JP2017116410A - Inspection device and manufacturing method of the same, and inspection kit and transfer medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection device that is excellent in coloration intensity and visibility.SOLUTION: An inspection device comprises a porous channel member for causing a specimen to flow and a resin layer at least at one place on the channel member; a solid phase agent on a surface of the resin layer facing the channel member contains an antibody reacting with the specimen; the ratio of the wettability B of water on the opposite side of a side of the channel member including the resin layer to the wettability A of water on the side of the channel member including the resin layer (B/A) is 1.5 or more and 2.0 or less.SELECTED DRAWING: Figure 2

Description

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

  In recent years, a measuring method for the purpose of POCT (Point of Care Testing) has rapidly spread. As a typical example, an inspection apparatus called a immunochromatography method using a measurement method combining a principle of a sandwich ELISA (Enzyme Linked Immuno Solvent Assay) method and a principle of chromatography is used.

Examples of the test apparatus include a sample pad as a test solution receiver, a conjugate pad for reacting the test solution supplied from the sample pad, and the test solution supplied from the conjugate pad. The thing which has a membrane film to flow is proposed (for example, refer patent document 1).
In addition, in order to improve the color development of the reagent and the developability of the reagent, the reagent that reacts with the sample is immobilized on the resin layer, and the resin layer is fixed to the flow path member so that the solidified sample surface is sandwiched. There has been proposed an inspection apparatus using a detection unit (see, for example, Patent Document 2).

  An object of this invention is to provide the inspection apparatus excellent in color intensity and visibility.

  The test apparatus according to the present invention as means for solving the problems includes a porous flow path member for flowing a specimen, and a resin layer at least at one position on the flow path member. The solid phase reagent on the surface of the layer facing the flow path member contains an antibody that reacts with the specimen, and the flow path member has a wettability A on the side having the resin layer of the flow path member. The ratio B / A of water wettability B on the side opposite to the side having the resin layer is 1.5 or more and 2.0 or less.

  According to the present invention, it is possible to provide an inspection apparatus having excellent color intensity and visibility.

FIG. 1 is a top view showing an example of the inspection apparatus of the present invention. 2 is a cross-sectional view taken along line AA of the inspection apparatus of FIG. 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 the transfer medium of the present invention. FIG. 8B is a schematic cross-sectional view showing another example of the transfer medium of 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 the inspection apparatus according to the first embodiment. 10B is a cross-sectional view taken along line BB in FIG. 10A. FIG. 11A is a top view showing another example of the inspection apparatus of the present invention. 11B is a cross-sectional view taken along the line CC of FIG. 11A. FIG. 12A is a top view showing another example of the inspection apparatus of the present invention. 12B is a cross-sectional view taken along the line DD of FIG. 12A.

(Inspection equipment)
The inspection apparatus of the present invention includes a porous flow path member for flowing a specimen, and a resin layer at least at one location on the flow path member, and a surface of the resin layer facing the flow path member The solid phase reagent contains an antibody that reacts with the specimen, and the wettability of water on the side having the resin layer of the flow path member is opposite to the side having the resin layer of the flow path member. The ratio B / A of the wettability B of water is 1.5 or more and 2.0 or less.
The inspection apparatus according to the present invention is superior to the conventional immunochromatography kit in the inspection apparatus described in Patent Document 2, but on the surface of the flow path member (hereinafter also referred to as “flow path”). Since the capture antibody is concentrated, the sample may not flow near the surface of the flow channel member due to the thickness of the flow channel member, and the sample does not contact the capture antibody. This is based on the knowledge that the color intensity decreases by passing through, or unevenness of color development occurs and visibility deteriorates.

  In the inspection apparatus of the present invention, when the ratio B / A is less than 1.5, the specimen is less likely to flow on the surface of the flow path member where the capture antibody is unevenly distributed, and the specimen and the capture antibody Since the reaction rate does not increase, a determination line having excellent color intensity and visibility cannot be obtained. When the ratio B / A exceeds 2.0, the development speed of the specimen is slowed down, so that development failure occurs and the test cannot be realized.

  The water wettability is based on the time from when 20 μL of purified water is dripped perpendicularly to the surface of the flow path member until it is completely absorbed (hereinafter sometimes referred to as “water absorption time”). The shorter the water absorption time, the higher the water wettability, and the longer the water absorption time, the lower the water wettability.

  There is no restriction | limiting in particular as said water absorption time, Although it can select suitably according to the objective, 1 second or more and 60 seconds or less are preferable, and 8 seconds or more and 50 seconds or less are more preferable. When the water absorption time is 8 seconds or more, water absorption is not too early, and malfunctions (nonspecific reactions) are unlikely to occur. In addition, if the water absorption time is 50 seconds or less, it is advantageous in that the time for the specimen to develop on the flow path member is not too late and it is quick.

The water wettability of the flow path member is preferably increased from the opposite side of the flow path member having the resin layer toward the side of the flow path member having the resin layer. An antibody in which a specimen (antigen) contained in a test solution is present in the resin layer when the flow path member has a wettability gradient on the opposite side of the side having the resin layer and the side having the resin layer This is advantageous in that the antigen-antibody reaction is likely to occur.
In addition, it is difficult to make appropriate physical properties such as pores and porosity due to manufacturing problems only by reducing the thickness of the flow path member. For this reason, as a method for inducing the flow of the specimen in the fluid member, it can be applied not only to the thick channel member but also to various channel members. It is preferable to have

There is no particular limitation on the method for making the wettability of water on the side having the resin layer of the flow path member higher than the wettability of water on the side opposite to the side of the flow path member having the resin layer. For example, there may be mentioned a method in which the side opposite to the side having the resin layer is subjected to a hydrophobic treatment, a method in which the side having the resin layer is subjected to a hydrophilic treatment, and the like. Among these, since the material used as the flow path member is generally hydrophilic, a method of subjecting the side opposite to the side having the resin layer to a hydrophobic treatment is preferable.
The hydrophobic treatment on the side opposite to the side having the resin layer means that the side opposite to the side having the resin layer is not completely hydrophobic and repels moisture, and is more hydrophobic than the side having the resin layer. This means processing that ensures the wettability of water. If the side opposite to the side having the resin layer is made completely hydrophobic, the specimen will not flow and will not function as an immunochromatography method.

There is no restriction | limiting in particular as said hydrophobic process, According to the objective, it can select suitably, For example, a fluorine process, a wax coat process, the process which plugs the pore of the said flow-path member, etc. are mentioned. Among these, fluorine treatment is preferable.
Examples of the fluorine treatment include a method of applying a fluorine compound.
The fluorine compound is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a fluorine compound having a trifluoromethyl group and a fluorine compound having a perfluoroalkyl group.
Examples of the application include application by spraying, application by brush, immersion, and the like. Among these, application by spraying is preferable.
When performing the application by spraying, a commercially available product may be used. Examples of the commercially available products include waterproof spray (AMEDAS 420, manufactured by Columbus Co., Ltd.), water repellent spray (water repellent spray, manufactured by Asahi Pen Co., Ltd.), water repellent waterproof spray (Tarago water repellent nano protector, Tarrago Brands International, S. L.).

Examples of the wax coating treatment include a method of applying a wax.
There is no restriction | limiting in particular as said wax, According to the objective, it can select suitably, For example, vegetable wax, natural wax, petroleum wax etc. are mentioned.
Examples of the vegetable wax include carnauba wax.
Examples of the natural wax include candelilla wax.
Examples of the petroleum wax include paraffin wax.

  A method for controlling the developing speed of the test solution is not particularly limited and may be appropriately selected depending on the purpose. For example, a method of changing the average thickness of the flow path member with a constant pore diameter, a hybrid material And the like.

Here, the inspection apparatus of the present invention will be described with reference to the drawings.
1, 2, 3, 4A, 4B, 5A, and 5B are drawings showing the overall configuration of the inspection apparatus. FIG. 1 is a top view showing an example of the inspection apparatus of the present invention. 2 is a cross-sectional view taken along line AA of the inspection apparatus of FIG. 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, FIG. 4B, FIG. 5A, and FIG. 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 as a flow path for flowing the test liquid 30 (an example of a specimen), and resin layers (15a, 15b, 15c) provided on the flow path member 12. On the surface of the resin layer (15a, 15b, 15c) facing the flow path member 12, the labeled antibody 16, the capture antibody 17, or the capture antibody 18 that reacts with the antigen contained in the test solution 30 is solid-phased. . As a result, 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 and immobilization of the reagent.

In the present embodiment, in the inspection apparatus 10, the 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. The present invention is not limited to such a form.
In this embodiment, providing on the flow path member 12 means providing on the flow path member 12 irrespective of the direction 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 (17, 18) may be solid-phased by any chemical bond such as covalent bond, hydrogen bond, metal bond, etc., or any interaction such as adhesion, adhesion, adsorption, van der Waals bond, etc. That's fine.

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, since the lower part of the flow path member 12 (the side opposite to the side having the resin layer 15 of the flow path member 12) is subjected to hydrophobic treatment, the upper part of the flow path member 12 (resin of the flow path member 12). The test solution 30 can easily flow to the side having the layer 15.
Further, in the inspection apparatus 10, the resin layer 15a (second resin layer) contains an amphiphilic resin 151 having a large number of hydrophilic groups 152, and is preferably configured as a main component (content is 50% by mass or more). Has been.
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 has a hydrophilic portion 16g, and is thereby immobilized on the surface of the resin layer 15a facing the flow path member 12. 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 inhibition of the binding between the antigen and the antibody, the amphiphilic resin 151 is preferably a water-insoluble resin.

  Here, the water-insoluble means that it is substantially insoluble in water. Further, the fact that it is substantially insoluble in water means that the mass change amount of the resin when it is sufficiently dried by a method such as vacuum drying after being immersed in a large amount of water at 25 ° C. for 24 hours is 1% by mass. It means the following. This is because 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 lower part of the flow path member 12 (the side opposite to the side having the resin layer 15 of the flow path member 12) is subjected to hydrophobic treatment. The test solution 30 easily flows to the side having the 12 resin layers 15.
In the inspection apparatus 10, the resin layer 15 b (first resin layer) is preferably a resin having a hydrophobic group 153. Specifically, the resin layer 15b contains the hydrophobic resin 155 or the amphiphilic resin 154 having many hydrophobic groups 153, and the hydrophobic resin 155 or the amphiphilic resin 154 is a main component (content is included). 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, the capture antibody 17 is solid-phased on the surface of the resin layer 15b facing the flow path member 12. . 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 colored, the resin layer 15 b can be used as a line for determining the presence or absence of the antigen 31. In order to prevent bleeding of the line, each of the hydrophobic resin 155 and the amphiphilic resin 154 is preferably a water-insoluble resin.

As shown in FIGS. 5A and 5B, the lower part of the flow path member 12 (the side opposite to the side having the resin layer 15 of the flow path member 12) is subjected to hydrophobic treatment, so that the upper part of the flow path member 12 (flow path member) The test solution 30 easily flows to the side having the 12 resin layers 15.
In the inspection apparatus 10, the resin layer 15 c (first resin layer) contains the hydrophobic resin 155 or the amphiphilic resin 154 having many hydrophobic groups 153, and the hydrophobic resin 155 or the amphiphilic resin is contained. It is preferable that 154 is 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 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 depending on the purpose. Examples thereof include an antibody that specifically binds to the labeled antibody 16. Thereby, since the labeled antibody 16 is immobilized and colored, the resin layer 15c can be used as a control line indicating that the labeled antibody 16 has reached. In order to prevent bleeding of the control line, the hydrophobic resin 155 and the amphiphilic resin 154 are each preferably a water-insoluble resin.

  The resin layer is preferably a non-porous material. The non-porous body is a non-porous structure that does not substantially include voids, and is a structure that is contrary to a porous material including voids provided to promote absorption of liquid such as a membrane. Means. 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 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 said inspection apparatus 10 is demonstrated in detail.
<Base material>
There is no restriction | limiting in particular as a material of the base material 11, It can select according to the objective, For example, an organic material, an inorganic material, or a metal material 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 organic material is not particularly limited and may be appropriately selected depending on the intended purpose. For example, polyvinyl chloride, polyethylene terephthalate, polypropylene, polystyrene, polyvinyl acetate, polycarbonate, polyacetal, modified polyphenyl ether, polybutylene Examples thereof include phthalate and B / AS 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 mechanical strength as the substrate 11 is good, and when it is 0.5 mm or less, the flexibility of the substrate is good and it can be suitably used as a sensor. it can.
Here, the average thickness refers to, for example, a thickness of the measurement object in five places in the longitudinal direction, three places in the width direction, and a total of 15 places in a micrometer (MDH-25M so that the measurement places are substantially evenly spaced. , Manufactured by Mitutoyo Co., Ltd.). 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 has a ratio B / A of the water wettability B on the side opposite to the side having the resin layer 15 of the flow path member 12 to the water wettability A on the side having the resin layer 15. 1.5 or more and 2.0 or less. When the ratio B / A of the flow path member 12 is less than 1.5, the sample hardly flows on the surface of the flow path member 12 where the capture antibody is unevenly distributed, and the reaction rate between the sample and the capture antibody is high. Since it does not increase, a determination line having excellent color intensity and visibility cannot be obtained. When the ratio B / A exceeds 2.0, the development speed of the specimen is slowed down, so that development failure occurs and the test cannot be realized.
The flow path member 12 is not particularly limited as long as the test solution 30 can be flowed, and can be appropriately selected according to the purpose. Examples thereof include a hydrophilic porous material.

  There is no particular limitation on the method for making the water wettability of the flow path member 12 side having the resin layer 15 higher than the water wettability of the flow path member 12 opposite to the side having the resin layer 15. For example, there may be mentioned a method of subjecting the side opposite to the side having the resin layer 15 to a hydrophobic treatment, a method of subjecting the side having the resin layer 15 to a hydrophilic treatment, and the like. Among these, since the material used as the flow path member 12 is generally hydrophilic, a method of subjecting the side opposite to the side having the resin layer 15 to a hydrophobic treatment is preferable.

There is no restriction | limiting in particular as said hydrophobic process, According to the objective, it can select suitably, For example, a fluorine process, a wax coat process, the process which plugs the pore of the flow path member 12, etc. are mentioned.
The fluorine treatment can be suitably performed by a fluorine resin coating means.
There is no restriction | limiting in particular as said fluororesin application | coating means, According to the objective, it can select suitably, You may use a commercial item. As said commercial item, a fluorine coat spray (AMeDAS 420, Columbus Co., Ltd. product) etc. are mentioned, for example.

  The water wettability of the flow path member 12 is preferably increased as it approaches the side of the flow path member 12 having the resin layer 15 from the opposite side of the flow path member 12 having the resin layer 15.

The flow path member 12 made of the hydrophilic porous material has gaps (12a, 12b), and the flow path is formed by the test liquid 30 flowing in the gaps (12a, 12b).
In FIG. 3 to FIG. 5B, the gap 12a is a gap formed in each cross section, and the gap 12b is a gap on the back side of the cross section. It is preferable that bubbles are present inside the hydrophilic 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 hydrophilic porous material, Although it can select suitably according to the objective, The material which shows hydrophilic property and has a high porosity is used suitably.
The hydrophilic porous material means a porous material that can be easily penetrated by an aqueous solution. “Easy to penetrate” refers to a water permeability evaluation test in which 0.01 mL of pure water is dropped onto the surface of a plate-like test piece dried at 120 ° C. for 1 hour. All of 0.01 mL of pure water penetrates within 10 minutes. To do.

The porosity of the hydrophilic porous material is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 40% or more and 90% or less, and more preferably 65% or more and 80% or less. When the porosity is 90% or less, the mechanical strength of the substrate is good. Further, when the porosity is 40% or more, the permeability of the test solution is improved.
Here, the said porosity can be calculated | required by the following formula 1 from the basic weight (g / m < ² >), thickness (micrometer), and composition specific gravity of a hydrophilic porous material, for example.
[Calculation Formula 1]
Porosity (%) = {1- [basis weight (g / m ² ) / thickness (μm) / specific gravity of composition]} × 100

The hydrophilic porous material is not particularly limited and may be appropriately selected depending on the intended purpose. For example, filter paper such as membrane membrane, plain paper, fine paper, watercolor paper, Kent paper, synthetic paper, synthetic resin film , Special paper having a coating layer, fabric, textiles, film, inorganic substrate, glass and the like. These may be used individually by 1 type and may use 2 or more types together. Among these, filter paper such as membrane membrane and fabric are preferable, and filter paper such as membrane membrane is more preferable from the viewpoint of high porosity and good hydrophilicity.
Examples of the fabric include artificial fibers such as rayon, Bemberg, acetate, nylon, polyester, and vinylon, natural fibers such as cotton and silk, blended fibers thereof, and nonwoven fabrics thereof.

There is no restriction | limiting in particular about the shape of the said hydrophilic porous material, Although it can select suitably according to the objective, A sheet form is preferable.
There is no restriction | limiting in particular as average thickness of the said hydrophilic porous material, Although it can select suitably according to the objective, 0.01 mm or more and 0.3 mm or less are preferable. The intensity | strength of a base material is favorable in the said average thickness being 0.01 mm or more. Further, when the average thickness is 0.3 mm or less, the required amount of the test solution can be optimized.
The porous pore diameter of the hydrophilic porous material is preferably 0.45 μm or more and 10 μm or less. When the pore diameter is 0.45 μm or more and 10 μm or less, the development of the test solution becomes appropriate, and a rapid test becomes possible, and the capture antibody easily catches the antigen in the test solution and the test sensitivity is improved. Is advantageous.
The porous pore diameter can be measured from an image obtained by observing a cross section of the hydrophilic porous material using, for example, a scanning electron microscope (FE-SEM) (MERLIN, manufactured by Zeiss).

<Resin layer>
The function of the resin layer 15 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, when 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 test time will be longer due to the decrease in water absorption of the test solution from the sample pad. Cost. For this reason, the fiber F1 which can be used as a conjugate pad is limited. Further, in the conventional inspection apparatus, the labeled antibody 16 is immobilized on the fiber F1 forming the conjugate pad (see FIG. 6). The labeled antibody 16 that can be released from the conjugate pad is limited to those having a weak binding force with the fiber F1. That is, the conventional inspection apparatus is limited in usable fiber F1 and labeled antibody 16 by design.
Similarly, in the conventional inspection apparatus, the capture antibody 17 is solid-phased on the fiber F2 constituting the membrane (see FIG. 7). For this reason, the capture antibody 17 that can be immobilized on the membrane is limited to those having a strong binding force to the fiber F2. That is, the conventional inspection apparatus is limited in usable fiber F2 and capture antibody 17 in design.

  In the inspection apparatus 10 of this embodiment, reagents such as the labeled antibody 16, the capture antibody 17, and the capture antibody 18 are immobilized on the resin layer 15 (15a, 15b, 15c). For this reason, it is possible to control the release or immobilization of the capture antibody in accordance with the strength of the interaction between the resin layer 15 and the capture antibody and the affinity with the test solution 30.

  As a method for adjusting the strength of interaction between the resin layer 15 and the capture antibody and the affinity with the test solution 30, for example, the type of resin constituting the resin layer 15 and the composition ratio of the resin may be changed to the corresponding capture antibody. The method of changing according to it is mentioned. For example, the greater the proportion of hydrophobicity in the resin constituting the resin layer 15, the easier it is for the resin layer 15 to immobilize a capture antibody 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, Try to reduce the contact area as much as possible. As a result, the hydrophobic molecular species come to gather close to each other, and this is a phenomenon that appears to have a binding force acting between the molecules.
When the hydrophilic ratio in the resin constituting the resin layer 15 is large, the interaction between the resin layer 15 and the hydrophilic capture antibody becomes strong, but when the binding portion comes into contact with the hydrophilic test solution 30, It is estimated that the reagent is likely to be released into the test liquid 30 in affinity with the test liquid 30.

  The resin constituting the resin layer 15 is preferably a water-insoluble resin. When a water-insoluble resin is used for the resin layer 15, 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 alcohols, 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, vinyl acetate Examples thereof include nyl-maleic acid ester copolymer, vinyl acetate-crotonic acid copolymer, vinyl acetate-acrylic acid copolymer, 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.

  Examples of the hydrophobic resin constituting the resin layer 15b and the resin layer 15c include polystyrene resins such as polystyrene and acrylonitrile-butadiene-styrene copolymers, polyolefin resins such as polypropylene resins, polyethylene resins, and ethylene-propylene copolymers. Resin or cyclic polyolefin resin, polycarbonate resin, polyethylene terephthalate resin, methacrylic resin such as polymethyl methacrylate resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone Resin, polyetherimide resin, fluorine resin such as polytetrafluoroethylene, polymethylpentene resin, acrylic resin such as polyacrylonitrile, propionate tree Such as cellulose resins and the like. These may be used alone or in combination of two or more.

Examples of compounds other than the hydrophobic resin 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, Synthetic waxes such as microcrystalline wax, oxidized wax, ozokerite, ceresin, ester wax, polyethylene wax, oxidized polyethylene wax; higher fatty acids such as margaric acid, lauric acid, myristic acid, palmitic acid, stearic acid, furonic acid, behenic acid; Examples include higher alcohols such as stearic alcohol and behenyl alcohol; esters such as sorbitan fatty acid ester; amides such as stearamide and oleinamide. These may be used alone or in combination of two or more.
Among the compounds constituting the resin layer 15b and the resin layer 15c, polystyrene resin, polyolefin resin, carnauba wax, and polyethylene wax are preferable because of strong hydrophobic interaction.

  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. Gold colloid-labeled antibodies such as colloidal gold-labeled Anti-human IgG, labeled antibodies against various allergens, and particles that label other antibodies.
The particles for labeling the other antibodies are not particularly limited and can be appropriately selected depending on the purpose. For example, metal colloids other than gold colloid, enzyme-labeled particles containing an enzyme, and colored particles containing a dye. , Fluorescent particles containing a fluorescent substance, magnetic substance-containing particles containing a magnetic substance, and the like. 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 antibodies such as Anti-human IgG and antibodies against various allergens.
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 antibodies against labeled antibody 16 such as Human IgG, the antibodies listed above, and the like. Moreover, the antigen itself with which the labeled antibody 16 reacts 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. A method in which a solution containing a reagent is applied to the resin layer 15 and then rapidly dried to dry up, and a solution containing the reagent is applied to the resin layer 15 and left in a humid environment (incubation) so that the coating solution does not dry. Then, a method of washing and drying components other than antibodies such as inorganic salts with distilled water and the like can be mentioned.

When the reagent such as the capture antibody is handled as a coating solution, it is not particularly limited and may be appropriately selected depending on the intended purpose. However, it is usually preferable to dilute with a buffer solution (buffer agent) called a buffer.
The buffer solution is not particularly limited as long as it does not inhibit the antigen-antibody reaction of the capture antibody, and includes a buffer solution generally used for antibody dilution. Examples of the buffer include phosphate buffered saline (PBS), Tris buffered saline (PBS), trishydroxymethylaminomethane (Tris) -HCl, and Good buffer. These may be used alone or in combination of two or more. Among these, phosphate buffered saline (PBS) and Good buffer are preferable.

There is no restriction | limiting in particular as a composition of the said phosphate buffered saline (PBS), According to the objective, it can select suitably, For example, NaCl is 8.0 g / L, KCl is 0.2 g / L, NaH Examples include a composition in which ₂ PO ₄ is 1.44 g / L and KH ₂ PO ₄ is 0.24 g / L, a composition not containing potassium, and a composition containing calcium and magnesium.
There is no restriction | limiting in particular as pH of said PBS, Although it can select suitably according to the objective, 4-10 is preferable and 6-8 is more preferable.
Examples of the good buffer include 2- (N-monophorino) ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-2-sulfonic acid (HEPES), and the like.

The inorganic salt means salts and ions that can be measured by ion chromatography or the like, and specifically, cations such as sodium ion, potassium ion, ammonium ion, magnesium ion, calcium ion; chlorine ion, PO ₄ ion , Anions such as SO ₄ ions, or salts thereof.

  In the present embodiment, 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 14 is not particularly limited as long as it is a member that absorbs water, and can be appropriately selected from known materials.
Examples of the absorbing member 14 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. Can be mentioned.

<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 intended to prevent 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 12. 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 15 can be provided on the flow path member 12 by various methods. As an example, a case where a thermal transfer method is used will be described. Hereinafter, a method for manufacturing a transfer medium and an inspection apparatus used in the thermal transfer method will be described.

(Transfer medium)
In the first embodiment, the transfer medium of the present invention comprises a support, a release layer provided on the support, and a reagent-immobilized layer provided on the release layer, and the reagent The surface of the solid phase layer has an antibody that reacts with the specimen.

  In the second embodiment, the transfer medium of the present invention has a support and a release layer / reagent-immobilized layer on the support, and the surface of the release layer / reagent-immobilized layer is Has an antibody that reacts with the analyte.

  Here, a transfer medium used when a resin layer is provided on the flow path member will be described with reference to the drawings.

  8A and 8B are schematic cross-sectional views showing an example of the transfer medium of the present invention. When the thermal transfer method is used, by using the transfer medium 100 to which the capture antibody is uniformly attached in advance, the concentration difference of the capture antibody (17, 18) in the test line or the control line can be reduced. 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 to which a high concentration capture antibody is attached in advance.

As shown in FIG. 8A, the transfer medium 100 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. The transfer medium 100 further includes other layers such as a back layer 104 as necessary.
As shown in the 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.

<Support>
There is no restriction | limiting in particular about the shape, a structure, a magnitude | size, a material, etc. as the support body 101, 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 10 and the like.

  There is no restriction | limiting in particular as a material of the support body 101, According to the objective, it can select suitably, For example, polyesters, 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 101 is preferably subjected to a surface activation treatment in order to improve adhesion with a layer provided on the support 101. Examples of the surface activation treatment include glow discharge treatment and corona discharge treatment.

  The support 101 may be left as it is after the reagent-immobilized layer 103 is transferred to the flow path member 12, and after the reagent-immobilized layer 103 is transferred, the support 101 and the like are peeled off by the release layer 102. And may be removed. When the release layer / reagent solid phase layer 105 is used, the release layer / reagent solid phase layer 105 is completely transferred to the flow path member 12 or the trial release layer / reagent solid phase layer 105 The part including the surface on which the antibody is immobilized is transferred, but a part of the release layer / reagent-immobilized layer 105 may remain on the support 101 side.

As the support 101, an appropriately synthesized product or a commercially available product may be used.
There is no restriction | limiting in particular as average thickness of the support body 101, 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 102 has a function of improving the peelability between the support 101 and the reagent-immobilized layer 103 during transfer. Further, when the release layer 102 is heated by a heating and pressurizing means such as a thermal head, it is melted into a low-viscosity liquid, and the reagent-immobilized layer 103 can be easily cut in the vicinity of the interface between the heated part and the non-heated part. It has a function to make.

  The release layer 102 includes a wax and a binder resin, and further includes other components as necessary.

  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, furonic 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, butadiene rubber, and acrylonitrile-butadiene copolymer. . 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 peeling layer 102, 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 as average thickness of the peeling layer 102, Although it can select suitably according to the objective, 0.5 micrometer or more and 50 micrometers or less are preferable.
The coating weight of the release layer 102 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 material of the reagent-immobilized layer 103 is not particularly limited as long as it contains a resin constituting the resin layer 15 in the inspection apparatus 10 and can be appropriately selected according to the purpose.
The method for forming the reagent-immobilized layer 103 is not particularly limited and may be appropriately selected depending on the purpose. For example, a hot-melt coating method or a reagent in which the resin constituting the resin layer 15 is dispersed in a solvent The coating solution is formed by applying the reagent-immobilized layer coating solution on the support 101 or the release layer 102 by a general coating method such as gravure coater, wire bar coater, roll coater, and drying. Can do.

  There is no restriction | limiting in particular as average thickness of the reagent solid-phased layer 103, 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 resin layer is improved and the resin 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 visibility is improved.

The adhesion amount of the reagent coating solution in the reagent immobilization layer 103 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 the resin layer is not damaged. 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 resin layer.

<Peeling layer and reagent solid phase layer>
The release layer / reagent-immobilized layer 105 has the functions of both a release layer and a reagent-immobilized layer, and the release property between the support 101 and the release layer / reagent-immobilized layer 105 is improved during transfer. By including the wax and binder resin to be improved and the resin that forms the resin layer 15 in the inspection apparatus 10, it is possible to solidify a reagent such as the capture antibody 17 or the capture antibody 18 while improving the peelability. It is.

  When the release layer / reagent-immobilized layer 105 is heated by a heating and pressurizing means such as a thermal head, the side surface in contact with the support 101 is thermally melted to become 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 solid-phased layer 105 contains a wax and a binder resin, 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 peeling layer 102 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 binder resin, According to the objective, it can select suitably, For example, the thing similar to the peeling layer 102 is mentioned. 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 peeling layer and reagent solid-phased layer 105, According to the objective, it can select suitably, For example, the hot-melt coating method, the said wax, and the said binder resin were disperse | distributed to the solvent. The method of apply | coating a coating liquid etc. are mentioned.
There is no restriction | limiting in particular as average thickness of the peeling layer and reagent solid-phased layer 105, Although it can select suitably according to the objective, 0.5 micrometer or more and 50 micrometers or less are preferable. When the average thickness is 0.5 μm or more, the durability of the release layer / reagent-immobilized layer 105 (resin layer 15) is improved, and the resin 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 visibility is improved.
The coating weight of the release layer and the reagent immobilization layer 105 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 application amount is appropriate, and the occurrence of defects in the release layer / reagent solid phase layer 105 (resin layer 15) is reduced. Moreover, when the adhesion amount is 50 g / m ² or less, the drying time is appropriate, and unevenness in the release layer / reagent-immobilized layer 105 is reduced.

-Immobilization of reagents-
After the coating solution is dried and the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 is formed, a labeled antibody is formed on the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105. 16 or a solution containing the capture antibody (17, 18) is applied to form a uniform coating film. Subsequently, the labeled antibody 16 or the capture antibody (17, 18) can be immobilized on the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 by drying the coating film.

-Immobilization of labeled antibody-
The method for immobilizing the labeled antibody is not particularly limited and may be appropriately selected depending on the purpose. For example, the labeled antibody may be applied to the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105. Examples thereof include a method in which a coating film is applied to form a water film and then dried up by natural drying, reduced pressure drying, freeze drying, or the like to form a solid phase.
The water film is preferably applied so as to have a uniform thickness.
The amount of the labeled antibody applied is not particularly limited and can be appropriately selected according to the purpose. For example, when a gold colloid labeled antibody is used as the labeled antibody, the OD (optical density) is 1.0 or more. It is preferable to apply 20 μL or less to 20 μL or more and 600 μL or less per unit area (cm ² ) of the resin layer. When the coating amount is 20 μL or more, the amount of colloidal gold labeled antibody is appropriate, and the color intensity of the line is good. When the coating amount is 600 μL or less, the amount of colloidal gold labeled antibody is appropriate, and the color intensity of the line is good.
There is no restriction | limiting in particular as a drying method, According to the objective, it can select suitably, For example, aeration drying, vacuum drying, natural drying, freeze-drying etc. are mentioned. Among these, natural drying under low humidity or drying under reduced pressure is preferable.
The humidity during drying is preferably 30% or less in terms of relative humidity. When the relative humidity is 30% or less, drying is appropriate and the antibody can be sufficiently solid-phased.
The drying temperature is preferably room temperature (20 ° C.) or more and 50 ° C. or less and the drying time is 30 minutes or more and 24 hours or less. When the drying temperature is room temperature (20 ° C.) or higher, the drying time is appropriate and productivity is improved. Further, when the drying time is 50 ° C. or less, the reagent can be prevented from being denatured by heat. When the drying time is 30 minutes or longer, drying can be performed appropriately, and when it is 24 hours or shorter, productivity is improved and discoloration can be prevented.

-Immobilization of capture antibody-
The method for immobilizing the capture antibody is not particularly limited and can be appropriately selected according to the purpose. For example, the capture antibody is immobilized on the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105. Apply a coating solution to form a water film and dry it up by natural drying, vacuum drying, freeze drying, etc. (dry-up method), or in a humid environment to prevent the coating solution from drying. Examples of the method include a method in which the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 is washed with distilled water and dried to be solid-phased (method of drying after adsorption). In any case, the coating film is preferably applied so as to have a uniform thickness.
The concentration of the buffer for diluting the capture antibody is not particularly limited and can be used in the composition of a buffer solution generally used for antibody dilution, preferably 10 μg / mL to 5,000 μg / mL, 100 μg / mL or more and 1,000 μg / mL or less is more preferable. When the coating concentration is 10 μg / mL or more, the amount of liquid to be applied per unit area (cm ² ) to the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 is appropriate. Inorganic salts and other components are reduced, and the antibody can be sufficiently immobilized. Further, when the coating concentration is 5,000 μg / mL or less, the wettability and viscosity of the coating solution are appropriate, and the coating onto the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 is good. In addition, since the amount of the coating solution is appropriate, a uniform water film can be formed.

The drying method when the capture antibody is solid-phased by the dry-up method is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include aeration drying, vacuum drying, natural drying, and freeze drying. Can be mentioned. Among these, natural drying under low humidity or drying under reduced pressure is preferable.
The humidity during drying is preferably 30% or less in terms of relative humidity. If the relative humidity exceeds 30%, drying may be insufficient and the antibody may not be sufficiently immobilized.
The drying temperature is preferably room temperature (20 ° C.) to 50 ° C. and the drying time is preferably 30 minutes to 24 hours. When the drying temperature is room temperature (20 ° C.) or higher, the drying time is appropriate and productivity is improved. Further, when the drying temperature is 50 ° C. or lower, the reagent is not denatured by heat. When the drying time is 30 minutes or more, drying can be performed appropriately, and when it is 24 hours or less, productivity is improved and discoloration can be prevented.

As stationary conditions in the case where the capture antibody is solid-phased by adsorption and then dried, the temperature is preferably 0 ° C. or higher and 40 ° C. or lower and the relative humidity is 30% or higher. When the temperature is 0 ° C. or higher, the capture antibody can be appropriately immobilized. Further, when the temperature is 40 ° C. or lower, the capture antibody can be prevented from being denatured. When the relative humidity is 30% or more, moisture volatilization during standing is small, so that a large amount of unnecessary components other than antibodies can be prevented from being solid-phased.
The washing method after standing is not particularly limited and may be appropriately selected depending on the purpose. For example, 20 μL or more per unit area (cm ² ) on the solid phase surface using a shaker or the like. Examples include a method of pouring 100 μL or less of distilled water or the like and then gently washing at room temperature (20 ° C.) for washing.

There is no restriction | limiting in particular as the drying method after washing | cleaning, According to the objective, it can select suitably, For example, aeration drying, vacuum drying, natural drying, freeze-drying etc. are mentioned. Among these, natural drying under low humidity or drying under reduced pressure is preferable.
The humidity during drying is preferably 30% or less in terms of relative humidity. When the relative humidity is 30% or less, drying is appropriate and the antibody can be sufficiently solid-phased. The drying temperature is preferably from room temperature (20 ° C.) to 50 ° C., and the drying time is preferably from 30 minutes to 24 hours.
When the drying temperature is room temperature (20 ° C.) or higher, the drying time is appropriate and productivity is improved. Further, when the drying temperature is 50 ° C. or lower, the reagent can be prevented from being denatured by heat. Further, when the drying time is 30 minutes or more, drying can be performed appropriately, and when it is 24 hours or less, productivity is improved and discoloration of the resin can be prevented.

The amount of immobilized capture antibody is preferably 500 ng / cm ² or more. When the immobilized amount of the capture antibody is 500 ng / cm ² or more, the immobilized amount is appropriate, and the color intensity of the line is sufficiently obtained.
Here, examples of the method for analyzing the amount of the immobilized antibody present on the surface of the resin layer 15 include X-ray photoelectron spectroscopy (XPS).

<Back layer>
The transfer medium 100 is preferably provided with a back layer 104 on the surface of the support 101 opposite to the surface on the peeling layer 102 side. At the time of transfer, heat is directly applied to the opposite surface in accordance with the shape of the resin layer by a thermal head or the like. Therefore, the back layer 104 preferably has resistance to high heat and resistance to friction with a thermal head or the like.

  The back layer 104 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 back layer 104, 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 back layer 104, 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 101 and the release layer 102, between the release layer 102 and the reagent-immobilized layer 103, or between the support 101 and the release-cum-reagent-immobilized layer 105. 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 103, the release layer 102, and the release layer / reagent-immobilized layer 105 It can be used.

<Protective film>
A protective film is preferably provided on the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 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 removed from the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105, and can be appropriately selected according to the purpose. Examples include silicone paper, polyolefin sheets such as polypropylene, and polytetrafluoroethylene sheets.
There is no restriction | limiting in particular as 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.

(Inspection device manufacturing method)
In the first embodiment of the manufacturing method of the inspection apparatus of the present invention, the step of subjecting one surface of the porous flow path member to a hydrophobic treatment, that is, the wetting of the water on the side of the flow path member having the resin layer A step of increasing the wettability of the flow path member on the side opposite to the side having the resin layer (hereinafter, also referred to as “water wettability adjustment step of the flow path member”), A step of bringing the surface of the reagent-immobilized layer of the transfer medium of the invention into contact with the non-hydrophobic surface of the flow path member and transferring the reagent-immobilized layer to the flow path member (hereinafter referred to as "Sometimes referred to as a" reagent-immobilized layer transfer step "), and other steps as necessary.

  In the second embodiment of the manufacturing method of the inspection apparatus of the present invention, the wettability of the water on the side having the resin layer of the flow path member is set to the water on the opposite side of the flow path member on the side having the resin layer. The surface of the release layer / reagent-immobilized layer of the transfer medium of the present invention and the flow of the flow medium. A step of contacting the surface of the path member that has not been subjected to hydrophobic treatment and transferring the release layer / reagent-immobilized layer to the flow path member (hereinafter referred to as “transfer step of release layer / reagent-immobilized layer”) And may include other steps as necessary.

<Water wettability adjustment process of flow path member>
As a method for adjusting the wettability of water in the flow path member, the wettability of water on the side of the flow path member having the resin layer may be adjusted to the wettability of water on the side of the flow path member opposite to the side having the resin layer. If it is higher than the property, there is no particular limitation, and it can be appropriately selected according to the purpose. For example, a method of performing a hydrophobic treatment on the side opposite to the side having the resin layer, a method for performing a hydrophilic treatment on the side having the resin layer Etc. Among these, since the material used as the flow path member is generally hydrophilic, a method of subjecting the side opposite to the side having the resin layer to a hydrophobic treatment is preferable.

<Reagent-immobilized layer transfer step or release layer / reagent-immobilized layer transfer step>
As a method for thermally transferring the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 to the flow path member 12, the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 of the reagent transfer medium 100 can be used. And a method in which the reagent-immobilized layer 103 or the separation layer / reagent-immobilized layer 105 is transferred to the channel member 12 by bringing the channel member 12 into contact with the channel member 12.

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 103 or the release layer / reagent-immobilized layer 105 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 101 and the thermal head are not contaminated.

-Application of inspection device-
There is no restriction | limiting in particular as an application of the test | inspection apparatus 10, According to the objective, it can select suitably, For example, in the biochemical sensor (sensing chip) for blood test and DNA test, the quality control use of foodstuffs, drinks, etc. Examples include small 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 if necessary by centrifugation or the like, and then subjected to cell destruction treatment by, for example, enzyme treatment, heat treatment, surfactant treatment, ultrasonic treatment, a combination thereof or the like. It may be given in advance.

Since the flow path member 12 works as a stationary phase, the inspection apparatus 10 of the present embodiment also has a function of chromatography (separation and purification) of the inspection liquid. In this case, the flow path member 12 having open cells whose inner wall is hydrophilic is a 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 more hydrophilic component is more easily adsorbed to the porous portion, which is the stationary phase, and the number of repetitions of desorption is larger, and the rate of permeation through the flow path is slower. On the other hand, the 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 30 by using the difference in moving speed in the test liquid, the test apparatus 10 can be used as a highly functional sensor for chemical or biochemical applications. it can.

<Inspection method>
The inspection method according to the present invention is not particularly limited and can be appropriately selected according to the purpose. The step of supplying a hydrophilic inspection liquid to the flow path member 12 of the inspection apparatus 10 and the solid phase on the resin layer 15a. And a step of releasing the labeled antibody 16 (an example of a reagent) from the resin layer 15a by bringing it into contact with the test solution 30, and further including other steps as necessary.

  As a method for testing using the test apparatus 10, the step of supplying the test liquid 30 to the flow path member 12 of the test apparatus 10, and when the test liquid 30 contains the antigen 31, A part of the example) may be captured by the capture antibody 17 immobilized on the resin layer 15b.

  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 affinity with 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 according to the present invention, an instrument for collecting a sample (an example of a sample collecting unit), and at least one liquid 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.
As said other member, an instruction manual etc. are mentioned, for example.

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 embodiment, the example in which the support 101 and the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 in the transfer medium 100 are peeled off by heat at the time of transfer has been described. It is not limited to the 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 pasted 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. Examples of the test liquid 30 include solvophilic solvents including organic solvents 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). It 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-
Silicone rubber emulsion (KS779H, manufactured by Shin-Etsu Chemical Co., Ltd., solid content: 30% by mass) 16.8 parts by mass, chloroplatinic acid catalyst 0.2 parts by mass, and toluene 83 parts by mass are mixed to form a back layer. A liquid was prepared.

(Preparation Example 2)
-Preparation of coating solution for release layer and reagent-immobilized layer (for fixation)-
90 parts by weight of carnauba wax, 1 part by weight of ethylene-vinyl acetate copolymer, 4 parts by weight of styrene-butadiene copolymer, 4 parts by weight of butadiene rubber, 1 part by weight of acrylonitrile-butadiene copolymer, and toluene / methyl ethyl ketone (volume ratio) 7/3) A coating solution composed of a solvent (B110AX stripping solution, manufactured by Ricoh Co., Ltd.) was used as a peeling layer / reagent-immobilized layer (fixing) coating solution.

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

(Preparation Example 4)
-Preparation of reagent coating solution for test line-
Anti-human IgG antibody (I1886, manufactured by Sigma-Aldrich) with Dulbecco's phosphate buffered saline (Ca, Mg-free, D-PBS (−), manufactured by Nacalai Tesque, Inc., 14249-95) as an antibody diluent. In addition, the reagent coating solution for test line was prepared to 100 μg / mL.

(Preparation Example 5)
-Preparation of reagent coating solution for control line-
The aforementioned D-PBS (−) was added as an antibody diluent to Human IgG (I2511-10MG, manufactured by Sigma Aldrich) to give 100 μg / mL, and a control line reagent coating solution was prepared.

(Preparation Example 6)
-Preparation of reagent coating solution for labeled antibody-
Gold colloid-labeled Anti-Human IgG antibody (Gold, manufactured by BAW, average particle size 40 nm, OD = 15) was prepared as a reagent coating solution for labeled antibody.

(Preparation Example 7)
-Fabrication of channel member with one surface treated with hydrophobic treatment-
Fluorine-coated spray (Amedas 420, Columbus Co., Ltd.) was sprayed on the surface of membrane film JM (hydrophilic PTFE, Merck Millipore, average thickness 100 μm, pore diameter 5 μm) for 2.0 seconds from a distance of 50 cm, A flow path member having one surface subjected to hydrophobic treatment was produced.

-Wettability evaluation-
In order to compare the wettability of the surface sprayed with the water and the wettability of the surface not sprayed, a micropipette on each surface in an environment with a temperature of 23 ° C. and a relative humidity of 60%. Then, 20 μL of purified water was dropped vertically onto the surface of the flow path member and the water absorption time until the water droplet was completely absorbed was measured, and this was defined as “water wettability”. Water absorption time was 30 seconds on the surface where the spray was not sprayed, and water absorption time was 47 seconds on the surface where the spray was sprayed. In addition, it expresses that the wettability of water is so high that water absorption time is early, and the wettability of water is low, so that water absorption time is slow. Further, “until water droplets are completely absorbed” means a state in which it can be determined that all the water droplets have disappeared from the flow path member and have been absorbed by the flow path member. When a water droplet of 20 μL is dropped on the flow path member, the surface tension of the water drop is stronger than the absorption capacity of the flow path member, and the water drop is gradually absorbed from around the water drop. Therefore, at the moment of absorption, the moment when the shape of the water droplet formed by the surface tension disappears can be visually confirmed, and the moment is regarded as “the water droplet has been completely absorbed”.
From this result, the water wettability B of the surface sprayed with the spray is lower than the water wettability A of the surface sprayed with the water, and the water wettability ratio B / A is 47 seconds / It was confirmed that 30 seconds = 1.6.

(Preparation Example 8)
-Fabrication of channel member with one surface treated with hydrophobic treatment-
Fluorine coat spray (AMEDAS 420, Columbus Co., Ltd.) was sprayed for 2.0 seconds from one side of membrane film PES (Polyethersulfone, manufactured by ASONE CORPORATION, average thickness 120 μm, pore diameter 0.45 μm) from 50 cm away. Thus, a flow path member having one surface subjected to hydrophobic treatment was produced.

-Water wettability evaluation-
When evaluated in the same manner as in Preparation Example 7, the water absorption time was 9.7 seconds on the surface where the spray was not sprayed, and the water absorption time was 17.5 seconds on the surface where the spray was sprayed.
From this result, the water wettability B of the surface sprayed with the spray is lower than the water wettability A of the surface sprayed with the spray, and the water wettability ratio B / A is 17.5. It was confirmed that sec / 9.7 sec = 1.8.

(Preparation Example 9)
-Production of flow path member-
A non-hydrophobic nitrocellulose membrane (HF180, manufactured by Merck Millipore, average thickness 135 μm, pore diameter 5 μm or more and 10 μm or less) was prepared as a flow path member.

-Water wettability evaluation-
When evaluated in the same manner as in Preparation Example 7, the water absorption time was 5.0 seconds on each surface, and the water wettability ratio B / A was 5.0 seconds / 5.0 seconds = 1.0. It was confirmed.

(Preparation Example 10)
-Production of flow path member-
A membrane film JM (hydrophilic PTFE, manufactured by Merck Millipore, average thickness: 100 μm, pore diameter: 5 μm) not subjected to hydrophobic treatment was prepared as a flow path member.

-Water wettability evaluation-
When evaluated in the same manner as in Preparation Example 7, the water absorption time was 30.0 seconds for all surfaces, and the water wettability ratio B / A was 30.0 seconds / 30.0 seconds = 1.0. It was confirmed.

(Preparation Example 11)
-Fabrication of channel member with one surface treated with hydrophobic treatment-
Fluorine coat spray (AMEDAS 420, Columbus Co., Ltd.) was sprayed on one side of membrane film JM (hydrophilic PTFE, manufactured by Merck Millipore, average thickness 100 μm, pore diameter 5 μm) for 50 seconds from 4.0 cm, A flow path member having one surface subjected to hydrophobic treatment was produced.

-Water wettability evaluation-
When evaluated in the same manner as in Preparation Example 7, the water absorption time was 30 seconds on the surface not sprayed with the spray, and the water absorption time was 70 seconds on the surface sprayed with the spray.
From this result, the water wettability (A) of the surface not sprayed with the spray becomes higher than the water wettability (B) of the surface sprayed with the spray, and the water wettability ratio B / It was confirmed that A was 70 seconds / 30 seconds = 2.3.

(Preparation Example 12)
-Production of flow path member-
A membrane film PES (polyethersulfone, manufactured by As One Corporation, average thickness 120 μm, pore diameter 0.45 μm) not subjected to hydrophobic treatment was produced as a flow path member.

-Water wettability evaluation-
When evaluated in the same manner as in Preparation Example 7, the water absorption time of each surface was 9.7 seconds, and the water wettability ratio B / A was 9.7 seconds / 9.7 seconds = 1.0. It was confirmed.

(Preparation Example 13)
-Fabrication of channel member with one surface treated with hydrophobic treatment-
Fluorine-coated spray (AMEDAS 420, Columbus Co., Ltd.) was sprayed for 4.0 seconds from one side of the membrane film PES (Polyethersulfone, manufactured by ASONE CORPORATION, average thickness 120 μm, pore diameter 0.45 μm) from a distance of 50 cm. Thus, a flow path member having one surface subjected to hydrophobic treatment was produced.

-Water wettability evaluation-
When evaluated in the same manner as in Preparation Example 7, the water absorption time was 9.7 seconds on the surface not sprayed with the spray, and the water absorption time was 30 seconds on the surface sprayed with the spray.
From this result, the water wettability A of the surface not sprayed with the spray is higher than the water wettability B of the surface sprayed with the spray, and the water wettability ratio B / A is 30. It was confirmed that 0 sec / 9.7 sec = 3.1.

(Preparation Example 14)
-Production of flow path member-
A membrane film JG (hydrophilic PTFE, manufactured by Merck Millipore, average thickness 55 μm, pore diameter 0.2 μm) not subjected to hydrophobic treatment was prepared as a flow path member.

-Water wettability evaluation-
When evaluated in the same manner as in Preparation Example 7, it was confirmed that the water absorption time was 150 seconds on each surface, and the water wettability ratio B / A was 150 seconds / 150 seconds = 1.0.

Example 1
<Preparation of test line transfer media>
-Formation of back layer-
The back layer coating solution of Preparation Example 1 was applied to one side of a polyethylene terephthalate (PET) film (Lumirror (registered trademark) F57, manufactured by Toray Industries, Inc.) having an average thickness of 4.5 μm as a support, and the coating was applied at 80 ° C. for 10 seconds. It was dried to form a back layer having an average thickness of 0.02 μm.

-Formation of release layer and reagent-immobilized layer (for immobilization)-
On the surface of the PET film opposite to the surface on which the back layer is formed, the release layer / reagent-immobilized layer (for immobilization) coating solution of Preparation Example 2 is applied and dried at 40 ° C. for 10 minutes, A release layer / reagent-immobilized layer (for fixation) having an average thickness of 20 μm was formed.

-Production of transfer media for test lines-
On the release layer / reagent-immobilized layer (for fixation), the reagent coating solution for test line of Preparation Example 4 was applied so as to be 12 μL per unit area (cm ² ) to form a water film, The transfer medium was placed in a container maintained at a relative humidity of 80% so that the water film was not dried, and allowed to stand at 25 ° C. for 10 minutes. After standing, the transfer medium was peeled off from the water film, and the surface of the release layer / reagent-immobilized layer was washed under the following conditions to prepare a test line transfer medium.

-Cleaning of transfer media-
The transfer medium peeled off from the water film was attached to a shaker (Shake-XR equipped with WR-3636, both manufactured by Taitec Co., Ltd.) with the reagent-immobilized side as the front (table). Next, distilled water was poured onto the solid phased surface so as to be 100 μL per unit area (cm ² ), and then gently shaken at 25 ° C. with a shaking speed of 20 rpm / minute for 1 minute. After completion of shaking, the supernatant liquid after washing was removed. This operation was repeated a total of 5 times. Finally, the supernatant liquid after washing was thoroughly cut from the surface of the transfer medium, and the transfer medium was washed (5 washings).

-Immobilization of reagents-
After washing, it was dried for 15 minutes in a desiccator at a temperature of 25 ° C. and a relative humidity of 20% to immobilize the reagent on the release layer / reagent immobilization layer (for immobilization). Thus, a test line transfer medium of Example 1 was obtained.
Here, when the surface of the release layer / reagent-immobilized layer was analyzed by XPS (AXIS ULTRA, manufactured by Shimadzu Corporation), it was confirmed that the capture antibody was immobilized at 728 ng / cm ² .

<Preparation of transfer medium for control line>
In the production of the test line transfer medium, except that the test line reagent coating liquid of Preparation Example 4 was changed to the control line reagent coating liquid of Preparation Example 5, A control line transfer medium of Example 1 was produced.

<Preparation of labeled antibody transfer medium>
A back layer and a release layer are formed on one side of a polyethylene terephthalate (PET) film (Lumirror (registered trademark) F57, manufactured by Toray Industries, Inc.) having an average thickness of 4.5 μm as a support in the same manner as the test line transfer medium. After forming the reagent-immobilized layer (for fixation), the reagent-immobilized layer (for release) coating solution of Preparation Example 3 is applied onto the release layer / reagent-immobilized layer (for fixation), It was dried at 40 ° C. for 10 minutes to form a reagent-immobilized layer (for release) having an average thickness of 5 μm.
Next, the reagent coating solution for labeled antibody of Preparation Example 6 is applied onto the reagent-immobilized layer (for release) so as to be 14 μL / cm ^2, and dried in a vacuum dryer at 25 ° C. for 5 hours. Then, the reagent was immobilized on the reagent-immobilized layer (for release), and the labeled antibody transfer medium of Example 1 was produced.

<Production of inspection device>
The inspection apparatus shown in FIGS. 10A and 10B was produced as follows.
FIG. 10A is a top view of the inspection apparatus of the embodiment. FIG. 10B is a cross-sectional view of the inspection apparatus of FIG. 10A along the line BB. Note that the inspection apparatus illustrated in FIGS. 10A and 10B is merely an example, and the inspection apparatus illustrated in FIGS. 11A and 11B and the inspection apparatus illustrated in FIGS. 12A and 12B may be configured.

-Production of substrate and flow path member-
Polyester hot melt adhesive (Aronmelt PES375S40, Toa Gosei Co., Ltd.) as a thermoplastic resin on top of a PET film (Lumirror (registered trademark) S10, manufactured by Toray Industries, Inc., average thickness 50 μm) cut to a width of 40 mm and a length of 80 mm Manufactured) was heated to 190 ° C. so as to have an average thickness of 50 μm on the PET film using a roll coater, and coated to form an adhesive layer.
After the PET film on which the adhesive layer was formed was allowed to stand for 2 hours or longer, the hydrophilic PTFE membrane film prepared in Preparation Example 7 was cut into a width of 40 mm and a length of 70 mm to prepare a flow path member. One end of the long axis side and one end of the long axis side of various members (this end is the upstream end and the opposite side is the downstream end) so that the sprayed surface of the membrane film is aligned with the adhesive layer surface And a load of 1 kgf / cm ² was applied at 150 ° C. for 10 seconds. Finally, the substrate 11 was cut along the long axis direction so as to have a width of 4 mm × a length of 80 mm, and a base material 11 having a surface on which the spray of the membrane film was not sprayed was produced.

-Transcription of labeled antibody-
After the base material and the side of the labeled antibody transfer medium on which the reagent is solid-phased are faced and overlapped, using a thermal transfer printer, as shown in FIG. 10A and FIG. The labeled antibody transfer medium was transferred in a pattern of width 3 mm × length 10 mm at a position 20 mm away from the end (resin layer 15 a).
The thermal transfer printer has a thermal head (manufactured by TDK Corporation) with a dot density of 300 dpi, and has built an evaluation system with a printing speed of 8.7 mm / second and a printing energy of 0.35 mJ / dot.

-Transfer of test line and control line-
As shown in FIGS. 10A and 10B, the test line transfer medium was transferred in a line of 4 mm height × 1 mm length at a position 15 mm away from the transfer position of the transfer medium for the labeled antibody (test line). 15b). Further, the control line transfer medium was transferred in a line shape having a height of 4 mm and a length of 1 mm at a position 5 mm away from the transfer position of the test line transfer medium (control line 15c). Each line was formed under the same transfer conditions as the above-mentioned “transfer of labeled antibody”.

-Production of absorbent member-
An absorption member 14 (Surewick C248, manufactured by Merck Millipore) was prepared, and as shown in FIGS. 10A and 10B, the prepared absorption member 14 was provided to obtain the immunochromatographic assay (inspection apparatus 10) of Example 1. .

<Line evaluation>
-Preparation of test solution-
As a developing solution, a D-PBS (−) solution of 0.1 mass% Tween 20 (P9416-50ML, Sigma-Aldrich) was prepared.
Next, the developing solution was added to Human IgG to prepare a test solution of 500 μg / mL.

-Reaction-
10 μL of the test solution was dropped at the upstream end of the immunochromatographic assay shown in FIG. 10A and FIG. 10B for reaction, and after 15 minutes, the test line was observed.

-Measurement of color intensity (line density)-
The immunochromatographic assay after completion of the reaction is placed in a measurement housing case, measured using a chromatographic reader (DiaScan 10, manufactured by Otsuka Electronics Co., Ltd.), and the color intensity (color density) of the line is determined from the readings. It was evaluated with. The results are shown in Table 2. The larger the reading value, the stronger the color intensity (the darker the line density).
[Evaluation criteria]
◎: Reading value is 150 or more ○: Reading value is less than 150 and 120 or more Δ: Reading value is less than 120 and 70 or more ×: Reading value is less than 70 or cannot be confirmed as a line and cannot be measured

-Line visibility evaluation-
For the immunochromatographic assay test line after the reaction was completed, the visibility of the line was evaluated according to the following evaluation criteria. The results are shown in Table 2. Examples of evaluation criteria are shown in Table 1. Table 2 is a digital image of the test line after evaluation.
[Evaluation criteria]
○: There is no uneven color density in the width direction of the line, and the whole is uniformly colored. Δ: The color density of the line is shaded. ×: The line is in a line shape, but part of the line is interrupted. , Where the color is missing inside the line

(Example 2)
In Example 1, except that the flow path member of Preparation Example 7 was replaced with the flow path member of Preparation Example 8, the inspection apparatus of Example 2 was manufactured in the same manner as in Example 1, and the same as in Example 1 Evaluated. The results are shown in Table 2.

(Comparative Example 1)
In Example 1, except that the flow path member of Preparation Example 7 was replaced with the flow path member of Preparation Example 9, the inspection apparatus of Comparative Example 1 was manufactured in the same manner as in Example 1, and the same as in Example 1 Evaluated. The results are shown in Table 2.

(Comparative Example 2)
In Example 1, except that the flow path member of Preparation Example 7 was replaced with the flow path member of Preparation Example 10, the inspection apparatus of Comparative Example 2 was manufactured in the same manner as in Example 1, and the same as in Example 1 Evaluated. The results are shown in Table 2.

(Comparative Example 3)
In Example 1, except that the flow path member of Preparation Example 7 was replaced with the flow path member of Preparation Example 11, the inspection apparatus of Comparative Example 3 was manufactured in the same manner as in Example 1, and the same as in Example 1 Evaluated. The results are shown in Table 2.

(Comparative Example 4)
In Example 1, except that the flow path member of Preparation Example 7 was replaced with the flow path member of Preparation Example 12, the inspection device of Comparative Example 4 was manufactured in the same manner as in Example 1, and the same as in Example 1 Evaluated. The results are shown in Table 2.

(Comparative Example 5)
In Example 1, except that the flow path member of Preparation Example 7 was replaced with the flow path member of Preparation Example 13, the inspection device of Comparative Example 5 was manufactured in the same manner as in Example 1, and the same as in Example 1 Evaluated. The results are shown in Table 2.

(Comparative Example 6)
In Example 1, except that the flow path member of Preparation Example 7 was replaced with the flow path member of Preparation Example 14, the inspection apparatus of Comparative Example 6 was manufactured in the same manner as in Example 1, and the same as in Example 1 Evaluated. The results are shown in Table 2.

  From the results of Table 2, in Examples 1 and 2, in the flow path member, the water wettability on the side opposite to the side having the resin layer was made lower than the water wettability on the side having the resin layer, so that the resin Because the test solution containing the antigen easily flows on the side having the layer, and the antigen-antibody reaction of the line transferred to the side having the resin layer proceeds, the color intensity is strong and the entire line is uniformly colored. It was confirmed that the visibility was excellent.

In Comparative Example 1, it was found that the nitrocellulose membrane that has been conventionally used as a flow path member has a low color intensity and poor visibility as compared with Examples 1 and 2.
In Comparative Examples 2, 4, and 6, there is no difference between the wettability of water on the side opposite to the side having the resin layer and the wettability of water on the side having the resin layer in the flow path member. Was low and visibility was poor. Furthermore, in Comparative Example 6, since the flow path member itself became thinner and the pore diameter became smaller, it was difficult for the test solution to flow.
In Comparative Examples 3 and 5, in the flow path member, when the ratio of the water wettability on the side opposite to the side having the resin layer and the water wettability on the side having the resin layer exceeds 2.0, the inspection is performed. The flow of the liquid deteriorated as a whole, the line did not develop color, and the visibility was poor.

Aspects of the present invention are as follows, for example.
<1> A porous flow path member for flowing a sample, and a resin layer at least at one location on the flow path member, and solidifying the resin layer on a surface facing the flow path member The reagent contains an antibody that reacts with the specimen, and the water wettability A on the side of the flow path member having the resin layer, and the water wettability of the flow path member on the side opposite to the side having the resin layer. The inspection apparatus is characterized in that a ratio B / A to B is 1.5 or more and 2.0 or less.
<2> In the above <1>, the water wettability of the flow path member increases from the opposite side of the flow path member having the resin layer toward the side of the flow path member having the resin layer. It is an inspection apparatus of description.
<3> The inspection device according to any one of <1> to <2>, wherein a side opposite to the side having the resin layer of the flow path member is subjected to a hydrophobic treatment.
<4> The inspection apparatus according to any one of <1> to <3>, wherein the hydrophobic treatment is a fluorine treatment.
<5> The inspection apparatus according to any one of <1> to <4>, wherein the flow path member is a hydrophilic porous material.
<6> The inspection apparatus according to <5>, wherein a porosity of the hydrophilic porous material is 40% or more and 90% or less.
<7> The inspection apparatus according to any one of <5> to <6>, wherein an average thickness of the hydrophilic porous material is 0.01 mm or more and 0.3 mm or less.
<8> The inspection apparatus according to any one of <5> to <7>, wherein a porous pore diameter of the hydrophilic porous material is 0.45 μm or more and 10 μm or less.
<9> The inspection apparatus according to any one of <1> to <8>, wherein the resin layer is a non-porous body.
<10> The inspection apparatus according to any one of <1> to <9>, wherein the antibody is at least one of a labeled antibody and a capture antibody.
<11> The inspection apparatus according to <10>, wherein an amount of the capture antibody immobilized is 500 ng / cm ² or more.
<12> The inspection apparatus according to any one of <1> to <11>, a sample collection unit for collecting the sample, and at least one selected from a liquid for processing the sample. It is the inspection kit characterized by having.
<13> a support, a release layer provided on the support, and a reagent-immobilized layer provided on the release layer, wherein the surface of the reagent-immobilized layer is < A transfer medium comprising the antibody that reacts with the specimen according to any one of <1> to <11>.
<14> The transfer medium according to <13>, wherein the release layer has an average thickness of 0.5 μm or more and 50 μm or less.
<15> The transfer medium according to any one of <13> to <14>, wherein an adhesion amount of the release layer is 0.5 g / m ² or more and 50 g / m ² .
<16> The transfer medium according to any one of <13> to <15>, wherein an adhesion amount of the reagent coating solution in the reagent-immobilized layer is 0.2 g / m ² or more and 50 g / m ² .
<17> The transfer medium according to any one of <13> to <16>, wherein applied energy in thermal transfer is 0.05 mJ / dot or more and 0.5 mJ / dot or less.
<18> a step of subjecting one surface of a porous flow path member to a hydrophobic treatment; a surface of the reagent-immobilized layer of the transfer medium according to any one of <13> to <17>; and the flow path member A method of manufacturing an inspection apparatus, comprising the step of bringing the reagent-immobilized layer into contact with a non-hydrophobic-treated surface and transferring the reagent-immobilized layer to the flow path member.
<19> A support and a release layer / reagent-immobilized layer on the support, wherein the surface of the release layer / reagent-immobilized layer is any one of <1> to <18> A transfer medium comprising an antibody that reacts with the specimen described in 1.
<20> a step of subjecting one surface of the porous flow path member to a hydrophobic treatment; and a hydrophobic treatment of the surface of the release layer and reagent-immobilized layer of the transfer medium according to <19> and the flow path member. And a step of bringing the release layer / reagent-immobilized layer into contact with the surface of the flow path member.

  The inspection apparatus according to any one of <1> to <11>, the inspection kit according to <12>, the transfer medium according to any one of <13> to <17>, and <19>, According to the method for manufacturing an inspection apparatus according to any one of <18> and <20>, the above-described 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)
19 Conjugate area 30 Test solution (example of specimen)
DESCRIPTION OF SYMBOLS 50 Inspection kit 100 Transfer medium 101 Support body 102 Release layer 103 Reagent immobilization layer 105 Release layer and reagent immobilization layer

JP 2010-256309 A Japanese Patent Laying-Open No. 2015-007604

Claims (9)

A porous channel member for flowing a specimen;
A resin layer in at least one place on the flow path member,
The solid phase reagent on the surface of the resin layer facing the flow path member contains an antibody that reacts with the specimen,
A ratio B / A of water wettability B on the side opposite to the side having the resin layer of the flow path member to water wettability A on the side having the resin layer of the flow path member is 1.5 or more. Inspection apparatus characterized by being 2.0 or less.   The inspection apparatus according to claim 1, wherein water wettability of the flow path member increases as the flow path member approaches the side of the flow path member having the resin layer from the side opposite to the side having the resin layer. .   The inspection apparatus according to claim 1, wherein a side opposite to the side having the resin layer of the flow path member is subjected to a hydrophobic treatment.   The inspection apparatus according to claim 3, wherein the hydrophobic treatment is a fluorine treatment. An inspection apparatus according to any one of claims 1 to 4,
A test kit comprising: a sample collecting means for collecting the sample; and at least one selected from a liquid for processing the sample. A support;
A release layer provided on the support;
A reagent-immobilized layer provided on the release layer,
5. A transfer medium, wherein a surface of the reagent-immobilized layer has the antibody that reacts with the specimen according to any one of claims 1 to 4. A step of subjecting one surface of the porous channel member to a hydrophobic treatment;
The surface of the reagent-immobilized layer of the transfer medium according to claim 6 is brought into contact with the surface of the flow path member that has not been subjected to a hydrophobic treatment to transfer the reagent-immobilized layer to the flow path member. The manufacturing method of the test | inspection apparatus characterized by including the process. A support;
A release layer and reagent-immobilized layer on the support;
5. A transfer medium, wherein a surface of the release layer / reagent-immobilized layer has the antibody that reacts with the specimen according to any one of claims 1 to 4. A step of subjecting one surface of the porous channel member to a hydrophobic treatment;
The surface of the release layer / reagent-immobilized layer of the transfer medium according to claim 8 is brought into contact with the surface of the flow path member that has not been subjected to a hydrophobic treatment, and the release layer / reagent-immobilized layer is brought into contact with the surface. And a step of transferring to the flow path member.