonding using an adhesive,
A method of fusing by thermal processing and others can be used.

FIG. 1 is an enlarged perspective view showing a specific configuration example of a stationary phase base material 10 when a capillary material is used. In this example, a plurality of thin cylindrical capillary members 14 are fixed to one surface of one flat sheet-like base member 12 so as to extend in the same direction, respectively, so that the stationary phase in a state of being integrally connected is formed. The base material 10 is configured.

Further, a base material for a stationary phase can be formed by forming a through-hole for defining an elongated space extending like a thread in an appropriate material for forming a sheet-like substrate without using a capillary material. In this case, the stationary phase base material is a sheet-like base material in which capillaries are integrally formed. FIG. 2 is an enlarged perspective view showing a specific configuration example of the stationary phase base material 20 according to this embodiment. In this example, the thickness t
Is formed with a circular through-hole 24 having a circular cross-sectional shape having an inner diameter d smaller than the thickness t of the sheet-like base 22, thereby forming a capillary with an elongated space. As a method of forming the through hole 24, for example, a method by precision machining, a method of molding with a die, a method of forming by etching using a resist, or the like can be used.

Further, by combining a plurality of materials, it is possible to form a stationary phase base material in the form of a sheet in which an elongated space extending in a thread form is formed. FIG. 3 is an enlarged perspective view showing a specific configuration example of the stationary phase base material 30 made of such a composite material. In this example, two flat sheet-like substrates 30A, 30B are arranged to face each other so as to extend in parallel, and between the sheet-like substrates 30A, 30B, elongated prisms 32, 32 each having a prismatic shape in the illustrated example are provided. They are arranged so as to extend in parallel in a state where they are separated from each other, and are integrally fixed to the sheet-like substrates 30A and 30B, thereby forming the stationary phase base material 30. In this example, two opposing inner surfaces of the sheet-like substrates 30A, 30B and two adjacent spacers 32, 32
And the two inner surfaces opposing each other, define an elongated space 34 extending like a thread, thereby forming a capillary.

The base material for the stationary phase is not limited to those described above, and the specific structure of the sheet-like substrate and the capillary provided integrally therewith can be various structures.
For example, a configuration in which, for example, a capillary forming member having a groove is coupled to the surface of a flat sheet-like substrate, a configuration in which a sheet-like substrate material having a groove serving as a capillary formed in the surface, or another configuration can be adopted. . Also, various changes can be made in the above specific examples of the stationary phase base material. For example, in the example shown in FIG. 1 or FIG. 2, the cross-sectional profile of the elongated space extending in a thread shape by the capillary need not be circular, but may be square or another shape. In the example shown in FIG. 3, the spacer may have a circular cross-sectional profile.

In the base material for the stationary phase, the inner diameter of the capillary is 10 to 500 μm, preferably 20 to 400 μm.
m. In the case of a capillary having an inner diameter of less than 10 μm, particles that can be used as a mobile phase to be developed are greatly restricted, and smooth movement may be hindered by slight aggregation of particles. On the other hand, the inner diameter is 500 μm
With capillaries exceeding 2, there is a risk that it will be difficult to reliably develop the mobile phase by capillary action.

As described above, the cross-sectional shape of the capillary is not limited to a circular shape. If the shape is not circular, it is sufficient if the capillary phenomenon similar to that of a circular shape occurs. The “inner diameter of the capillary” when the profile of the cross section of the capillary is not circular means the size of the gap at the minimum width where the capillary phenomenon occurs. Therefore, in the case of a capillary having a rectangular cross-sectional shape, the smaller diameter of the vertical side or the horizontal side of the square is used, and in the case of a capillary formed by a slit-shaped space, the diameter of the opening width of the slit is `` the capillary's Inner diameter ".

The material constituting the capillary is not particularly limited, and examples thereof include inorganic materials such as glass, silica glass, quartz glass, and ceramics; synthetic plastic materials such as polystyrene and polypropylene;
A metal material which may be an alloy can be used.

The above-mentioned base material for a stationary phase is used, when an immunoassay is carried out, on the inner surface of the capillary, when a functional substance specifically binding to a target substance to be detected, for example, a certain antigen is used as a target substance. In this method, an antibody reactive with the antigen is carried, and a substrate for a stationary phase in which the inner surface of the capillary is sensitized with an antibody is used as a stationary phase for immunoassay. In order to support the functional substance on the inner surface of the capillary, physical adsorption or chemical bonding can be used. When the inner surface of the capillary is made of a synthetic plastic material, a method using physical adsorption is simple,
When it is made of an inorganic material such as glass, it is preferable to carry out a surface activation treatment using a silane coupling agent or the like and then to carry the functional substance by chemical bonding.

The particles for the mobile phase used in combination with the stationary phase have an average particle size of 0.05 to 5 μm.
Preferably, magnetic solid particles containing particles of an organic polymer substance in the range of m, in which a magnetic substance powder is dispersed in the particles, are used. The magnetic solid particles are particles obtained by dispersing a magnetic powder in an organic polymer substance as a binder. The shape of the magnetic solid particles is not particularly limited, and may be, for example, spherical or polyhedral.

The organic high-molecular substance forming the magnetic solid particles is not particularly limited. For example, monomers such as styrene, divinylbenzene, acrylate and methacrylate are polymerized by a polymerization initiator. The resulting polymer can be prepared alone or in combination. As the magnetic powder contained in the magnetic solid particles, for example, a superparamagnetic powder or a dispersion can be used. Generally, the particle diameter of the magnetic powder is 10 to 50 nm. In the magnetic solid particles, the ratio of the organic polymer substance is, for example, 10 to 90% by mass, and the ratio of the powder of the superparamagnetic substance is, for example, 10 to 90%.
% By mass. By containing the magnetic substance powder in this way, the magnetic solid phase particles usually exhibit black or brown, which is the color of the magnetic substance powder. If necessary, the magnetic solid particles may contain additives such as a colorant, a dispersant, and an emulsifier.

The magnetic solid particles have an average particle size of 0.05 to 5
μm, preferably 0.1 to 2.5 μm. If the average particle size is too small, the surface area of the particles of a constant mass becomes extremely large and the surface energy becomes large, so that various restrictions are imposed on handling in order to maintain dispersion stability. This is not preferable because it increases the number of times. On the other hand, if the average particle size is too large, the mass of each particle increases, so that in the measurement of a sample in a low concentration region, the capture of the magnetic solid particles by a binding reaction such as an antigen-antibody reaction is sufficiently ensured. May not be done.

The magnetic solid phase particles constituting the mobile phase preferably have a particle size distribution of 40% or less. When magnetic solid particles having a particle size distribution exceeding 40% or less are used, the number or density of binding sites per unit particle of the magnetic solid particles tends to be insufficient in the measurement of a sample in a low concentration region. Since it is difficult to capture magnetic solid particles having a diameter as compared with magnetic solid particles having a small particle size, a sharp colored line is not formed, and thus a clear determination may be difficult.
Not preferred.

The magnetic solid particles are applied to the surface of the particles during the actual immunoassay or beforehand.
A functional substance that specifically binds to the target substance to be detected, for example, when an antigen-antibody reaction is used, carries a certain kind of antibody, and the sensitized substance is transferred for immunoassay. Use as phase. The functional substance used here is not particularly limited as long as it specifically binds to the target substance, and may be the same as the one supported on the inner surface of the capillary of the stationary phase base material described above. , May be different.

In the immunoassay method of the present invention, the above-mentioned stationary phase and mobile phase are used, and the mobile phase brought into contact with the sample is developed on the stationary phase. Specifically, a magnetic solid phase particle carrying a functional substance is brought into contact with an analyte, or a mixture of a magnetic solid particle and an analyte is brought into contact with an end opening of a capillary of a stationary phase base material, and the capillary of the capillary is contacted. It is deployed using the phenomenon and moves inside the capillary. In this developing operation, it is not necessary to add a development accelerator to the mobile phase, but addition of the development accelerator is not prohibited.

In this expanding operation, if necessary,
Apply a magnetic field of controlled strength in the direction of movement of the mobile phase particles, that is, in the direction parallel to the direction in which the capillary extends,
Thereby, the moving speed of the particles can be adjusted to an appropriate size. Therefore, when a high-viscosity specimen such as blood or sputum is to be measured, the moving speed of the particles can be increased, and the time required to complete the required development can be reduced. The strength of the magnetic field to be actually applied can be appropriately selected depending on the magnetic properties, particle size, and mass of the magnetic solid particles used, the viscosity of the sample used, and other conditions.

The magnetic solid particles moving by the operation of developing the mobile phase are brought into a state where the target substance of the sample is bound to the functional substance carried on the surface of the magnetic solid particles by contact with the sample. I have. Then, the magnetic solid particles move through the capillary of the base material for the stationary phase by capillary action.On the inner surface of the capillary, a functional substance that specifically binds to the target substance of the specimen is supported. The target substance bound to the magnetic solid particles binds to the functional substance of the stationary phase, whereby the magnetic solid particles are captured by the functional substance-supporting portion of the capillary.

Since the magnetic solid particles are colored black or brown by the magnetic powder contained therein, a colored line is formed in the capillary by the captured magnetic solid particles. It will be. Since the number of captured magnetic solid particles corresponds to the amount of the target substance contained in the sample, the number of the magnetic solid particles is determined in advance by measuring the color density of this colored line with a colorimeter. By collating with the calibration curve or equivalent collation data, the amount of the target substance captured in the capillary can be known, and the concentration of the target substance in the specimen substance is determined based on the concentration information of the specimen. Detection can be performed.

The detection of the magnetic solid particles can be performed by detecting the color state of the magnetic solid particles fixed to the capillary of the stationary phase base material. Quantitative detection of a target substance can be performed with high accuracy without being affected by external factors. In addition, for the detection of the magnetic solid particles, the intended quantitative detection can be performed also by a method of measuring magnetic properties. In this case, since the magnetic characteristics of the magnetic solid phase particles do not basically change with time, the target substance can be reliably detected even after a long storage period.

The type of target substance to be detected by the capillary immunoassay performed as described above is not particularly limited, but typical examples thereof include, for example, animals such as humans and antigenic antigens. Or an antibody thereof. Therefore, as the specimen, a body fluid such as blood, a body fluid component such as plasma, a body fluid-derived substance such as serum, or excretions such as urine, sweat, saliva, runny nose, and sputum can be used. These biological specimens can be subjected to the developing operation without any special pretreatment, for example, dilution with an appropriate diluent, treatment for generating a binding reaction with high sensitivity, various additions It is not prohibited to add an agent.

The functional substance to be carried on the inner surface of the capillary of the base material for the stationary phase and the surface of the particles for the mobile phase is not particularly limited as long as it specifically binds to the target substance. is not. For example, by sensitizing a single-stranded nucleic acid or a double-stranded nucleic acid, a nucleic acid complementary thereto or a protein substance that specifically binds thereto can be used as a target substance for detection. In this case, it is also possible to capture the magnetic solid particles using a double hybrid reaction via the nucleic acid to be detected.

In the capillary immunoassay device described above, the development path of the mobile phase particles in the base material for the stationary phase is formed by a filament formed by a capillary having an inner diameter defined in a specific range of 10 to 500 μm. Since the mobile phase is a magnetic solid phase particle having a specific average particle size of 0.05 to 5 μm, the mobile phase in the stationary phase base material is The unfolding movement is stably and smoothly performed under certain conditions by the capillary phenomenon caused by the capillary. That is, since the mobile phase has a uniform shape of the moving path, the magnetic solid particles do not agglomerate for reasons other than the intended reaction depending on the conditions of the moving path, and the intended development movement is inhibited. Nothing. Therefore, since a specific reaction by the target substance surely occurs, an accurate and highly reliable immunoassay can be performed, and an accurate determination result can be obtained.

In addition, since individual capillaries are in the form of thin threads, it is considerably difficult to handle each of them independently. In the above-mentioned capillary immunoassay device, the base material for the stationary phase is a sheet made of a capillary. Since the sheet-shaped substrate is provided integrally with the sheet-shaped substrate, the dimensions of the sheet-shaped substrate can be set to an appropriate size in consideration of operability by hand, so that the sheet can be easily handled. Therefore, the desired immunoassay can be performed reliably and easily, and an accurate detection result can be obtained.

In the present invention, the shape and dimensions of the sheet-like substrate are not particularly limited, but in practice, the dimension of the larger one of the vertical side and the horizontal side is, for example, 10 m.
If it is not less than m, there is no great difficulty in handling. With a sheet-shaped substrate having such dimensions, it is sufficiently possible to integrally provide a capillary which usually requires a length of 10 mm or more.

Although the present invention has been described in detail, various changes can be made in the present invention. For example, the solid particles constituting the mobile phase are not limited to magnetic solid particles, and need not be magnetic. In this case, the solid particles need to be colored in order to detect the captured particles by an optical method.

[0036]

Hereinafter, embodiments of the present invention will be described.
The present invention is not limited to these.

<Device Preparation Example 1>
A laminate of two commercially available polystyrene sheets having a width of 20 mm and a thickness of 0.5 mm is used as a sheet-like substrate, and a diameter of 0.25 mm and a length of 20 m is provided between the two sheets.
A plurality of stainless steel wires having a circular cross section of m are arranged so that each of them extends in the width direction of the sheet-shaped substrate and is arranged at intervals of 10 mm in the length direction, and the spacer width (thickness) is 1.0 mm. Is set in the auto-frace in a state of being stored in the frame of the plate-shaped processing spacer of
Heating at 100 ° C. for 10 minutes under a load of kg / cm ² , then slowly cooling to room temperature, and slowly pulling out a stainless steel wire to form a through hole extending in the width direction of the sheet, Thereby, inner diameter 0.25m
A capillary immunoassay device having m capillaries (referred to as “device A”) was prepared. The volume of each capillary in this device A is 1 μl (microliter).

<Device Preparation Example 2> Inner diameter 0.25 m
m, a fused silica electrophoresis capillary tube having an outer diameter of 0.35 mm was cut into a length of 20 mm to obtain a tubular capillary material. The volume of each capillary material is about 1 μl.
On a sheet-like substrate made of a nitrocellulose sheet having a length of 150 mm, a width of 17 mm, and a thickness of 1 mm, the above-mentioned capillary material is extended in the width direction of the sheet-like substrate, and
With about 1.5 mm of the leading end of each end protruding outward from the outer edge of the sheet, 10 mm in the length direction
The capillaries were fixed to the sheet with an adhesive tape in this state, and a capillary immunoassay device (hereinafter referred to as “device B”) was prepared.

<Preparation of Antibody-Sensitized Magnetic Solid Phase Particles> The following abbreviations are as follows. PBS solution: sodium phosphate buffer AFP antibody: α-fetoprotein monoclonal antibody (manufactured by Cosmo Bio) BSA: bovine serum albumin (manufactured by Sigma)

An average particle diameter of 0.5 μm made of polystyrene in which a superparamagnetic powder is dispersed at a content of 50% by mass.
Was washed three times with a 10 mM PBS solution (sodium chloride 150 mM) containing 1.0% by mass of the magnetic solid particles, and then the AFP antibody having a concentration of 10 μg / ml was washed. 100 μl of PBS solution
, And stirred for 1 hour at room temperature to obtain AFP
Antibody-sensitized magnetic solid particles were obtained. Thereafter, the magnetic solid particles are washed three times with a PBS solution of 0.1% by mass of BSA,
Finally, the particles were dispersed in 5 ml of a PBS solution so that the solid content of the magnetic solid particles became 0.01% by mass.

<Preparation of Substrate for Antibody-Sensitized Stationary Phase> A concentration of 1 μg was added to the capillaries of Device A and Device B.
/ Ml of AFP antibody solution, and allowed to stand for 10 minutes, then dextran at a concentration of 0.1% by mass and a concentration of 0.1% by mass.
Using 50 μl of a PBS solution containing 2% by mass of polyvinylpyrrolidone (molecular weight: 400,000), one open end of the capillary is immersed in a PBS solution, and the other open end is closed with a water-absorbing pad, thereby utilizing capillary action. By carrying out a flow-type washing operation, a substrate for an antibody-sensitized stationary phase was prepared.

<Creation of Data for Verification> In the capillaries of the devices A and B prepared as described above,
AFP antigen prepared from bovine serum (Cosmo Bio)
Diluents of various concentrations (concentrations 0, 0.01, 0.1,
When a mixed solution of 5 μl of 1, 10 (unit: pg / ml) and 5 μl of the above-mentioned particle dispersion was loaded into the capillary as a sample liquid, the sample liquid passed through the capillary from the loading side by capillary action. It moved and reached the other outlet, and the phenomenon that it was absorbed by the water absorbent pad occurred. The time until the transfer of 10 μl of the sample solution was completed was about 5 minutes. Next, flow-type washing was performed using 50 μl of a PBS solution.

In the stationary phase substrate on which the immunoassay was performed as described above, a colored line was formed in the capillary. The color density of the colored line was measured by a non-contact colorimeter (Minolta). The results are shown in Table 1 below. Here, the numerical values in Table 1 are the Y values of the colorimeter at each antigen concentration. These numerical values are used as collation data.

[0044]

[Table 1]

<Immunoassay Using Clinical Specimens> The same operation as described above was performed using eight clinical sera instead of the AFP antigen. The results are shown in Table 2 below. Here, the numerical values in Table 2 are the Y values of the colorimeter.

[0046]

[Table 2]

From the above results, the use of the capillary immunoassay device provided the following for each clinical serum:
It is apparent that the AFP antigen can be sufficiently developed in a short time, and the AFP antigen as its target substance can be quantitatively detected. Then, by collating the obtained numerical value with the collation data, it is detected that, for example, in the specimen of clinical serum 1, the concentration of the AFP antigen as the target substance is slightly lower than about 0.1 pg / ml. Further, the above-mentioned substrate for an antibody-sensitized stationary phase was stored in a dry state at room temperature for one year and subjected to the same measurement, but no deterioration in performance was observed.

<Immunoassay under the action of a magnetic field>
3000 having a shape of 10 mm in length and 10 mm in width at a position 10 mm from one end of device A and device B
The same operation as the immunoassay using the clinical specimen described above was performed except that the development was performed in a state where a magnetic field was applied in a direction parallel to the direction in which the capillary was extended by installing a Gaussian permanent magnet. In this development, 1
The time required to complete the transfer of the 0 μl sample solution is about 3.5.
Minutes. After loading the sample solution which is a mixture of the specimen and the magnetic solid particles, the magnet was removed by washing with 50 μl of the PBS solution in the same manner as described above. The color density of the colored line formed on the stationary phase substrate on which the immunoassay was performed in this manner was measured. The results are shown in Table 3 below. Here, the numerical values in Table 3 are the Y values of the colorimeter at each antigen concentration.

[0049]

[Table 3]

[0050]

According to the capillary immunoassay device of the present invention, the moving path of the mobile phase has an inner diameter of 10 to 500.
Since the moving path of the mobile phase particles is uniform and the continuity is uniform because of being formed by the capillary of μm, the solid phase particles having an average particle diameter of 0.05 to 5 μm can be smoothly moved by the capillary phenomenon. The required deployment can be performed reliably and stably, so that accurate and reliable detection results can be obtained. In addition, since the stationary phase substrate is constituted by a sheet-like substrate integrally provided with a capillary, by using an appropriate size as the sheet-like substrate, handling convenience or operation is improved. It is easy to make it easy, so that an accurate and reliable immunoassay can be easily performed.

[Brief description of the drawings]

FIG. 1 is an explanatory perspective view showing an example of a configuration of a stationary phase substrate in a capillary immunoassay device of the present invention.

FIG. 2 is an explanatory perspective view showing another example of the structure of the stationary phase base material.

FIG. 3 is an explanatory perspective view showing still another example of the configuration of the base material for the stationary phase.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 10 Stationary phase base material 12 Flat sheet base 14 Capillary material 20 Stationary phase base 22 Sheet base 24 Through hole 30 Stationary phase base 30A, 30B Flat sheet base 32 Spacer 34 Slender space

Claims (3)

[Claims] 1. A stationary phase base material comprising a sheet-shaped substrate integrally provided with a capillary having an inner diameter of 10 to 500 μm,
A capillary immunoassay device comprising mobile phase particles comprising solid phase particles having an average particle size of 0.05 to 5 μm. 2. A functional substance which specifically binds to a target substance to be detected is provided on an inner surface of a capillary of a stationary phase substrate in which a capillary having an inner diameter of 10 to 500 μm is integrally provided on a sheet-like substrate. The solid phase particles having an average particle diameter of 0.05 to 5 μm and having a functional substance which specifically binds to the target substance are supported on the surface thereof are used as a stationary phase, and the mobile phase is used as a stationary phase. The mobile phase is developed in the stationary phase by moving the solid phase particles of the mobile phase in the stationary phase capillary, and the solid phase particles captured on the inner surface of the capillary by the reaction between the functional substance and the target substance are removed. An immunoassay method comprising detecting a target substance for the sample by detecting the target substance. 3. The immunoassay according to claim 2, wherein the solid particles are magnetic solid particles.