JP2018100888A - Transfer medium for inspection device, inspection device, method for manufacturing inspection device, and inspection kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transfer medium for an inspection device, the transfer medium allowing the manufacture of an inspection device having a detection unit capable of obtaining a sufficient color optical density.SOLUTION: The transfer medium for an inspection device includes: a supporting body; and a porous reagent solid-phasing layer on the supporting body. The reagent solid-phasing layer includes an antibody, a wax, and at least one type of thermoplastic elastomer including at least one type of hydrophobic thermoplastic elastomer. The ratio of the mass of the at least one type of thermoplastic elastomer to the total amount of the wax and the at least one type of thermoplastic elastomer is in the range of 8% by mass to 25% by mass, both inclusive.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a transfer medium for an inspection apparatus, an inspection apparatus, a manufacturing method thereof, and an inspection kit.

  Conventionally, as an immunoassay method for detecting and quantifying an antigen or antibody using a reaction between an antigen and an antibody, for example, for the purpose of testing the specimen such as blood, DNA, food, beverage, etc. An inspection apparatus in which a flow path is formed is used.

  The test apparatus includes, for example, a sample pad as a test solution receiver, a conjugate pad that reacts the test solution supplied from the sample pad, and a membrane film that flows the test solution supplied from the conjugate pad. It is configured. In such an inspection apparatus, a reagent such as a capture antibody is usually fixed to a fiber or the like inside the flow path member. The determination line (test line and control line) that is a detection unit is formed by directly applying a solution in which the capture antibody is dissolved to a flow path member made of a porous material. It exists in a diffuse manner. Therefore, there is a problem that the density of the accumulated labeled antibody is uneven, or both end portions of the determination line are blurred and the color becomes unclear. On the other hand, for example, an inspection apparatus has been proposed in which a reagent is immobilized on the surface of a reagent-immobilized layer and transferred onto a flow path member as a transfer medium (see, for example, Patent Document 1).

  An object of the present invention is to provide a transfer medium for an inspection apparatus capable of manufacturing an inspection apparatus having a detection unit that can obtain a sufficient color density.

The transfer medium for an inspection apparatus of the present invention as a means for solving the above problems has a support and a porous reagent-immobilized layer on the support,
The reagent-immobilized layer contains an antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer,
The mass ratio of the thermoplastic elastomer to the total amount of the wax and the thermoplastic elastomer is 8% by mass or more and 25% by mass or less.

  According to the present invention, it is possible to provide a transfer medium for an inspection apparatus that can manufacture an inspection apparatus having a detection unit that can obtain a sufficient color density.

FIG. 1 is a top view showing an example of the inspection apparatus of the present invention. FIG. 2 is a cross-sectional view taken along the line A-A ′ of the inspection apparatus of FIG. 1. FIG. 3 is a cross-sectional view illustrating an example of an inspection device at a facing portion between the flow path member and the resin layer. FIG. 4A is a cross-sectional view illustrating another example of the inspection device in the facing portion between the flow path member and the resin layer. FIG. 4B is a cross-sectional view illustrating another example of the inspection device in the facing portion between the flow path member and the resin layer. FIG. 5A is a cross-sectional view showing another example of the inspection device at the facing portion of the flow path member and the resin layer. FIG. 5B is a cross-sectional view illustrating another example of the inspection device in the facing portion between the flow path member and the resin layer. FIG. 6 is a conceptual diagram of a conjugate pad in a conventional inspection apparatus. FIG. 7 is a conceptual diagram of a membrane in a conventional inspection apparatus. FIG. 8A is a schematic cross-sectional view showing an example of a transfer medium for an inspection apparatus according to the present invention. FIG. 8B is a schematic cross-sectional view showing another example of the transfer medium for an inspection apparatus according to the present invention. FIG. 9 is a schematic view showing an example of a test kit of the present invention. FIG. 10A is a top view illustrating an example of the inspection apparatus used in the example. FIG. 10B is a cross-sectional view taken along B-B ′ of FIG. 10A. FIG. 11A is a top view illustrating another example of the inspection apparatus used in the example. FIG. 11B is a cross-sectional view taken along the line H-H ′ of FIG. 11A. 12A is a cross-sectional SEM image of the thermal transfer medium for test line in Example 1. FIG. 12B is a cross-sectional SEM image of the test line thermal transfer medium in Comparative Example 2. FIG. FIG. 12C is a cross-sectional SEM image of the thermal transfer medium for test line in Comparative Example 3.

(Transfer media for inspection equipment)
The transfer medium for an inspection apparatus of the present invention has a support and a porous reagent-immobilized layer on the support,
The reagent-immobilized layer contains an antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer,
The mass ratio of the thermoplastic elastomer to the total amount of the wax and the thermoplastic elastomer is 8% by mass or more and 25% by mass or less, and further includes other members as necessary.

  In the transfer medium for an inspection apparatus according to the present invention, in the conventional transfer medium, a sufficient amount of capture antibody capable of binding to the antigen is not attached to the transfer medium. For this reason, it is based on the knowledge that the color density of the judgment line (test line and control line) as a detection part obtained by combining antigen, labeled antibody, and capture antibody in the finished inspection device was not sufficient It is.

<Support>
There is no restriction | limiting in particular about the shape, a structure, a magnitude | size, a material, etc. as said support body, According to the objective, it can select suitably.
The structure of the support may be a single layer structure or a laminated structure.
The size of the support can be appropriately selected according to the size of the inspection apparatus.
The material of the support is not particularly limited and may be appropriately selected according to the purpose. For example, polyester such as polyethylene terephthalate (PET) and polyethylene naphthalate (PEN), polycarbonate, polyimide resin (PI), Examples thereof include polyamide, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, styrene-acrylonitrile copolymer, and cellulose acetate. These may be used individually by 1 type and may use 2 or more types together. Among these, polyethylene terephthalate (PET) and polyethylene naphthalate (PEN) are particularly preferable.

  The surface of the support is preferably subjected to a surface activation treatment in order to improve adhesion with a layer provided on the support. Examples of the surface activation treatment include glow discharge treatment and corona discharge treatment.

  The support may be left as it is after transferring the reagent-immobilized layer to the flow path member, or after transferring the reagent-immobilized layer, the support or the like may be peeled off and removed by the release layer. Good. When using a release layer / reagent-immobilized layer, the release layer / reagent-immobilized layer is completely transferred to the flow path member, or the antibody is immobilized on the release layer / reagent-immobilized layer. The portion including the surface thus transferred is transferred, but a part of the release layer / reagent-immobilized layer may remain on the support side.

The support is not particularly limited and may be appropriately synthesized or a commercially available product may be used.
There is no restriction | limiting in particular as average thickness of the said support body, Although it can select suitably according to the objective, 3 micrometers or more and 50 micrometers or less are preferable.

<Peeling layer>
The release layer has a function of improving the peelability between the support and the reagent-immobilized layer during transfer. In addition, when the release layer is heated by a heating and pressurizing means such as a thermal head, it melts into a low-viscosity liquid, and the reagent-immobilized layer can be easily cut in the vicinity of the interface between the heated part and the non-heated part. Has the function of

The release layer contains a wax and a binder resin, and further contains other components as necessary.
The function of the wax in the release layer includes melting by appropriate thermal energy, releasing the reagent-immobilized layer from the support, and transferring it to a transfer medium. In order to transfer the release layer using a printer, it is necessary to melt the wax by the thermal energy to the printer head. Therefore, the melting point of the release layer needs to be selected in consideration of the thermal energy given by the printer. There is.

  The wax is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include natural waxes such as beeswax, carnauba wax, whale wax, wood wax, candelilla wax, rice bran wax, and montan wax; paraffin Synthetic waxes such as wax, microcrystalline wax, oxidized wax, ozokerite, ceresin, ester wax, polyethylene wax, oxidized polyethylene wax; higher grades such as margaric acid, lauric acid, myristic acid, palmitic acid, stearic acid, furoic acid, behenic acid Fatty acids; higher alcohols such as stearic alcohol and behenyl alcohol; esters such as fatty acid esters of sorbitan; amides such as stearamide and oleinamide. These may be used alone or in combination of two or more. Among these, carnauba wax and polyethylene wax are preferable from the viewpoint of excellent peelability.

  There is no restriction | limiting in particular as said binder resin, According to the objective, it can select suitably, For example, ethylene-vinyl acetate copolymer, partially saponified ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer , Ethylene-sodium methacrylate copolymer, polyamide, polyester, polyurethane, polyvinyl alcohol, methyl cellulose, carboxymethyl cellulose, starch, polyacrylic acid, isobutylene-maleic acid copolymer, styrene-maleic acid copolymer, polyacrylamide, polyvinyl Examples include acetal, polyvinyl chloride, polyvinylidene chloride, isoprene rubber, styrene-butadiene copolymer, ethylene-propylene copolymer, butyl rubber, acrylonitrile-butadiene copolymer, and the like. These may be used alone or in combination of two or more.

There is no restriction | limiting in particular as a formation method of the said peeling layer, According to the objective, it can select suitably, For example, the method of apply | coating the coating liquid which disperse | distributed the hot-melt coating method and the said wax and the said binder resin to the solvent Etc.
There is no restriction | limiting in particular in the average thickness of the said peeling layer, Although it can select suitably according to the objective, 0.5 micrometer or more and 50 micrometers or less are preferable.
Adhesion amount of the release layer is not particularly limited and may be appropriately selected depending on the purpose, 0.5 g / ^{m 2} or more 50 g / ^{m 2} or less.

<Reagent solid phase layer>
The reagent-immobilized layer needs to contain an antibody, a wax, and a thermoplastic elastomer.
Examples of the function of the wax in the reagent-immobilized layer include immobilization with an antibody by hydrophobic interaction. There is no restriction | limiting in particular as said wax, It can select according to the objective similarly to the wax used for the said peeling layer.
In order to fix more antibodies, it is necessary to make a structure with many voids in order to increase the surface area of the release layer. For this purpose, the wax is removed by pulverizing and dispersing so that the median diameter of the wax is 0.1 μm or more and 10 μm or less (laser diffraction / scattering particle size distribution measuring device, manufactured by HORIBA, LA960). By dispersing in the form of granules and coating and drying on the support, the solvent is volatilized, resulting in a structure having voids. When the median diameter of the wax is 0.1 μm or more and 10 μm or less, a structure with many voids can be obtained, many antibodies can be fixed, and a sufficient color density can be obtained.

The function of the thermoplastic elastomer in the reagent-immobilized layer includes a role of binding waxes and fixing them to a transfer medium for an inspection apparatus. Moreover, since it is necessary to melt | dissolve with a thermal energy, and to transfer | transfer to a flow-path member from a support body and to bind, it needs to be the thermoplasticity which softens by heating.
As a result of intensive studies by the present inventors, it has been found that the thermoplastic elastomer needs to be hydrophobic in order to make the cross-section of the reagent-immobilized layer have a void structure.
Here, the hydrophobicity is defined as a result of measuring the contact angle when the thermoplastic elastomer alone is applied smoothly on an arbitrary film with a bar coater or the like using distilled water and is 75 ° or more. Less than ° is defined as hydrophilic. The contact angle can be measured with a portable contact angle meter (PG-X, manufactured by Fibro System).

The hydrophobic thermoplastic elastomer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include butadiene rubber, styrene-butadiene rubber, high styrene rubber, isoprene rubber, acrylic rubber, epichlorohydrin rubber, and butyl rubber. Synthetic rubber such as ethylene-propylene rubber, natural rubber, and the like can be mentioned.
Moreover, since at least one of the hydrophobic thermoplastic elastomers is a thermoplastic elastomer having a styrene skeleton, a void in a cross section can be obtained more easily. This is presumably because the styrene skeleton contains a benzene ring, so that aggregation of waxes is less likely to occur.

  The mass ratio of the thermoplastic elastomer to the total amount of the wax and the thermoplastic elastomer in the reagent-immobilized layer is 8% by mass or more and 25% by mass or less. When the mass ratio is 8% by mass or more, the thermoplastic elastomer can appropriately bind the wax, and after the release layer is applied on the base material, the release layer is prevented from being partially peeled by an external force. it can. Moreover, when it is 25% by mass or less, the balance between the thermoplastic elastomer and the wax is appropriate, and a target void structure is obtained.

  The method for forming the reagent-immobilized layer is not particularly limited and can be appropriately selected depending on the purpose. For example, a hot-melt coating method or a resin constituting the reagent-immobilized layer is dispersed in a solvent. The reagent coating solution can be formed by coating on a support or a release layer and drying by a general coating method such as a gravure coater, wire bar coater, roll coater or the like.

  The porosity of the reagent-immobilized layer before immobilizing the reagent is preferably 10% or more and 45% or less. The porosity varies slightly depending on the material used, but is preferably 10% or more in order to obtain sufficient color development, and is preferably 45% or less in order to maintain the mechanical strength of the porous body itself. However, it is not limited to this when the shape of the porous body is maintained even if the material itself has high mechanical strength and the porosity is large.

The method for measuring the porosity can be selected without particular limitation depending on the measurement sample, and can be obtained by obtaining a cross-section of the material using a method obtained from the density of the material or a scanning electron microscope (SEM). And a method of obtaining the image by analyzing the obtained image.
As a method of obtaining from the density, it can be obtained by the following formula 1 from the basis weight (g / m ² ), average thickness (μm), and composition specific gravity of the material.
[Calculation Formula 1]
Porosity (%) = {1- [basis weight (g / m ² ) / average thickness (μm) / specific gravity of composition]} × 100

As a method for measuring the porosity by image analysis, there is a method in which image data obtained by observing the cross section of the reagent-immobilized layer is binarized and calculated.
As an example, image analysis using image processing software (Image J) will be described. First, the cross section of the reagent-immobilized layer is enlarged at a magnification of 5,000 times using a scanning electron microscope (SEM) and recorded as a digital image. Next, the image is taken into Image J, and 8 bits are selected from Type in the Image menu. Next, select “Threshold” from “Adjust” in the “Image” menu, and adjust the threshold so that the void area is extracted from the cross section of the reagent-immobilized layer, and binarize and distinguish the void and the other parts. . Finally, the area of the void is calculated by selecting “Analyze Particles” in the Analyze menu, and the void ratio can be obtained by the following calculation formula 2.
[Calculation Formula 2]
Porosity (%) = (Area of void part) / (Area of the whole reagent-immobilized layer) × 100
In addition, the porosity here means ten average cross-sectional images, and means the average of the porosity obtained from them.

  The average pore size of the voids in the reagent-immobilized layer is not particularly limited, but the lower limit value varies depending on the particle size of the colloidal gold or fluorescent resin particles used for the labeled antibody to be used, but these ranges are approximately A pore size three times or more that of the particles is required. If it is less than 3 times, problems such as particle clogging may occur and the antigen-antibody reaction inside the test line may be significantly impaired. As for the upper limit value, the larger the pore diameter, the smaller the surface area of the void, so the amount of capture antibody that can be immobilized is reduced and the effect is lowered. About 1/3 is preferable. As an example, in the case of a reagent-immobilized layer having an average thickness of 6 μm, the upper limit is 2 μm.

  Examples of the method for measuring the average pore size of the voids include a method in which a cross section of the reagent-immobilized layer is observed with a scanning electron microscope (SEM) and the obtained image is analyzed. Specifically, the average particle diameter of the void, the average radius of the major axis, and the average radius of the minor axis can be obtained when the void is regarded as an irregular particle, which can be obtained by the same procedure as the image analysis using Image J. be able to. In addition, the hole diameter of a space | gap here means the average particle diameter acquired from 10 arbitrary cross-sectional images, and obtained from those distribution.

  The method for generating the voids is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the hydrophobic material is dispersed in a poor solvent (such as water) to form granules, The solvent is volatilized by coating and drying, and as a result, a reagent-immobilized layer that is an aggregate of hydrophobic material-derived particles having voids can be formed.

  There is no restriction | limiting in particular as average thickness of the said reagent solid-phase layer, Although it can select suitably according to the objective, 200 nm or more and 50 micrometers or less are preferable. When the average thickness is 200 nm or more, the durability of the reagent-immobilized layer is improved, and the reagent-immobilized layer can be prevented from being damaged by friction or impact. Further, when the average thickness is 50 μm or less, heat from the thermal head can be uniformly transmitted, and the sharpness is improved.

The adhesion amount of the reagent coating solution in the reagent immobilization layer is not particularly limited and may be appropriately selected depending on the purpose, 0.2 g / m ² or more 50 g / m ² or less. When the adhesion amount is 0.2 g / m ² or more, the application amount is appropriate, and no defect occurs in the reagent solid phase layer. Moreover, when the said adhesion amount is 50 g / m < ² > or less, drying time is suitable and a nonuniformity does not arise in a reagent solid-phase-ized layer.

<Peeling layer and reagent solid phase layer>
The release layer / reagent-immobilized layer combines the functions of both the release layer and the reagent-immobilized layer, and improves the releasability between the support and the reagent-immobilized layer during transfer. In addition, it is possible to immobilize a reagent such as a capture antibody by immobilizing a resin constituting the reagent immobilization layer in the inspection apparatus.

When the release layer / reagent-immobilized layer is heated by a heating and pressurizing means such as a thermal head, the side surface in contact with the support is melted into a low-viscosity liquid (heating portion). On the other hand, the side surface on which the reagent is solid-phased is in a solid state or a state close thereto (non-heated portion), and has a function of facilitating cutting near the interface between the heated portion and the non-heated portion.
The release layer / reagent-immobilized layer contains a wax and a hydrophobic thermoplastic elastomer, and further contains other components as necessary.

  There is no restriction | limiting in particular as said wax, According to the objective, it can select suitably, For example, the thing similar to the said peeling layer is mentioned. These may be used alone or in combination of two or more. Among these, carnauba wax and polyethylene wax are preferable because they are excellent in releasability and capture antibody immobilization ability (hydrophobicity).

There is no restriction | limiting in particular as said hydrophobic thermoplastic elastomer, According to the objective, it can select suitably, For example, the thing similar to the said reagent solid-phase layer is mentioned. These may be used alone or in combination of two or more.
The mass ratio of the thermoplastic elastomer to the total amount of the wax and the thermoplastic elastomer in the release layer and reagent-immobilized layer is the same as that of the reagent-immobilized layer, and is 8% by mass or more and 25% by mass or less. preferable.

The method for forming the release layer / reagent-immobilized layer is not particularly limited and may be appropriately selected depending on the purpose. For example, the hot melt coating method, the wax and the binder resin are dispersed in a solvent. The method of apply | coating a coating liquid etc. are mentioned.
There is no restriction | limiting in particular as average thickness of the said peeling layer and reagent solid-phased layer, Although it can select suitably according to the objective, 0.5 to 50 micrometer is preferable. When the average thickness is 0.5 μm or more, the durability of the release layer / reagent-immobilized layer is improved, and damage to the release layer / reagent-immobilized layer due to friction or impact can be prevented. Further, when the average thickness is 50 μm or less, heat from the thermal head can be uniformly transmitted, and the sharpness is improved.
As the coating weight of the release layer and the reagent immobilization layer is not particularly limited and may be appropriately selected depending on the purpose, 0.5 g / m ² or more 50 g / m ² or less. When the adhesion amount is 0.5 g / m ² or more, the coating amount is appropriate, and the release layer / reagent-immobilized layer is not damaged. Moreover, when the adhesion amount is 50 g / m ² or less, the drying time is appropriate, and unevenness is less likely to occur in the release layer / reagent-immobilized layer.

<Back layer>
The transfer medium for an inspection apparatus is preferably provided with a back layer on the surface of the support opposite to the surface on the peeling layer side. At the time of transfer, heat is directly applied to the opposite surface in accordance with the shape of the reagent-immobilized layer or the release layer / reagent-immobilized layer with a thermal head or the like. For this reason, the back layer preferably has resistance to high heat and resistance to friction with a thermal head or the like.

  The back layer contains a binder resin, and further contains other components as necessary.

  There is no restriction | limiting in particular as said binder resin, According to the objective, it can select suitably, For example, silicone modified urethane resin, silicone modified acrylic resin, silicone resin, silicone rubber, fluorine resin, polyimide resin, epoxy resin, phenol Resins, melamine resins, nitrocellulose and the like can be mentioned. These may be used alone or in combination of two or more.

  There is no restriction | limiting in particular as said other component, According to the objective, it can select suitably, For example, inorganic fine particles, such as a talc, a silica, and an organopolysiloxane, a lubricant, etc. are mentioned.

There is no restriction | limiting in particular as a formation method of the said back layer, According to the objective, it can select suitably, For example, a gravure coater, a wire bar coater, a roll coater etc. are mentioned.
There is no restriction | limiting in particular as average thickness of the said back layer, Although it can select suitably according to the objective, 0.01 to 1.0 micrometer is preferable.

<Under layer>
An under layer is provided between the support and the release layer, between the release layer and the reagent-immobilized layer, or between the support and the release / reagent-immobilized layer. Can do.
The under layer contains a resin, and further contains other components as necessary. The resin is not particularly limited and may be appropriately selected depending on the purpose. For example, various resins used in the reagent-immobilized layer, the release layer, and the release layer / reagent-immobilized layer may be used. It can be used.

<Protective film>
A protective film is preferably provided on the reagent-immobilized layer or the release layer / reagent-immobilized layer in order to protect it from contamination and damage during storage.
The material of the protective film is not particularly limited as long as it can be easily peeled off from the reagent-immobilized layer or the release layer and reagent-immobilized layer, and can be appropriately selected according to the purpose. And polyolefin sheets such as silicone paper and polypropylene, and polytetrafluoroethylene sheets.
There is no restriction | limiting in particular in the average thickness of the said protective film, Although it can select suitably according to the objective, 5 micrometers or more and 100 micrometers or less are preferable, and 10 micrometers or more and 30 micrometers or less are more preferable.

<Antibody>
The capture antibody to be immobilized on the reagent-immobilized layer or release layer / reagent-immobilized layer (hereinafter referred to as resin layer) has a hydrophobic site and reacts with an antigen. If there is no restriction | limiting in particular, according to the objective, it can select suitably, For example, IgG antibody etc. are mentioned.
The antibody may be in any form of monoclonal antibody, polyclonal antibody, chimeric antibody, Fab antibody, and (Fab) ₂ antibody.
The capture antibody to be immobilized on the resin layer is not particularly limited as long as it has a hydrophobic group and reacts with the labeled antibody, and can be appropriately selected according to the purpose. And the antibodies listed above, and the like. Moreover, the antigen itself which reacts with a labeled antibody may be sufficient.

The method for impregnating the solution containing the antibody (antibody coating solution) inside the void is not particularly limited and can be appropriately selected depending on the purpose. For example, the pressure impregnation method, the vacuum impregnation method, the vacuum pressure method Examples include an impregnation method.
The resin layer is made of a hydrophobic material, and the wall surface inside the gap exhibits hydrophobicity, so that even if the antibody coating solution is applied as it is, it does not easily penetrate into the gap. In addition, the gas remaining inside the gap may interfere with the penetration of the coating solution. Therefore, by applying the antibody coating solution to the resin layer or impregnating the resin layer in the antibody coating solution and then applying pressure, the solution is pushed into the gap and the inside of the gap is filled with the antibody coating solution. Is possible.
The pressure of the pressurization is not particularly limited and may be appropriately selected depending on the purpose. However, in order to sufficiently impregnate the antibody coating solution, the pressure when the atmospheric pressure is 0 is preferably 3 MPa or more, 3 MPa or more and 5 MPa or less are preferable. In addition, although the pressurization may be higher if originally, due to the problem of the apparatus, this time, the examination was performed at 3 MPa or more and 5 MPa or less.

There is no restriction | limiting in particular as time of pressurization, Although it can select suitably according to the objective, 10 minutes or more are preferable starting from the time which reached | attained the target pressure. When the pressurization time is 10 minutes or more, it is a sufficient time for the antibody coating solution to reach the inside of the gap.
The number of times of pressurization is not particularly limited and can be appropriately selected according to the purpose. However, when the pressure is applied twice or more (pressurization after pressurization and breakage), the antibody coating solution is mixed. As a result, a new antibody is supplied to the inside of the void, and the solid-phase efficiency of the antibody can be increased. In addition, this process makes it easy to push out the bubbles remaining inside the gap and to fill the gap with the coating liquid.

Here, the configuration of the transfer medium for the inspection apparatus will be described with reference to the drawings. FIG. 8A is a schematic cross-sectional view showing an example of a transfer medium for an inspection apparatus according to the present invention. FIG. 8B is a schematic cross-sectional view showing another example of the transfer medium for an inspection apparatus according to the present invention.
When the thermal transfer method is used, since the transfer medium 100 for an inspection apparatus to which the capture antibody is uniformly attached in advance can be used, the concentration difference of the capture antibody (17, 18) in the determination line (test line and control line) is small. Become. In addition, when the capture antibody is applied and arranged by a conventional method, it is necessary to dilute the capture antibody with a solvent until the viscosity is such that it can be applied (for example, can be ejected by an inkjet printer). When the capture antibody is disposed by thermal transfer, a high concentration capture antibody can be disposed in the flow path by using a transfer medium for an inspection apparatus to which a high concentration capture antibody is attached in advance.

As shown in FIG. 8A, the transfer medium 100 for an inspection apparatus includes a support 101, a release layer 102 provided on the support 101, and a reagent-immobilized layer 103 provided on the release layer 102. The reagent is immobilized on the surface of the reagent-immobilized layer 103. Further, the inspection apparatus transfer medium 100 further includes other layers such as a back layer 104 as necessary.
As shown in the inspection apparatus transfer medium 110 in FIG. 8B, the release layer 102 and the reagent-immobilized layer 103 can also serve as the release layer / reagent-immobilized layer 105.

(Inspection equipment)
The inspection apparatus of the present invention has a porous flow path member having a flow path for flowing a specimen, and a resin layer provided at least in one place on the flow path member,
The resin layer contains an antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer,
The mass ratio of the thermoplastic elastomer to the total amount of the wax and the thermoplastic elastomer is 8% by mass or more and 25% by mass or less.

The inspection apparatus according to the present invention is based on the knowledge that the conventional inspection apparatus cannot effectively utilize the color development region in the thickness direction of about 5 μm to 10 μm from the surface layer where light scattering does not occur.
In addition, since the inspection apparatus of the present invention uses a porous body made of a hydrophilic material as the resin layer in the conventional inspection apparatus, the reagent (capture antibody) cannot be sufficiently adsorbed, and the resin layer is porous. This is based on the knowledge that the color density is insufficient despite the use of the body.

In the inspection apparatus of the present invention, the resin layer contains an antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer. Can be immobilized on the resin layer, so that determination lines (test line and control line) with sufficient color density can be obtained.
The inspection apparatus includes a porous flow path member in which a flow path for flowing a specimen is formed,
A first resin layer provided on the flow path member, and a second resin layer,
The first resin layer contains a capture antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer;
The mass ratio of the thermoplastic elastomer to the total amount of the wax and the thermoplastic elastomer is 8% by mass or more and 25% by mass or less,
The second resin layer preferably has a labeled antibody on the surface facing the flow path member.
It is preferable to provide a plurality of the first resin layers. A plurality of the first resin layers is advantageous in that it can be easily confirmed that the sample is deployed without any problem in the flow path member.

  In the inspection apparatus, the capture antibody is solid-phased on the side of the resin layer facing the flow path member, as in the conventional ELISA (Enzyme Linked Immuno Solvent Assay) method, whereby the specimen is placed on the resin layer side. However, in the present invention, since more antibodies are immobilized on the transfer medium, the specimen can be detected with high sensitivity.

There is no restriction | limiting in particular as said resin layer, Although it can select suitably according to the objective, It is preferable that it is a porous body which consists of hydrophobic materials.
As a measure of the hydrophobicity, a contact angle between the material constituting the resin layer and distilled water can be used, and a substance having a large contact angle can be regarded as having high hydrophobicity. In this case, for example, the contact angle is measured from the coordinates of the droplet image by the ATAN1 / 2θ method and the proximity method based on the young formula, which is the contact angle measurement model used internationally in many fields as a contact angle calculation formula. In this case, a material having a contact angle of 75 degrees or more with distilled water at room temperature (25 ° C.) can be treated as hydrophobic.

As an example of the method for evaluating the contact angle, there is a method of measuring the contact angle of the surface of the sample for measurement obtained by smoothing the material of the resin layer (porous body, sheet, film, etc.) by heat or pressure.
Here, using a contact angle meter, in a 50% RH environment at 25 ° C., 4.0 μL of distilled water is dropped on the measurement sample, and the contact angle is measured.

The hydrophobic material is preferably a resin having a hydrophobic group described later. Here, the hydrophobic group is an atomic group that is not easily compatible with water, and means that it has a low affinity for water and is hardly soluble in water or mixed with water.
In the present invention, the porous body refers to a structure in which bubbles are present and the bubbles are connected to form an open cell, and is distinguished from a non-porous body. The open cell is distinguished from the closed cell in which the bubbles are not connected to each other. In the continuous bubble, since a small hole is opened in the wall between the bubbles, it has a function of sucking liquid or allowing gas to pass therethrough by a capillary phenomenon.

Here, the non-porous material is a non-porous structure that does not substantially include voids, and is contrary to a porous material including voids provided to promote absorption of liquid such as a membrane. Means a structure.
As the non-porous body, for example, a bubble that has been included in the manufacturing process accidentally and contains a small amount of bubbles that do not contribute to the promotion of the liquid absorption action is the non-porous body. Included in the category. In the present invention, the resin layer is made of a hydrophobic material, but since the capture antibody is solid-phased inside the void, the wall surface of the bubble exhibits hydrophilicity. Thereby, based on the same principle as that of the flow path member described later, the test solution permeates into the resin layer by using the capillary phenomenon, so that it can react with the labeled antibody to show color.

The thickness of the resin layer is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1.0 μm or more in order to obtain sufficient color development. 0 to 20 μm is more preferable, and 1.0 to 10 μm is even more preferable.
The method for impregnating the solution containing the antibody inside the void (antibody coating solution) is not particularly limited and may be appropriately selected according to the purpose. For example, the pressure impregnation method, the vacuum impregnation method, the vacuum pressure impregnation method Etc.

  Conventional determination lines (test lines and control lines) are generally formed by directly applying a solution in which a capture antibody is dissolved to a channel member made of a porous material. Therefore, the capture antibody diffuses into the porous material as the liquid penetrates. However, the color development of labeling particles such as gold colloid particles that bind to the capture antibody present inside the porous material cannot be actually detected because light scattering occurs. That is, most of the capture antibodies are not effectively used. In general, the color developing particles that can be detected with a porous material have a depth of about 5 μm to 10 μm from the surface. In order to immobilize the capture antibody necessary for detection in this region, it is necessary to apply a large amount of the capture antibody in consideration of diffusion in the thickness direction. That is, the amount of the capture antibody applied increases in proportion to the average thickness of the porous material.

  On the other hand, in the inspection apparatus of the present invention, a resin layer as a porous body containing a large amount of hydrophobic groups is used for immobilizing the capture antibody, and a large amount of the capture antibody is solid-phased at a high density. It is disposed on the flow path member. Further, the resin layer is adjusted to a thickness that can detect the color development of the colored particles, and the capture antibody is efficiently used. As a result, there is no extra capture antibody in the thickness direction, and there is an advantage that the amount of the capture antibody applied may be small.

  Here, the inspection apparatus of the present invention will be described with reference to the drawings. 1-3, FIG. 4A and FIG. 4B, and FIG. 5A and FIG. 5B are drawings which show the whole structure of an inspection apparatus. FIG. 1 is a top view showing an example of the inspection apparatus of the present invention. FIG. 2 is a cross-sectional view taken along the line A-A ′ of the inspection apparatus of FIG. 1. FIG. 3 is a cross-sectional view illustrating an example of an inspection device at a facing portion between the flow path member and the resin layer. 4A to 5B are cross-sectional views showing other examples of the inspection device at the facing portions of the flow path member and the resin layer.

  As shown in FIGS. 1 to 5B, the test apparatus 10 is hydrophilic, such as blood, cerebrospinal fluid, urine, or a specimen extract (for example, a liquid containing a specimen collected by a specimen collecting means such as a stick). It has a porous flow path member 12 in which a flow path for flowing the test solution 30 (an example of a specimen) is formed, and resin layers (15a, 15b, 15c) provided on the flow path member 12. Yes. On the side of the resin layer (15a, 15b, 15c) facing the flow path member 12, a labeled antibody 16 that reacts with the antigen contained in the test solution 30, a capture antibody 17 that captures the antigen, and a capture that captures the labeled antibody. Each antibody (or antigen) 18 is immobilized. Thereby, the strength of the interaction between the resin layer (15a, 15b, 15c) and the reagent can be adjusted for each resin layer (15a, 15b, 15c), so that the flow path member 12 is arbitrarily selected according to the purpose. Even in this case, it becomes easier to control the release of the labeled antibody 16 and the immobilization of the capture antibodies 17 and 18.

  In the inspection apparatus 10, a case where the flow path member 12 is provided on the base material 11 and the absorption member 14 is provided on the base material 11 and the flow path member 12 will be described. It is not limited. Providing on the flow path member 12 means providing in contact with the flow path member 12 regardless of the orientation when the inspection apparatus 10 is arranged. Moreover, when showing arbitrary resin layers among resin layers (15a, 15b, 15c), it represents with the resin layer 15. FIG. The capture antibody 18 may be solid-phased by an arbitrary chemical bond such as a covalent bond, a hydrogen bond, or a metal bond, or an arbitrary interaction such as adhesion, adhesion, adsorption, or van der Waals bond.

Hereinafter, the case where the test liquid 30 is a hydrophilic test liquid such as blood, spinal fluid, urine, or a sample extract (for example, a liquid containing a sample collected by a sample collecting means such as a stick) will be described. to continue.
As shown in FIG. 3, the resin layer 15 a (second resin layer) of the inspection apparatus 10 contains an amphiphilic resin 151 having many hydrophilic groups 152.
As content of the hydrophilic group 152, 50 mass% or more is preferable with respect to the resin layer 15a whole quantity.
Here, the hydrophilic group is an atomic group that forms a weak bond such as a hydrogen bond with a water molecule, and means that there is an affinity for water. The amphiphilic means that both water and the organic solvent have affinity.

  The labeled antibody 16 is solid-phased on the surface of the resin layer 15a facing the flow path member 12 by having the hydrophilic portion 16g. On the other hand, when the test solution 30 is filled in the gap formed between the flow path member 12 and the resin layer 15a, the hydrophilic portion 16g of the labeled antibody 16 and the hydrophilic test solution 30 are compatible. The labeled antibody 16 is released from the amphiphilic resin 151. Further, when the test solution 30 contains the antigen 31, the released labeled antibody 16 and the antigen 31 react and bind by the antigen-antibody reaction. In order to prevent the binding between the labeled antibody 16 and the antigen 31 from being inhibited, the amphiphilic resin 151 is preferably a water-insoluble resin. Here, the water-insoluble means that it is substantially insoluble in water. “Substantially insoluble in water” means that the mass change amount of the resin layer when immersed in a large amount of water at 25 ° C. for 24 hours and then sufficiently dried by a method such as vacuum drying is 1% by mass or less. It means that. The reason why the mass of the resin changes is that by-products (such as monomer components) contained in the resin product are dissolved in water and the mass is reduced.

As shown in FIGS. 4A and 4B, the resin layer 15b (first resin layer) is preferably a thermoplastic elastomer having a hydrophobic group 153. Specifically, the resin layer 15b contains a hydrophobic thermoplastic elastomer 155 or an amphiphilic resin 154 having many hydrophobic groups 153, and the hydrophobic thermoplastic elastomer 155 or the amphiphilic resin is contained in the resin layer 15b. It is preferable that 154 is comprised as a main component (content is 50 mass% or more).
Here, the hydrophobic group is an atomic group that is not easily compatible with water, and means that it has a low affinity for water and is hardly soluble in water or mixed with water.

The capture antibody 17 has a hydrophobic portion 17g. When the hydrophobic portion 17g is bonded by intermolecular force, a solid phase is formed on the surface of the resin layer 15b facing the flow path member 12 and inside the void. It becomes. When the test solution 30 is filled in the gap formed between the flow path member 12 and the resin layer 15b, the capture antibody 17 captures the antigen 31 bound to the labeled antibody 16. Thereby, since the antigen 31 and the labeled antibody 16 are fixed and color is developed, the resin layer 15 b can be used as a line for determining the presence or absence of the antigen 31.
The hydrophobic thermoplastic elastomer 155 and the amphiphilic resin 154 are not particularly limited and may be appropriately selected depending on the intended purpose, but each of them is preferably the water-insoluble resin. When the hydrophobic thermoplastic elastomer 155 and the amphiphilic resin 154 are each the water-insoluble resin, it is advantageous in that line bleeding can be prevented.

  As shown in FIGS. 5A and 5B, the resin layer 15c (first resin layer) contains a hydrophobic thermoplastic elastomer 155 or an amphiphilic resin 154 having a large number of hydrophobic groups 153. The thermoplastic elastomer 155 or the amphiphilic resin 154 is preferably used as a main component (content is 50% by mass or more).

  The capture antibody 18 is solid-phased on the surface of the resin layer 15c facing the flow path member 12 and the inside of the gap by binding the hydrophobic site of the capture antibody 18 by intermolecular force. The capture antibody 18 is not particularly limited as long as it can capture the labeled antibody 16 and can be appropriately selected according to the purpose. Examples thereof include an antibody (antigen) that specifically binds to the labeled antibody 16. It is done. Thereby, since the labeled antibody 16 is immobilized and color develops, the resin layer 15c can be used as a control line indicating that the labeled antibody 16 has reached. The hydrophobic thermoplastic elastomer 155 and the amphiphilic resin 154 are not particularly limited and may be appropriately selected depending on the intended purpose, but each of them is preferably the water-insoluble resin. When the hydrophobic thermoplastic elastomer 155 and the amphiphilic resin 154 are each the water-insoluble resin, it is advantageous in that bleeding of the control line can be prevented.

  In the present embodiment, the inspection apparatus 10 for inspecting the presence or absence of the antigen 31 contained in the inspection liquid 30 will be described. However, the inspection apparatus of the present invention is not limited to an apparatus using an antigen-antibody reaction. For example, the inspection apparatus may inspect a specific component contained in the inspection liquid 30 by using a reagent whose hue changes due to a structural change as a reagent.

Hereinafter, each member which comprises the inspection apparatus 10 is demonstrated in detail.
<Base material>
There is no restriction | limiting in particular as the base material 11, It can select according to the objective, For example, the thing made from an organic, inorganic, or metal is mentioned.
There is no restriction | limiting in particular in the base material 11, Although it can select suitably according to the objective, It is preferable that at least 1 surface is covered with the hydrophobic resin.
When the inspection apparatus 10 is used for a sensor chip, it is preferable to use a synthetic resin that is lightweight, flexible and inexpensive.

According to this embodiment, since the base material 11 with high durability, such as a plastic sheet, can be selected, the durability of the inspection apparatus 10 is also improved as a result.
The material of the substrate 11 is not particularly limited and can be appropriately selected according to the purpose. For example, polyvinyl chloride, polyethylene terephthalate, polypropylene, polystyrene, polyvinyl acetate, polycarbonate, polyacetal, modified polyphenyl ether, Examples include polybutylene phthalate and ABS resin. Among these, polyethylene terephthalate is preferable because it is inexpensive and highly versatile.

There is no restriction | limiting in particular as a shape of the base material 11, Although it can select suitably according to the objective, A sheet form is preferable.
There is no restriction | limiting in particular in the average thickness of the base material 11, Although it can select suitably according to the objective, 0.01 mm or more and 0.5 mm or less are preferable. When the average thickness is 0.01 mm or more, the strength as the substrate 11 is good, and when it is 0.5 mm or less, the flexibility of the substrate is good and can be suitably used as a sensor. .
Here, the average thickness means, for example, that the thickness of the measurement object is 5 in the longitudinal direction (length direction), 3 in the width direction, and 15 in total so that the measurement locations are substantially evenly spaced. It can be set as the average value of thickness when measured with a meter (MDH-25M, manufactured by Mitutoyo Corporation). In the present embodiment, the thickness may be the length of the object in the direction perpendicular to the contact surface between the base material 11 and the flow path member 12.

<Flow channel member>
The flow path member 12 of the inspection apparatus 10 is not particularly limited as long as it is a member capable of flowing the inspection liquid 30, and can be appropriately selected according to the purpose. Examples thereof include a porous material. The flow path member 12 made of the porous material has gaps (12a, 12b), and the flow path is formed when the test liquid 30 flows through the gaps (12a, 12b).

3-5B, the space | gap 12a is a space | gap formed in each cross section, and the space | gap 12b is a space | gap of the back | inner side of a cross section. It is preferable that bubbles are present inside the porous material, and the bubbles are connected to form an open cell.
The open cell is distinguished from the closed cell in which the bubbles are not connected to each other. In the continuous bubble, since a small hole is opened in the wall between the bubbles, it has a function of sucking liquid or allowing gas to pass therethrough by a capillary phenomenon. Since the flow path member 12 uses the capillary phenomenon to transfer the test solution 30 in the gaps (12a, 12b), an external driving device such as a pump is not necessary.

  There is no restriction | limiting in particular as said porous material, According to the objective, it can select suitably, For example, the membrane film | membrane etc. which consist of nitrocellulose, hydrophilized PTFE, hydrophilized PVDF, nylon, vinylon etc. are mentioned.

There is no restriction | limiting in particular about the shape of the said porous material, Although it can select suitably according to the objective, A sheet form is preferable.
There is no restriction | limiting in particular in the average thickness of the said porous material, Although it can select suitably according to the objective, 0.01 mm or more and 0.3 mm or less are preferable. When the average thickness is 0.01 mm or more, it is advantageous in that the strength of the substrate is good. Moreover, when the average thickness is 0.3 mm or less, it is advantageous in that the required amount of the test solution can be optimized.

<Resin layer>
The function of the resin layer 15 in the present invention will be described in comparison with the conventional inspection apparatus shown in FIGS.
FIG. 6 is a conceptual diagram of a conjugate pad in a conventional inspection apparatus. FIG. 7 is a conceptual diagram of a membrane in a conventional inspection apparatus.

In the conventional inspection apparatus, if the hydrophilicity of the conjugate pad is too high, the inspection liquid tends to remain on the conjugate pad, and it is difficult to transfer to the membrane. Conversely, if the hydrophobicity of the conjugate pad is too high, the transfer of the test solution to the membrane will be faster, but the water absorption of the test solution from the sample pad will drop, so the test time will be longer or a large amount of test solution will be used. It was supposed to take. For this reason, the fiber F1 which can be used as a conjugate pad was limited. Furthermore, in the conventional inspection apparatus, since the labeled antibody 16 is solid-phased on the fiber F1 constituting the conjugate pad (see FIG. 6), as the labeled antibody 16 that can be released from the conjugate pad, It will be limited to the thing with a weak bond strength with the fiber F1. That is, the conventional inspection apparatus has a limited number of usable fibers F1 and labeled antibodies 16 in design.
In the conventional inspection apparatus, since the capture antibody 17 is solid-phased on the fiber F2 constituting the membrane (see FIG. 7), the capture antibody 17 that can be immobilized on the membrane is the fiber F2. It will be limited to those with strong binding power. That is, the conventional inspection apparatus has a limited number of usable fibers F2 and capture antibodies 17 in design.

  In the inspection apparatus 10, reagents such as a labeled antibody 16, a capture antibody 17, and a capture antibody 18 are immobilized on a resin layer 15 (15a, 15b, 15c). Therefore, the release of the labeled antibody 16 or the immobilization of the capture antibody 17 can be controlled according to the strength of the interaction between the resin layer 15 and the capture antibody 17 and the affinity with the test solution 30.

  Examples of a method for adjusting the strength of interaction between the resin layer 15 and the capture antibody 17 and the affinity with the test solution 30 include, for example, the type of resin or thermoplastic elastomer constituting the resin layer 15 and the resin composition ratio. Examples of the method include a method of changing according to the corresponding capture antibody 17. The greater the proportion of hydrophobicity in the resin or thermoplastic elastomer constituting the resin layer 15, the easier it is for the resin layer 15 to immobilize the capture antibody 17 having a hydrophobic group by hydrophobic interaction.

Here, the hydrophobic interaction means a cause (driving force) of a change in which a hydrophobic molecule or a hydrophobic group that does not adjust to water in water gathers. Specifically, when a hydrophobic molecule or a molecule having a hydrophobic group is put in water, in many cases, it does not simply dissolve, but the hydrophobic molecule or hydrophobic group is in contact with each other and contacts with the water molecule. Try to reduce the area as much as possible. As a result, hydrophobic molecules come to gather together, and this is a phenomenon that appears to have a binding force acting between the molecules.
If the ratio of hydrophilicity in the resin or thermoplastic elastomer constituting the resin layer 15 is large, the interaction between the resin layer 15 and the hydrophilic capture antibody becomes strong, but the bonded portion is in contact with the hydrophilic test solution 30. Then, it is estimated that the reagent is likely to be released into the test solution 30 in affinity with the test solution 30.

  The resin or thermoplastic elastomer constituting the resin layer 15 is preferably a water-insoluble resin. When the resin constituting the resin layer 15 is the water-insoluble resin, it is possible to prevent the resin from being dissolved in the test solution 30 and clogging the flow path or bleeding the control line or the test line.

There is no restriction | limiting in particular as amphiphilic resin which comprises the resin layer 15a, According to the objective, it can select suitably, For example, polyvinyl alcohol resin, polyvinyl acetal resin, polyacrylic acid, acrylic acid-acrylonitrile copolymer weight Polymer, vinyl acetate-acrylic acid ester copolymer, acrylic acid-acrylic acid ester copolymer, styrene-acrylic acid copolymer, styrene-methacrylic acid copolymer, styrene-methacrylic acid-acrylic acid ester copolymer, Styrene-α-methylstyrene-acrylic acid copolymer, styrene-α-methylstyrene-acrylic acid-acrylic acid ester copolymer, styrene-maleic acid copolymer, styrene-maleic anhydride copolymer, vinylnaphthalene- Acrylic acid copolymer, vinyl naphthalene-maleic acid copolymer, acetic acid Examples thereof include vinyl-maleic acid ester copolymers, vinyl acetate-crotonic acid copolymers, vinyl acetate-acrylic acid copolymers, and salts thereof. These may be used alone or in combination of two or more.
Among these, a copolymer of a monomer having a hydrophobic functional group and a monomer having a hydrophilic functional group, and a polymer composed of a monomer having both a hydrophobic functional group and a hydrophilic functional group are preferable. As the form of the copolymer, any form of a random copolymer, a block copolymer, an alternating copolymer, and a graft copolymer can be used.

The hydrophobic thermoplastic elastomer constituting the resin layer 15b and the resin layer 15c is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include butadiene rubber, styrene-butadiene rubber, and high styrene rubber. And synthetic rubber such as isoprene rubber, acrylic rubber, epichlorohydrin rubber, butyl rubber ethylene-propylene rubber, and natural rubber.
Moreover, since at least one of the hydrophobic thermoplastic elastomers is a thermoplastic elastomer having a styrene skeleton, a void in a cross section can be obtained more easily. This is presumably because the styrene skeleton contains a benzene ring, so that aggregation of waxes is less likely to occur.

  Examples of the wax constituting the resin layer 15b and the resin layer 15c include natural waxes such as beeswax, carnauba wax, whale wax, wood wax, candelilla wax, rice bran wax, and montan wax; paraffin wax, microcrystalline wax, and oxidation Examples thereof include synthetic waxes such as wax, ozokerite, ceresin, ester wax, polyethylene wax, and oxidized polyethylene wax. These may be used alone or in combination of two or more.

  As the resin constituting each resin layer (15a, 15b, 15c), it is also possible to use the same kind of resin. In this case, it is preferable that the resin constituting the resin layer 15a is more hydrophilic than the resin constituting the resin layers (15b, 15c). In addition, when using the same kind of resin, it is not necessary to measure the hydrophilicity, and it can be said that the higher the ratio of hydrophilic groups, the higher the hydrophilicity.

The labeled antibody 16 to be immobilized on the resin layer 15a is not particularly limited as long as it has a hydrophilic site and reacts with the antigen 31, and can be appropriately selected according to the purpose. And antibodies labeled with colloidal gold particles, particles that label other antibodies, and the like.
The particles for labeling the other antibodies are not particularly limited other than gold colloid, and can be appropriately selected according to the purpose. For example, metal colloid other than gold colloid, enzyme-labeled particles containing an enzyme, dye Examples thereof include colored particles containing a fluorescent substance, fluorescent particles containing a fluorescent substance, and magnetic substance-containing particles containing a magnetic substance. These may be used alone or in combination of two or more.
The antibody may be in any form of monoclonal antibody, polyclonal antibody, chimeric antibody, Fab antibody, and (Fab) ₂ antibody.

The capture antibody 17 to be immobilized on the resin layer 15b is not particularly limited as long as it has a hydrophobic site and reacts with the antigen 31, and can be appropriately selected according to the purpose. And IgG antibodies.
The antibody may be in any form of monoclonal antibody, polyclonal antibody, chimeric antibody, Fab antibody, and (Fab) ₂ antibody.

  The capture antibody 18 to be immobilized on the resin layer 15c is not particularly limited as long as it has a hydrophobic group and reacts with the labeled antibody 16, and can be appropriately selected according to the purpose. Examples thereof include an antibody against labeled antibody 16 such as IgG, and the antibodies listed above. Moreover, the antigen itself which reacts with the labeled antibody 16 may be sufficient.

  The method for immobilizing the reagent such as the labeled antibody 16 and the capture antibody (17, 18) on the resin layer 15 is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a solution containing the reagent After the resin layer 15 is applied / impregnated, it is dried quickly by drying, and after the solution containing the reagent is applied / impregnated to the resin layer 15, it is left in a humid environment (incubation) so that the coating solution does not dry. Then, a method of washing components other than antibodies such as inorganic salts with distilled water and the like, followed by drying, and the like can be mentioned.

  In particular, as a means for solidifying the reagent such as the capture antibody (17, 18) in the voids of the resin layers (15b, 15c), for example, pressure impregnation method, vacuum impregnation method, vacuum pressure impregnation Law. Since the resin layer is made of a hydrophobic material, and the wall surface inside the gap shows hydrophobicity, even if the antibody coating solution is applied as it is, it hardly penetrates into the gap. In addition, the gas remaining inside the gap may interfere with the penetration of the coating solution. Therefore, by applying an antibody coating solution to the resin layer or impregnating the resin layer in the antibody coating solution and then applying pressure, the solution is pushed into the gap and the inside of the gap is filled with the antibody coating solution. It becomes possible. After impregnating the resin layer with the solution containing the reagent by the above-described method, it can be performed by a dry-up method or incubation and solidification.

The resin layer 15 is preferably fixed on the flow path member 12.
The method for fixing the resin layer 15 on the flow path member 12 is not particularly limited as long as it is a method in which the reagent and the test liquid 30 are fixed in a state where they can be contacted at the time of inspection. For example, a method of thermally transferring the resin constituting the resin layer onto the flow path member 12 using a thermal transfer printer or the like, and transferring by applying pressure to the resin constituting the resin layer using a dot impact printer or the like. And a method of attaching a resin constituting the resin layer onto the flow path member 12 with a tape, an adhesive, an adhesive, or the like.

<Absorbing member>
The absorbing member is not particularly limited as long as it absorbs water, and can be appropriately selected from known materials.
Examples of the absorbent member include fibers such as paper and cloth, a polymer compound having a carboxyl group or a salt thereof, a partially crosslinked body of a polymer compound having a carboxyl group or a salt thereof, and a partially crosslinked body of a polysaccharide. .

<Other members>
There is no restriction | limiting in particular as said other member, According to the objective, it can select suitably, For example, a protection member, a labeled antibody holding pad, a sample dripping pad etc. are mentioned.

The protective member is a member for preventing contamination when a hand touches the flow path member.
There is no restriction | limiting in particular as said protective member, According to the objective, it can select suitably, For example, the housing etc. which cover the whole inspection apparatus, the film provided on a flow-path member, etc. are mentioned.
When the protective member is provided, it is preferable that an opening is provided in the upper portion of the dropping portion of the flow path member. Moreover, it is preferable that the protective member is provided with an opening for releasing the pressure in the flow path.
As described above, the resin layer can be provided on the flow path member by various methods. As an example, a case where a thermal transfer method is used will be described. Hereinafter, an inspection apparatus transfer medium used in the thermal transfer method and a method for manufacturing the inspection apparatus will be described.

(Inspection device manufacturing method)
The method for producing an inspection apparatus of the present invention comprises contacting the reagent-immobilized layer (or release layer and reagent-immobilized layer) of the transfer medium for an inspection apparatus of the present invention with a porous flow path member, Including a step of transferring the reagent-immobilized layer (or release layer / reagent-immobilized layer) to the flow path member (hereinafter also referred to as “reagent-immobilized layer transfer step”), and further necessary Depending on the process, other steps are included.

<Transfer process of reagent solid phase layer>
As a method for thermally transferring the reagent-immobilized layer (or release layer / reagent-immobilized layer) to the flow path member, the reagent-immobilized layer (or release layer / reagent-immobilized layer) of the transfer medium for the inspection apparatus can be used. And a method of transferring the reagent-immobilized layer (or release layer / reagent-immobilized layer) to the channel member by bringing it into contact with the channel member.

There is no restriction | limiting in particular as a printer used for the said thermal transfer, According to the objective, it can select suitably, For example, the thermal printer etc. which have a serial thermal head, a line-type thermal head, etc. are mentioned.
The energy applied in the thermal transfer is not particularly limited and can be appropriately selected according to the purpose, but is preferably 0.05 mJ / dot or more and 0.5 mJ / dot or less. When the applied energy is 0.05 mJ / dot or more, the reagent-immobilized layer or the release layer / reagent-immobilized layer can be efficiently melted. Further, when the applied energy is 0.5 mJ / dot or less, thermal denaturation of the reagent can be prevented, and dissolution of the support and thermal head are not contaminated.

-Application of inspection device-
The use of the test apparatus is not particularly limited and can be appropriately selected according to the purpose. For example, a biochemical sensor (sensing chip) for blood test or DNA test, small size for food or beverage quality control, etc. Analytical instruments (chemical sensors).

  The sample (specimen) used in the biochemical field test is not particularly limited and can be appropriately selected depending on the purpose. For example, pathogens such as bacteria and viruses, blood separated from living bodies, saliva, tissues Examples thereof include excrement such as diseased stool and manure. Furthermore, when performing prenatal diagnosis, fetal cells present in amniotic fluid, a part of dividing egg cells in a test tube, or the like may be used. In addition, these samples are concentrated as a sediment directly or as necessary by centrifugation or the like, and then, for example, cell destruction treatment by enzyme treatment, heat treatment, surfactant treatment, ultrasonic treatment, or a combination thereof. May be given in advance.

The inspection apparatus of the present invention also has a function of chromatography (separation and purification) of the inspection liquid because the flow path member functions as a stationary phase. In this case, the flow path member having open cells whose inner wall is hydrophilic is the stationary phase (carrier). Each component in the test solution undergoes a difference in the speed of flowing in the flow path due to a difference in interaction with the stationary phase, that is, a difference in hydrophilicity / hydrophobicity in the process of permeating the flow path.
This is because the higher the hydrophilic component, the easier it is to adsorb to the porous part, which is the stationary phase, and the greater the number of repeated desorption, the slower the rate of permeation through the flow path. On the other hand, a highly hydrophobic component penetrates without adsorbing to the stationary phase, and thus moves quickly in the flow path. By selectively extracting and reacting the target component of the test liquid using the difference in moving speed in the test liquid, the test apparatus can be used as a highly functional sensor for chemical or biochemical applications.

<Inspection method>
The inspection method according to the present invention includes a step of supplying a specimen to the flow path member of the inspection apparatus of the present invention,
And a step of releasing the reagent immobilized on the resin layer from the resin layer by bringing the reagent into contact with the specimen, wherein a part of the specimen is obtained by the reagent immobilized on the resin layer. It is preferable to include a step of capturing, and further include other steps as necessary.

  As a specific process, first, the hydrophilic test solution 30 is dropped and supplied to the dropping unit 12c (see FIG. 1) provided in the flow path member 12 of the test apparatus 10. Next, the supplied test solution 30 is brought into contact with the labeled antibody 16 immobilized on the resin layer 15a to release the labeled antibody 16 from the resin layer 15a. When the test solution 30 contains the antigen 31, the labeled antibody 16 released from the resin layer 15a reacts with and binds to the antigen 31 (see FIG. 3).

  Next, the test solution 30 containing the labeled antibody 16 and the antigen 31 is developed along the flow path member 12 and reaches the region where the resin layer 15b is disposed. The capture antibody 17 solid-phased on the surface of the resin layer 15b facing the flow path member 12 also binds to and captures the antigen 31 to which the labeled antibody 16 is bound. Since the capture antibody 17 is solid-phased on the resin layer 15b by the hydrophobic group 17g, even if it contacts the test solution 30, it does not have affinity with the test solution 30 and is not easily released. Even if a part of the capture antibody 17 is released into the test solution 30, it immediately binds to the fibers constituting the flow path member 12. As a result, the labeled antibody 16 is immobilized in the vicinity of the resin layer 15b, so that the test line is clearly colored (see FIGS. 4A and 4B).

The labeled antibody 16 that has passed without being captured in the resin layer 15b is developed along the flow path member 12 and reaches the region where the resin layer 15c is disposed. In the present embodiment, a capture antibody 18 having a hydrophobic group is immobilized on the surface of the resin layer 15c facing the flow path member 12. The labeled antibody 16 is captured by binding to the capture antibody 18.
Since the capture antibody 18 is solid-phased on the resin layer 15 c by a hydrophobic group, even if it contacts the test solution 30, it does not have an affinity for the test solution 30 and is not easily released. Even if a part of the capture antibody 18 is released into the test solution 30, it immediately binds to the fibers constituting the flow path member 12. As a result, the labeled antibody 16 is immobilized in the vicinity of the resin layer 15c, so that the control line is clearly colored (see FIGS. 5A and 5B).

(Inspection kit)
The test kit of the present invention comprises the test apparatus of the present invention, a sample collection means for collecting a sample, and at least one selected from a liquid for processing the sample, and further if necessary It has other members.

As shown in FIG. 9, the test kit includes the test apparatus 10 of the present invention, an instrument for collecting a sample (an example of a sample collecting unit), and at least one of liquids for processing the sample. Have.
Examples of the instrument for collecting the specimen include a sterilized cotton swab 51 for collecting the specimen from the pharynx or nasal cavity.
Examples of the liquid for processing the specimen include a diluting liquid 52 for diluting the specimen and an extracting liquid for extracting the specimen.
Examples of the other members include an instruction manual.

In the above embodiment, the case where the reagent immobilized on the resin layer 15 is an antigen or an antibody has been described, but the present invention is not limited to this embodiment. For example, it can be applied to an inspection apparatus using an indicator used in a chemical assay.
Here, the indicator used in the chemical assay refers to a reagent that indicates the chemical properties of the solution, and is not particularly limited and can be appropriately selected according to the purpose. For example, a pH indicator, lead ion, copper ion And various ionophores that change color by reacting with various ions such as nitrite ion, and reagents that change color by reacting with various agricultural chemicals.

In the above-described embodiment, an example has been described in which the support 101 and the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 in the transfer medium 100 for inspection apparatus are peeled off by heat during transfer. The invention is not limited to this embodiment. For example, the support 101 and the reagent-immobilized layer 103 or the separation layer / reagent-immobilized layer 105 may be separated by light. In this case, the release layer 102 or the release layer / reagent-immobilized layer 105 is mixed with a light absorber such as carbon black, and the light is absorbed thereby to generate heat, whereby the release layer 102 or the release layer. Alternatively, the reagent-immobilized layer 105 may be melted and the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 may be separated. Alternatively, the release layer 102 or the release layer / reagent-immobilized layer 105 is mixed with a material that is altered by light irradiation, and the release layer 102 is made brittle by absorbing light, whereby the reagent-immobilized layer 103 or The peeling layer / reagent solid phase layer 105 may be peeled off.
As a transfer method other than the thermal transfer, for example, a sheet comprising the reagent-immobilized layer 103 in which the reagent is immobilized or the release layer / reagent-immobilized layer 105 is stuck on the flow path member 12 with a tape or the like. The method of attaching is mentioned.

  In the said embodiment, although the flow path was formed in the whole flow path member 12, the present invention is not limited to this. As a method of forming a flow path in a part of the flow path member 12, for example, a flow wall serving as an outer edge of the flow path is obtained by filling the gap of the flow path member 12 with a hydrophobic material by a known method. The method of forming is mentioned.

  In the above embodiment, an example in which the resin layer 15 is provided at a plurality of locations on the flow path member 12 is shown. However, depending on the type of reagent, the resin layer 15 is provided at one location on the flow path member 12. May be. For example, the flow path member 12 provided with the resin layer 15a1 in which the reagent that specifically binds to the component A in the test liquid 30 is solid-phased, and the resin layers 15b1 and 15c1 in which the reagent for capturing them is solid-phased. Furthermore, when the resin layer 15a2 in which the reagent that specifically binds to the component B in the test solution is solid-phased and the resin layers 15b2 and 15c2 in which the reagent for capturing them is solid-phased are provided, An inspection apparatus capable of detecting multiple components at the same time can be obtained.

  In the embodiment, the case where the test liquid 30 is hydrophilic has been described, but the test liquid is not limited to hydrophilic. The test solution 30 is a solvophilic one containing an organic solvent such as alcohols such as methyl alcohol, ethyl alcohol, 1-propyl alcohol and 2-propyl alcohol, and ketones such as acetone and MEK (methyl ethyl ketone). There may be. In this case, “hydrophilicity” in the above embodiment is replaced with “hydrophobic”, and “hydrophobic” is replaced with “hydrophilic”.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Preparation Example 1)
-Preparation of back layer coating solution-
16.8 parts by mass of a silicone rubber emulsion (solid content 30% by mass), 0.2 parts by mass of chloroplatinic acid catalyst, and 83 parts by mass of toluene were mixed to prepare a back layer coating solution of Preparation Example 1.

(Preparation Example 2-1)
-Preparation of coating solution for release layer and reagent-immobilized layer (for fixation)-
A coating solution composed of 96 parts by weight of carnauba wax, 4 parts by weight of styrene-butadiene rubber, and a solvent of toluene / methyl ethyl ketone (volume ratio 7/3) was mixed, and the release layer and reagent-immobilized layer (fixed) of Preparation Example 2-1 A coating solution was prepared.

(Preparation Example 2-2)
-Preparation of coating solution for release layer and reagent-immobilized layer (for fixation)-
A coating solution composed of 92 parts by weight of carnauba wax, 8 parts by weight of styrene-butadiene rubber, and a solvent of toluene / methyl ethyl ketone (volume ratio 7/3) was mixed, and the release layer / reagent-immobilized layer (fixed) of Preparation Example 2-2 A coating solution was prepared.

(Preparation Example 2-3)
-Preparation of coating solution for release layer and reagent-immobilized layer (for fixation)-
A coating solution composed of 88 parts by mass of carnauba wax, 12 parts by mass of styrene-butadiene rubber, and a solvent of toluene / methyl ethyl ketone (volume ratio 7/3) was mixed, and the release layer / reagent-immobilized layer (fixed) of Preparation Example 2-3 A coating solution was prepared.

(Preparation Example 2-4)
-Preparation of coating solution for release layer and reagent-immobilized layer (for fixation)-
A coating solution composed of 84 parts by weight of carnauba wax, 16 parts by weight of styrene-butadiene rubber and a solvent of toluene / methyl ethyl ketone (volume ratio 7/3) was mixed, and the release layer / reagent-immobilized layer (fixed) of Preparation Example 2-4 A coating solution was prepared.

(Preparation Example 2-5)
-Preparation of coating solution for release layer and reagent-immobilized layer (for fixation)-
A coating solution composed of 75 parts by weight of carnauba wax, 25 parts by weight of styrene-butadiene rubber and a solvent of toluene / methyl ethyl ketone (volume ratio 7/3) was mixed, and the release layer / reagent-immobilized layer (fixed) of Preparation Example 2-5 A coating solution was prepared.

(Preparation Example 2-6)
-Preparation of coating solution for release layer and reagent-immobilized layer (for fixation)-
A coating solution composed of 65 parts by mass of carnauba wax, 35 parts by mass of styrene-butadiene rubber, and a solvent of toluene / methyl ethyl ketone (volume ratio 7/3) was mixed, and the release layer / reagent-immobilized layer (fixed) in Preparation Example 2-6 A coating solution was prepared.

(Preparation Example 2-7)
-Preparation of coating solution for release layer and reagent-immobilized layer (for fixation)-
A coating solution composed of 88 parts by mass of carnauba wax, 12 parts by mass of butadiene rubber, and a solvent of toluene / methyl ethyl ketone (volume ratio 7/3) was mixed, and the release layer / reagent solid phase layer of Preparation Example 2-7 (for immobilization) A coating solution was prepared.

(Preparation Example 2-8)
-Preparation of coating solution for release layer and reagent-immobilized layer (for fixation)-
A coating solution consisting of 88 parts by weight of carnauba wax, 12 parts by weight of acrylonitrile butadiene rubber, and a solvent of toluene / methyl ethyl ketone (volume ratio 7/3) was mixed, and the release layer / reagent-immobilized layer of Preparation Example 2-8 (for immobilization) ) A coating solution was prepared.

(Preparation Example 2-9)
―Preparation of coating solution for release layer and reagent-immobilized layer (for fixation) ―
Carnauba wax 90 parts by mass, ethylene-vinyl acetate copolymer 1 part by mass, styrene-butadiene rubber 4 parts by mass, butadiene rubber 4 parts by mass, acrylonitrile-butadiene rubber 1 part by mass, and toluene / methyl ethyl ketone (volume ratio 7/3) The solvent was mixed to prepare a peeling layer / reagent-immobilized layer (for immobilization) coating solution of Preparation Example 2-9.

Next, Table 1 summarizes the mixing ratio of the materials used in each coating solution of Preparation Example 2-1-2-9.
* In addition, in Preparation Example 2-9 in Table 1, description of 1 part by mass of ethylene-vinyl acetate copolymer is omitted.
The details of the wax and thermoplastic elastomer in Table 1 are as follows.
Carnauba wax: melting point 85 ° C
・ Styrene-butadiene rubber: Styrene ratio 40%, butadiene ratio 60%; Hydrophobic ・ Butadiene rubber: Butadiene ratio 100%, cis 1,4 bond 95%; Hydrophobic ・ Acrylonitrile-butadiene rubber; Hydrophilic

(Preparation Example 3)
-Preparation of reagent-immobilized layer (for release) coating solution-
5 parts by mass of polyvinyl butyral resin (Sekisui Chemical Co., Ltd., BL-1, butyralization degree 64 mol%) and 95 parts by mass of ethanol are mixed, and the reagent solid phase layer (for release) coating solution of Preparation Example 3 is mixed. Prepared.

(Preparation Example 4)
-Preparation of reagent coating solution for test line-
Dulbecco's phosphate buffered saline (Ca, Mg-free, D-PBS (-), Nacalai Tesque, 14249-95) is added to Anti-Rabbit IgG antibody (Sigma Aldrich, R5506) as a diluent. The concentration of the antibody was 1,000 μg / mL, and the test line reagent coating solution of Preparation Example 4 was prepared.

(Preparation Example 5)
-Preparation of reagent coating solution for control line-
The D-PBS (−) was added as a diluent to Rabbit IgG (Sigma Aldrich, I5006) to give 1,000 μg / mL, and a control line reagent coating solution of Preparation Example 5 was prepared.

(Preparation Example 6)
-Preparation of reagent coating solution for labeled antibody-
Gold colloid-labeled Anti-Rabbit IgG antibody (manufactured by BioAssay Works, Gold, average particle size 40 nm, optical density (OD) = 15) was used as the reagent coating solution for labeled antibody of Preparation Example 6.

Example 1
<Preparation of thermal transfer media for test lines>
-Formation of back layer-
The back layer coating solution of Preparation Example 1 was applied to one side of a polyethylene terephthalate (PET) film (Toray Co., Ltd., Lumirror F57) having an average thickness of 4.5 μm as a support, and dried at 80 ° C. for 10 seconds. A back layer having an average thickness of 0.02 μm was formed.

-Formation of release layer and reagent-immobilized layer (for immobilization)-
Next, the release layer / reagent-immobilized layer (fixing) coating solution of Preparation Example 2-2 was applied to the surface of the PET film opposite to the surface on which the back layer was formed, and the coating solution was 10 at 30 ° C. It was dried for 5 minutes to form a release layer / reagent solid phase layer having an average thickness of 5.4 μm.

-Production of thermal transfer media for test lines-
A PET film laminated with a back layer and a release layer / reagent solid-phased layer was cut into 1 cm × 3 cm strips. This was placed in the center of a glass petri dish having an inner diameter of 5 cm and a depth of 1 cm so that the PET film surface side was in contact with the petri dish. Further, a cubic SUS block having a side of 1 cm was placed on both ends of the film, and the film was pressed from above.
Next, a test line reagent coating solution was gently poured into the petri dish so that the height of the solution was 1.5 mm. This petri dish was put in a pressure vessel (TM5SRV, manufactured by Unicontrols Corporation), and pressurized with a compressor (model number DP-40C-AC100V, manufactured by VACUTRONICS) until the gauge pressure of the pressure vessel reached 4.5 MPa. The mixture was allowed to stand for 10 minutes while maintaining the pressurized state, and then returned to atmospheric pressure to immobilize the reagent.
After the solid phase is completed, the solid phase film is taken out from the petri dish, and the solid phase surface is faced (table) on a shaker (Shake-XR equipped with WR-3636, both manufactured by Taitec Co., Ltd.). After pouring distilled water on the solid-phased surface to 100 μL per unit area (cm ² ), gently shake for 1 minute at 25 ° C. with a shaking speed of 20 r / min. The coating solution was thoroughly cut and washed.
After washing, the solid-phased film was left as it was for 15 minutes in a desiccator with a temperature of 25 ° C. and a relative humidity of 20% to immobilize the reagent on the release layer / reagent solid-phase layer (for immobilization). . Thus, a thermal transfer medium for test line of Example 1 was obtained.

-Production of thermal transfer media for control lines-
In the production of the test line thermal transfer medium, except that the test line reagent coating solution of Preparation Example 4 was changed to the control line reagent coating solution of Preparation Example 5, The thermal transfer medium for control line of Example 1 was obtained.

<Preparation of thermal transfer medium for labeled antibody>
A back layer was formed on one side of a polyethylene terephthalate (PET) film (Lumirror F57, manufactured by Toray Industries, Inc.) having an average thickness of 4.5 μm as a support in the same manner as the production of the thermal transfer medium for test lines. Next, the release layer / reagent-immobilized layer (for immobilization) coating solution of Preparation Example 2-2 was applied, dried at 40 ° C. for 10 minutes, and the release layer / reagent-immobilized layer having an average thickness of 1.8 μm. Formed. Furthermore, the reagent-immobilized layer (for release) coating solution of Preparation Example 3 was applied on the release layer / reagent-immobilized layer (for immobilization), dried at 30 ° C. for 10 minutes, and an average thickness of 5 μm. A reagent-immobilized layer (for release) was formed.
Further, on the reagent-immobilized layer (for release), the labeled antibody reagent coating solution of Preparation Example 6 was applied at 25 μL / cm ² and dried in a vacuum dryer at 25 ° C. for 5 hours. The reagent was immobilized on the reagent-immobilized layer (for release). Thus, a thermal transfer medium for labeled antibody of Example 1 was obtained.

<Production of inspection device>
The inspection apparatus shown in FIGS. 10A and 10B was produced as follows. FIG. 10A is a top view illustrating an example of the inspection apparatus used in the example. 10B is a cross-sectional view taken along the line BB ′ of FIG. 10A.

-Fabrication of paper substrate-
A polyester hot melt adhesive (Aron Melt PES375S40, manufactured by Toa Gosei Co., Ltd.) is used as a thermoplastic resin on top of a PET film (Lumilar S10, manufactured by Toray Industries, Inc., average thickness 50 μm) 11 cut to a width of 40 mm × length of 80 mm. Using an roll coater, the film was heated to 190 ° C. and coated to form an adhesive layer so that the average thickness was 50 μm on the PET film.
A nitrocellulose membrane (made by Merck Millipore, HF180, 70% porosity) cut after leaving the PET film 11 with the adhesive layer formed thereon for 2 hours or longer and then cutting the adhesive layer surface into a width of 40 mm and a length of 70 mm. Are aligned so that one end on the long axis side of the adhesive layer surface and one end on the long axis side of various members (this end is the upstream end and the opposite side is the downstream end), and a temperature of 150 ° C. for 10 seconds, A load of 1 kgf / cm ² was applied. Finally, the paper substrate 12 was obtained by cutting along the long axis direction so as to be 4 mm wide × 80 mm long.

Here, the porosity of the paper substrate 12 was obtained from the basis weight (g / m ² ), average thickness (μm), and composition specific gravity of the paper substrate 12 according to the following calculation formula 1. The porosity was 70%. When the porosity of the paper substrate is 40% or more and 90% or less, it can be said that the paper substrate is porous.
[Calculation Formula 1]
Porosity (%) = {1− [basis weight (g / m ² ) / average thickness (μm) / specific gravity of composition]} × 100

-Transcription of labeled antibody-
After the paper substrate 12 and the side of the labeled antibody thermal transfer medium on which the reagent is solid-phased are faced and overlapped, the paper substrate 12 is used as shown in FIGS. 10A to 10B by using a thermal transfer printer. The labeled antibody thermal transfer medium was transferred in a pattern of 4 mm width × 10 mm length (resin layer 15 a) at a position 20 mm away from the upstream end.
The thermal transfer printer has a thermal head with a dot density of 300 dpi (manufactured by TDK Corporation), and has built an evaluation system with a printing speed of 8.7 mm / sec and an applied energy of 0.28 mJ / dot.

-Transfer of test line and control line-
As shown in FIGS. 10A to 10B, first, the test line thermal transfer medium was transferred in a line shape of 4 mm length × 0.8 mm width to a position 15 mm away from the transfer position of the labeled antibody thermal transfer medium. . Next, the control line thermal transfer medium was transferred in a line shape having a length of 4 mm and a width of 0.8 mm at a position 5 mm away from the transfer position of the test line thermal transfer medium. Each line was formed under the same printing conditions as the transfer of the labeled antibody.

-Production of absorbent member-
As shown in FIGS. 10A to 10B, the immunochromatographic assay (inspection apparatus 10) of Example 1 was obtained by providing the absorbing member 14 (Surewick C248, manufactured by Merck Millipore).

  Next, the porosity of the release layer / reagent-immobilized layer before solidifying the reagent, the average pore size of the release layer / reagent-immobilized layer before immobilizing the reagent, and before immobilizing the reagent The average thickness of the release layer and reagent-immobilized layer and the contact angle of the thermoplastic elastomer were measured by the following methods. The results are shown in Table 2-1 and Table 2-2.

<Porosity>
First, using a scanning electron microscope (SEM), the cross-section of the release layer and reagent-immobilized layer before immobilizing the reagent was enlarged at a magnification of 5,000 and recorded as a digital image. Next, using the image processing software (Image J), the digital image is converted into a monochrome image, and the laying area is extracted so that the void area is extracted from the cross section of the release layer / reagent-immobilized layer before immobilizing the reagent. And binarized image processing was performed. Finally, the area of the void area and the area of the cross-section of the material in an arbitrary area were calculated and obtained by the following formula 2.
[Formula 2]
Porosity (%) = (Area of void part) / (Area of entire release layer / reagent-immobilized layer) × 100

<Average pore diameter>
The average pore size of the release layer and reagent-immobilized layer before solidifying the reagent is extracted 10 arbitrary voids from the digital image of the cross-section of the release layer and reagent-immobilized layer obtained by calculating the porosity Calculated as an average value of pore diameters.

<Average thickness>
The average thickness of the release layer and reagent-immobilized layer before immobilizing the reagent is 5 in the longitudinal direction (length direction), 3 in the width direction, and approximately the same as the measurement location. It calculated as an average value of the thickness when measuring a total of 15 places with a micrometer (MDH-25M, Mitutoyo Corporation) so that it may become the interval of.

<Contact angle evaluation>
Coating film surface obtained by smoothly applying a toluene solution of the thermoplastic elastomer used in the peeling layer / reagent solid phase coating solution of Preparation Example 2-2 to a PET film with a bar coater so that the average thickness is 5.4 μm. Then, 4.0 μL of distilled water was dropped and the contact angle was measured with a portable contact angle meter (PG-X, manufactured by Fibro System), and the hydrophilicity / hydrophobicity was evaluated according to the following criteria. The results are shown in Table 2.
[Evaluation criteria]
"Hydrophilic": Contact angle with distilled water is less than 75 degrees "Hydrophobic": Contact angle with distilled water is 75 degrees or more

<Line evaluation>
-Preparation of test solution-
As a developing solution, a D-PBS (−) solution of 0.3 mass% Tween 20 (manufactured by Sigma Aldrich, P9416-50ML) was prepared.
Next, the developing solution was added to Rabbit IgG to obtain a test solution prepared at a concentration of 500 ng / mL.

-Reaction-
100 μL of the test solution was dropped onto the upstream end of the immunochromatographic assay shown in FIGS. 10A to 10B, and the state of development was observed.

-Measurement of line color density-
After completion of the reaction, the immunochromatographic assay was placed in a housing case for measurement, the reading was measured using an immunochromatographic reader (C10066, manufactured by Hamamatsu Photonics), and the color density of the line was evaluated according to the following criteria. The results are shown in Table 1. The larger the reading value, the higher the color density of the line, which is preferable.
[Evaluation criteria]
○: Reading is 300 or more Δ: Reading is 200 or more and less than 300 ×: Reading is less than 200 or cannot be measured as a line and cannot be measured

(Example 2)
In Example 1, in the step of forming the release layer / reagent-immobilized layer (for immobilization) in <Preparation of test line thermal transfer medium>, the exfoliation layer / reagent-immobilized layer (for immobilization) of Preparation Example 2-2 An immunochromatographic assay was prepared in the same manner as in Example 1 except that the coating solution was changed to the peeling layer / reagent-immobilized layer (fixing) coating solution in Preparation Example 2-3, and the same evaluation as in Example 1 was performed. Went. The results are shown in Table 2-1 and Table 2-2.

(Example 3)
In Example 1, in the step of forming the release layer / reagent-immobilized layer (for immobilization) in <Preparation of test line thermal transfer medium>, the exfoliation layer / reagent-immobilized layer (for immobilization) of Preparation Example 2-2 An immunochromatographic assay was prepared in the same manner as in Example 1 except that the coating solution was changed to the peeling layer / reagent-immobilized layer (fixing) coating solution of Preparation Example 2-4, and the same evaluation as in Example 1 was performed. Went. The results are shown in Table 2-1 and Table 2-2.

Example 4
In Example 1, in the step of forming the release layer / reagent-immobilized layer (for immobilization) in <Preparation of test line thermal transfer medium>, the exfoliation layer / reagent-immobilized layer (for immobilization) of Preparation Example 2-2 An immunochromatographic assay was prepared in the same manner as in Example 1 except that the coating solution was changed to the peeling layer / reagent-immobilized layer (fixing) coating solution in Preparation Example 2-5, and the same evaluation as in Example 1 was performed. Went. The results are shown in Table 2-1 and Table 2-2.

(Example 5)
In Example 1, in the step of forming the release layer / reagent-immobilized layer (for immobilization) in <Preparation of test line thermal transfer medium>, the exfoliation layer / reagent-immobilized layer (for immobilization) of Preparation Example 2-2 An immunochromatographic assay was prepared in the same manner as in Example 1 except that the coating solution was changed to the peeling layer / reagent-immobilized layer (fixing) coating solution in Preparation Example 2-7, and the same evaluation as in Example 1 was performed. Went. The results are shown in Table 2-1 and Table 2-2.

(Example 6)
In Example 1, in the step of forming the release layer / reagent-immobilized layer (for immobilization) in <Preparation of test line thermal transfer medium>, the exfoliation layer / reagent-immobilized layer (for immobilization) of Preparation Example 2-2 An immunochromatographic assay was prepared in the same manner as in Example 1 except that the coating solution was changed to the peeling layer / reagent-immobilized layer (fixing) coating solution of Preparation Example 2-9, and the same evaluation as in Example 1 was performed. Went. The results are shown in Table 2-1 and Table 2-2.

(Comparative Example 1)
In Example 1, in the step of forming the release layer / reagent-immobilized layer (for immobilization) in <Preparation of test line thermal transfer medium>, the exfoliation layer / reagent-immobilized layer (for immobilization) of Preparation Example 2-2 An immunochromatographic assay was prepared in the same manner as in Example 1 except that the coating solution was changed to the peeling layer / reagent-immobilized layer (fixing) coating solution of Preparation Example 2-1, and the same evaluation as in Example 1 was performed. Went. The results are shown in Table 2-1 and Table 2-2.

(Comparative Example 2)
In Example 1, in the step of forming the release layer / reagent-immobilized layer (for immobilization) in <Preparation of test line thermal transfer medium>, the exfoliation layer / reagent-immobilized layer (for immobilization) of Preparation Example 2-2 An immunochromatographic assay was prepared in the same manner as in Example 1 except that the coating solution was changed to the peeling layer / reagent-immobilized layer (fixing) coating solution of Preparation Example 2-6, and the same evaluation as in Example 1 was performed. Went. The results are shown in Table 2-1 and Table 2-2.

(Comparative Example 3)
In Example 1, in the step of forming the release layer / reagent-immobilized layer (for immobilization) in <Preparation of test line thermal transfer medium>, the exfoliation layer / reagent-immobilized layer (for immobilization) of Preparation Example 2-2 An immunochromatographic assay was prepared in the same manner as in Example 1 except that the coating solution was changed to the peeling layer / reagent-immobilized layer (fixing) coating solution of Preparation Example 2-8, and the same evaluation as in Example 1 was performed. Went. The results are shown in Table 2-1 and Table 2-2.

From the results of Table 2-1 and Table 2-2, the sufficient line concentration is obtained by Examples 1 to 6 and Comparative Examples 1 to 3 because of the thermoplastic elastomer relative to the total amount of wax and thermoplastic elastomer. It was found that the mass ratio was in the range of 8% by mass to 25% by mass.
Further, Example 6 is a mixture of a hydrophobic thermoplastic elastomer and a hydrophilic thermoplastic elastomer. This mixed thermoplastic elastomer exhibits hydrophobicity as a whole, and a sufficient line color density can be obtained. ing.

12A and 12B show cross-sectional SEM images (FIB / SEM Carl ZEISS (SII-NT) NVision 40, 5000 times magnification) of the thermal transfer medium for test lines in Example 1 and Comparative Example 2, respectively. Comparative Example No. 2 showed that the release layer / reagent-immobilized layer had no voids, and the difference in voids was significant.
From the results of Table 2, it is more preferable that the thermoplastic elastomer is hydrophobic and has a styrene skeleton in order to obtain a sufficient line color density in the present invention according to Examples 1 and 4 and Comparative Example 3. I understood it.
FIG. 12C shows a cross-sectional SEM image of the thermal transfer medium for test line in Comparative Example 3. When the thermoplastic elastomer is hydrophilic as in Comparative Example 3, a void structure is obtained in the release layer / reagent-immobilized layer. I found it impossible.

Aspects of the present invention are as follows, for example.
<1> A support and a porous reagent-immobilized layer on the support,
The reagent-immobilized layer contains an antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer,
The transfer medium for an inspection apparatus, wherein a mass ratio of the thermoplastic elastomer to a total amount of the wax and the thermoplastic elastomer is 8% by mass or more and 25% by mass or less.
<2> In the above <1>, the thermoplastic elastomer is at least one selected from butadiene rubber, styrene-butadiene rubber, high styrene rubber, isoprene rubber, acrylic rubber, epichlorohydrin rubber, and butyl rubber ethylene-propylene rubber. The transfer medium for an inspection apparatus described.
<3> The transfer medium for an inspection apparatus according to any one of <1> to <2>, wherein the thermoplastic elastomer includes a thermoplastic elastomer having a styrene skeleton.
<4> The inspection medium transfer medium according to any one of <1> to <3>, wherein the thermoplastic elastomer has a contact angle with distilled water at 25 ° C. of 75 ° or more.
<5> The transfer medium for an inspection apparatus according to any one of <1> to <4>, wherein a release layer is provided between the support and the reagent-immobilized layer.
<6> The transfer medium for an inspection apparatus according to any one of <1> to <5>, wherein the reagent-immobilized layer is a release layer / reagent-immobilized layer that also serves as a release layer.
<7> The reagent-immobilized layer of the transfer medium for an inspection apparatus according to any one of <1> to <6> is brought into contact with a porous flow path member so that the reagent-immobilized layer is It is a manufacturing method of an inspection device characterized by including the process of transferring to a porous channel member.
<8> a porous flow path member in which a flow path for flowing a specimen is formed;
A resin layer provided in at least one place on the flow path member,
The resin layer contains an antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer,
The inspection apparatus is characterized in that a mass ratio of the thermoplastic elastomer to a total amount of the wax and the thermoplastic elastomer is 8% by mass or more and 25% by mass or less.
<9> a porous flow path member in which a flow path for flowing a specimen is formed;
A first resin layer provided on the flow path member, and a second resin layer,
The first resin layer contains an antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer,
The mass ratio of the thermoplastic elastomer to the total amount of the wax and the thermoplastic elastomer is 8% by mass or more and 25% by mass or less,
The inspection apparatus according to <8>, wherein the second resin layer has a labeled antibody on a surface facing the flow path member.
<10> From the above <8>, wherein the thermoplastic elastomer is at least one selected from butadiene rubber, styrene-butadiene rubber, high styrene rubber, isoprene rubber, acrylic rubber, epichlorohydrin rubber, and butyl rubber ethylene-propylene rubber. <9> The inspection apparatus according to any one of the above.
<11> The inspection apparatus according to any one of <8> to <10>, wherein the thermoplastic elastomer includes a thermoplastic elastomer having a styrene skeleton.
<12> The inspection apparatus according to any one of <8> to <11>, wherein the thermoplastic elastomer has a contact angle with distilled water at 25 ° C. of 75 degrees or more.
<13> The inspection apparatus according to any one of <8> to <12>, a sample collection unit for collecting a sample, and at least one selected from a liquid for processing the sample It is the inspection kit characterized by this.

  <1> to <6> a transfer medium for an inspection apparatus according to any one of <1> to <6>, a manufacturing method of the inspection apparatus according to <7>, an inspection apparatus according to any one of <8> to <12>, And according to the test kit as described in <13>, the above-mentioned problems can be solved and the object of the present invention can be achieved.

DESCRIPTION OF SYMBOLS 10 Inspection apparatus 11 Base material 12 Flow path member 14 Absorbing member 15 Resin layer 15a Resin layer 15b Resin layer (test line)
15c Resin layer (control line)
16 Labeled antibody (example of reagent)
17 Capture antibody (example of reagent)
18 Capture antibody (example of reagent)
30 Test solution (example of specimen)
DESCRIPTION OF SYMBOLS 50 Inspection kit 100 Inspection apparatus transfer medium 110 Inspection apparatus transfer medium 101 Support body 102 Release layer 103 Reagent immobilization layer 104 Back layer 105 Release layer and reagent immobilization layer

Japanese Patent Laid-Open No. 2006-145789

Claims (8)

A support, and a porous reagent-immobilized layer on the support,
The reagent-immobilized layer contains an antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer,
A transfer medium for an inspection apparatus, wherein a mass ratio of the thermoplastic elastomer to a total amount of the wax and the thermoplastic elastomer is 8% by mass or more and 25% by mass or less.   The transfer medium for an inspection apparatus according to claim 1, wherein the thermoplastic elastomer includes a thermoplastic elastomer having a styrene skeleton.   The transfer medium for an inspection apparatus according to claim 1, further comprising a release layer between the support and the reagent-immobilized layer.   The transfer medium for an inspection apparatus according to claim 1, wherein the reagent-immobilized layer is a release layer / reagent-immobilized layer that also serves as a release layer.   5. The reagent-immobilized layer of the transfer medium for an inspection apparatus according to claim 1 and a porous flow path member are brought into contact with each other, so that the reagent-immobilized layer is made into the porous flow path member. A method for manufacturing an inspection apparatus, comprising a step of transferring to an inspection apparatus. A porous flow path member in which a flow path for flowing a sample is formed;
A resin layer provided in at least one place on the flow path member,
The resin layer contains an antibody, a wax, and a thermoplastic elastomer, and at least one of the thermoplastic elastomers is a hydrophobic thermoplastic elastomer,
The inspection apparatus, wherein a mass ratio of the thermoplastic elastomer to a total amount of the wax and the thermoplastic elastomer is 8% by mass or more and 25% by mass or less.   The inspection apparatus according to claim 6, wherein the thermoplastic elastomer includes a thermoplastic elastomer having a styrene skeleton. 8. A test comprising: the test apparatus according to claim 6; a sample collecting means for collecting a sample; and at least one selected from a liquid for processing the sample. kit.