JP2017125778A - Inspection device and method for manufacturing the same, and inspection kit and transfer medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection device that can gradually release an agent reacting with a specimen and can measure a detection object in the specimen with high sensitivity.SOLUTION: There is provided an inspection device that comprises a porous channel member for causing a specimen to flow and a resin layer provided at least at one place on the channel member, and has an antibody reacting with the specimen and polyethylene glycol converted into a solid phase between the resin layer and the channel member.SELECTED DRAWING: Figure 1

Description

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

  In recent years, in-vitro diagnostic apparatuses for the purpose of POCT (Point of Care Testing) have rapidly spread. As a typical example, a test apparatus using a measurement method called an immunochromatography method, which can be measured without pretreatment and detects an antigen in a test solution using a specific reactivity of an antibody, is used.

As such an inspection apparatus, for example, a sample pad as a receiving part for a test liquid, a conjugate pad for reacting the test liquid supplied from the sample pad, and a test liquid supplied from the conjugate pad are flowed Some are composed of membrane membranes.
Among these, a resin layer in which a reagent such as a capture antibody, a labeled antibody, or a detection indicator is non-porous on the upper part of a porous flow path member such as a membrane film in which a flow path for flowing a sample is formed, There has been proposed an inspection apparatus that is solid-phased on the opposing surface of a flow path member (see, for example, Patent Document 1).

  However, in the proposed inspection apparatus, it is difficult to release the labeled antibody from the resin layer in a sustained manner, and accordingly, there is a problem that the color development on the test line and the control line is weak.

  On the other hand, in the immunochromatography method, various studies have been made to increase the visibility and detection sensitivity of an inspection device. As a technique for that purpose, an antibody antigen reaction is promoted, a capture antibody, a labeled antibody, a detection indicator, etc. These reagents are stabilized in a dry state and the dispersibility of the labeled antibody during development is improved. For example, it is generally known that the antibody antigen reaction is enhanced with a water-soluble polymer, and in immunochromatography, a detection apparatus in which a reagent labeled with serum albumin and a linear water-soluble polymer has been proposed ( For example, see Patent Document 2). In addition, an immunochromatographic method in which a vinyl-based water-soluble polymer having an oxygen atom-containing polar group is present in a measurement system as a dispersant has been proposed in order to suppress aggregation of labeled antibodies (for example, see Patent Document 3).

  An object of the present invention is to provide an inspection apparatus that can release a reagent that reacts with a specimen in a controlled manner and can measure a detection target in the specimen with high sensitivity.

The test apparatus of the present invention as a means for solving the above problems includes a porous flow path member for flowing a specimen,
A resin layer in at least one place on the flow path member,
An antibody that reacts with the specimen and polyethylene glycol are immobilized between the resin layer and the flow path member.

  ADVANTAGE OF THE INVENTION According to this invention, the reagent which reacts with a test substance can be discharge | released slowly, and the test | inspection apparatus which can measure the detection target object in a test substance with high sensitivity can be provided.

FIG. 1 is a top view showing an example of the inspection apparatus of the present invention. FIG. 2 is a cross-sectional view taken along the line 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 an example of the inspection apparatus used in the experimental example. 10B is a cross-sectional view taken along the line BB of FIG. 10A. FIG. 11A is a top view illustrating another example of the inspection apparatus used in the experimental example. FIG. 11B is a cross-sectional view taken along the line CC of FIG. 11A.

(Inspection equipment)
An inspection apparatus according to 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 between the resin layer and the flow path member. The antibody that reacts with the specimen and polyethylene glycol are solid-phased, and further include other members as necessary.
Examples of the antibody that reacts with the specimen include a capture antibody and a labeled antibody.
In the inspection apparatus, an antibody that reacts with the sample and polyethylene glycol are solid-phased between the resin layer and the flow path member, so that the antibody that reacts with the sample is gradually released from the resin layer. The detection target in the specimen is released with high sensitivity.

  The effect of adding a water-soluble polymer in Patent Documents 2 and 3 is to enhance the antibody antigen reaction efficiency and to improve the color intensity by improving the dispersibility of the labeled antibody during development. The effect has not been confirmed. Further, these effects have been reported in a conventional inspection apparatus in which a capture antibody is solid-phased on a fiber or the like inside a flow path member. However, in the case of an inspection apparatus in which the capture antibody is solidified on the opposite surface of the flow channel member on the upper surface of the flow channel member as proposed in Patent Document 1 Is based on the knowledge that the color intensity of the judgment line (test line and control line) is low because the labeled antibody is placed on a fiber such as glass fiber together with a water-soluble polymer. It is.

The inspection apparatus includes a porous flow path member having a flow path for flowing a specimen;
A first resin layer provided in at least one location on the flow path member and having a capture antibody immobilized on a surface facing the flow path member;
It is preferable to have a second resin layer provided on the flow path member and having a polyethylene glycol and a labeled antibody immobilized on a surface facing the flow path member.
It is preferable to have a plurality of the first resin layers in order to confirm that the specimen has moved without any problem in the inspection apparatus.

In the inspection apparatus, a capture antibody is solid-phased on a surface of the first resin layer provided in at least one place on the flow path member that faces the flow path member.
By immobilizing the capture antibody on the surface of the first resin layer facing the flow path member, the specimen can be detected on the resin layer side. Thereby, the detection part can detect with high sensitivity by the resin layer by which the capture antibody was solid-phased in high density like the conventional ELISA (Enzyme Linked Immuno Solvent Assay) method. On the other hand, the test solution is developed in the flow path (flow path member) using the capillary phenomenon as a driving force, as in the conventional immunochromatography method, so that the reaction proceeds. A judgment line is obtained.

In the inspection apparatus, when the second resin layer has the labeled antibody, the labeled antibody is solid-phased with polyethylene glycol (PEG).
The polyethylene glycol is a polymer having a structure in which ethylene glycol is polymerized, and has high hydrophilicity. Therefore, by immobilizing the labeled antibody together with polyethylene glycol, the hydrophilicity of the second resin layer is increased, and the labeled antibody is gradually released from the resin layer, thereby improving the color development intensity. An inspection device can be obtained.

The polyethylene glycol preferably has a weight average molecular weight of 1,000 or more and 30,000 or less, and more preferably 5,000 or more and 10,000 or less. When the weight average molecular weight is 1,000 or more and 30,000 or less, the hydrophilicity of the second resin layer can be enhanced, and the labeled antibody is gradually released from the resin layer, and color development An inspection apparatus with improved strength can be obtained.
Examples of the method for measuring the weight average molecular weight of polyethylene glycol include a gel permeation chromatography (GPC) method, a terminal group determination method, and a viscosity method. Among these, the GPC method is preferable.

The content of the polyethylene glycol is preferably 0.015 times or more and 1.5 times or less, more preferably 0.03 times or more and 0.3 times or less by mass ratio with respect to the antibody or the labeled antibody. 0.03 times to 0.15 times is more preferable.
When the content of the polyethylene glycol is 0.015 times or more and 1.5 times or less in terms of mass ratio, the hydrophilicity of the second resin layer can be increased, and the antibody gradually increases from the resin layer. Thus, an inspection apparatus with improved color intensity can be obtained.
Therefore, the weight average molecular weight of the polyethylene glycol is 5,000 or more and 20,000 or less, and the content of the polyethylene glycol is 0.03 times or more and 1. Preferably, the polyethylene glycol has a weight average molecular weight of 5,000 or more and 10,000 or less, and the polyethylene glycol content is in a mass ratio with respect to the antibody or the labeled antibody. It is more preferably 0.03 times or more and 0.15 times or less, and the weight average molecular weight is 8,000, and the content of the polyethylene glycol is 0.03 times or more to 0 with respect to the antibody or the labeled antibody. More preferably, it is 15 times or less.
The content of the polyethylene glycol is calculated from the amount of the labeled antibody and the added amount of polyethylene glycol at the time of preparing the labeled antibody and at the time of immobilization.

  Here, the inspection apparatus of the present invention will be described with reference to the drawings. 1 to 5 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 the 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. 4 and 5 are cross-sectional views showing another example of the inspection apparatus at the facing portion of the flow path member and the resin layer.

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

In the 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 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.

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, in the inspection apparatus 10, the resin layer 15 a (second resin layer) contains an amphiphilic resin 151 having many hydrophilic groups 152, preferably a main component (content is 50). Mass% or more).
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.

Polyethylene glycol (not shown) and labeled antibody 16 are solid-phased on the surface of the resin layer 15a (second resin layer) facing the flow path member 12. The polyethylene glycol has a hygroscopic property, has a low drying property, and is excellent in maintaining the amount of moisture retained with respect to temperature changes. Therefore, by immobilizing the labeled antibody together with the polyethylene glycol, the hydrophilicity of the second resin layer is increased, and the labeled antibody is gradually released from the second resin layer. An inspection apparatus with improved strength can be obtained.
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.
The polyethylene glycol has a hydrophilic group, and is thereby solidified on the surface corresponding to the flow path member 12 in the resin layer 15a.
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.
When the test solution contains the antigen 31, the released labeled antibody 16 and the antigen 31 react and bind to each other 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 insolubility means substantially insoluble in water. Said substantially insoluble in water means that the amount of change in the mass 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 or less. It means that there is. 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, 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% by mass or more) is preferable.
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 fixed to the surface of the resin layer 15b facing the flow path member 12. Be phased. 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 15b 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, in the inspection apparatus 10, the resin layer 15 c (first resin layer) contains the hydrophobic resin 155 or the amphiphilic resin 154 having a large number of hydrophobic groups 153. It is preferable that the resin 155 or the amphiphilic resin 154 is configured as a main component (content is 50% by mass or more).
The capture antibody 18 is solid-phased on the surface of the resin layer 15c facing the flow path member 12 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 contain voids, and is a structure that is opposite to a porous material including voids provided to promote absorption of liquid such as a membrane. Point to. 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.

Next, the feature by the said resin layer being a non-porous body is demonstrated.
Conventional test lines and control lines are formed by directly applying a solution in which a capture antibody is dissolved to a flow path member made of a hydrophilic porous material. Therefore, the capture antibody diffuses into the porous material as the liquid penetrates. However, the color development of labeling particles such as gold colloid particles that bind to the capture antibody present inside the porous material cannot be actually detected because light scattering occurs. That is, most of the capture antibodies are not effectively used.
Generally, the color developing particles that can be detected with a porous material are about 5 μm deep from the surface. In order to immobilize the capture antibody necessary for the inspection in the region having a depth of 5 μm, a large amount of capture antibody must be applied in consideration of diffusion in the thickness direction. That is, the amount of the capture antibody applied increases in proportion to the thickness of the porous material.

  In the present invention, since the resin layer made of a non-porous material containing a large amount of hydrophobic groups is used for immobilizing the capture antibody, the capture antibody does not enter the resin layer, and the resin layer Immobilized only on the surface. The labeling particles are bound to the capture antibody immobilized on the surface of the resin layer, and the color is developed. However, since the color can be detected through a resin layer made of a non-porous material that does not cause light scattering, The utilization efficiency of coloring by particles can be greatly improved. Further, since there are no extra colored particles in the thickness direction, there is a merit that the amount of the capture antibody applied may be extremely small. For example, assuming that the flow path member made of a hydrophilic porous material has a thickness of 100 μm, assuming that only a color of a thickness of 5 μm can be used from the surface, the application of a capture antibody used to obtain the same color intensity The amount can be reduced to 1/20.

  Therefore, in the present invention, since a resin layer made of a non-porous material containing a large amount of hydrophobic groups is used for immobilizing the capture antibody, it is possible to greatly improve the efficiency of color development by the labeling particles. Since there are no extra colored particles in the direction, the coating amount of the capture antibody can be reduced as compared with the prior art.

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

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

  The material of the substrate 11 is not particularly limited and can be appropriately selected according to the purpose. For example, polyvinyl chloride, polyethylene terephthalate, polypropylene, polystyrene, polyvinyl acetate, polycarbonate, polyacetal, modified polyphenyl ether, Examples include polybutylene phthalate and ABS resin. Among these, polyethylene terephthalate is preferable because it is inexpensive and highly versatile.

There is no restriction | limiting in particular as a shape of the base material 11, Although it can select suitably according to the objective, A sheet form is preferable.
There is no restriction | limiting in particular in the average thickness of the base material 11, Although it can select suitably according to the objective, 0.01 mm or more and 0.5 mm or less are preferable. When the average thickness is 0.01 mm or more, the strength as the substrate 11 is good, and when it is 0.5 mm or less, the flexibility of the substrate is good and can be suitably used as a sensor. .
Here, the average thickness 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 is not particularly limited as long as it is a member capable of flowing the inspection liquid 30, and can be appropriately selected according to the purpose. Examples thereof include a hydrophilic porous material. It is done.
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).
3 to 5, the gap 12 a is a gap formed in each cross section, and the gap 12 b 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 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 in the 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.

<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, if the hydrophilicity of the conjugate pad is too high, the inspection liquid tends to remain on the conjugate pad, and it is difficult to transfer to the membrane. Conversely, if the hydrophobicity of the conjugate pad is too high, the transfer of the test solution to the membrane will be faster, but the test time will be longer due to the decrease in water absorption of the test solution from the sample pad. It came to require. For this reason, the fiber F1 which can be used as a conjugate pad was limited. Furthermore, in the conventional inspection apparatus, the labeled antibody 16 is solid-phased on the fiber F1 constituting 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 to the fiber F1. That is, the conventional inspection apparatus has a limited number of usable fibers F1 and labeled antibodies 16 in 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 has a limited number of usable fibers F2 and capture antibodies 17 in design.

  In the inspection apparatus 10 of this embodiment, reagents such as a labeled antibody 16, a capture antibody 17, a capture antibody 18, and polyethylene glycol are immobilized on the resin layer 15 (15a, 15b, 15c). Therefore, the release of the labeled antibody or the immobilization of the capture antibody can be controlled according to 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 refers to a cause (driving force) of a change in which hydrophobic molecules and hydrophobic groups that do not adjust to water in water gather. Specifically, when a hydrophobic molecule or a molecule having a hydrophobic group is put in water, in many cases, it does not simply dissolve, but the hydrophobic molecule or hydrophobic group is in contact with each other and contacts with the water molecule. Try to reduce the area as much as possible. As a result, hydrophobic molecular species come to gather close to each other, and this is a phenomenon that appears to have a binding force acting between 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, 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 other than gold colloid, and can be appropriately selected according to the purpose. For example, metal colloid other than gold colloid, enzyme-labeled particles containing an enzyme, dye Examples thereof include colored particles containing a fluorescent substance, fluorescent particles containing a fluorescent substance, and magnetic substance-containing particles containing a magnetic substance. These may be used alone or in combination of two or more.
The antibody may be in any form of monoclonal antibody, polyclonal antibody, chimeric antibody, Fab antibody, and (Fab) ₂ antibody.

The capture antibody 17 to be immobilized on the resin layer 15b is not particularly limited as long as it has a hydrophobic site and reacts with the antigen 31, and can be appropriately selected according to the purpose. , Antibodies such as Anti-human IgG, antibodies against various allergens, and the like.
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, and the antibodies listed above.

  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 and dried up. After a solution containing a reagent is applied to the resin layer 15, the solution is left to stand (incubate) in a humid environment so as not to dry. Then, a method of washing components other than antibodies such as inorganic salts with distilled water and the like, followed by drying, and the like can be mentioned.

When the reagent such as the capture antibody is handled as a coating solution, it is not particularly limited and can be appropriately selected according to the 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 can be appropriately selected according to the purpose. For example, a buffer solution generally used for antibody dilution Etc. Examples of the buffer include phosphate buffered saline (PBS), trishydroxymethylaminomethane (Tris) -HCl, and Good buffer. Among these, phosphate buffered saline (PBS) and Good buffer are preferable.

The phosphate buffered saline (PBS) is preferably pH 4-10, more preferably pH 6-8. The composition of the PBS is not particularly limited, and various compositions exist. Examples of the composition of the PBS include 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L NaH ₂ PO _{4 and} 0.24 g / L KH ₂ PO ₄ . There are also compositions that do not contain potassium and compositions that contain calcium and magnesium.
Examples of the good buffer include 2- (N-monophorino) ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-2-sulfonic acid (HEPES), and the like.

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 absorbent 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. It is done.

<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 transfer medium for an inspection apparatus used in the thermal transfer method and a method for manufacturing the inspection apparatus will be described.

(Transfer media for inspection equipment)
In the first embodiment of the transfer medium for an inspection apparatus according to the present invention, a support, a release layer provided on the support, and a capture antibody provided on a surface of the release medium are provided at at least one location of the release layer. 1 reagent-immobilized layer, and a second reagent-immobilized layer provided on one surface of the release layer and having a labeled antibody and polyethylene glycol on the surface, and, if necessary, other members It has.

  In the second embodiment of the transfer medium for an inspection apparatus of the present invention, a support, a first release layer / reagent-immobilized layer provided on at least one place of the support and having a capture antibody on the surface, A second release layer / reagent-immobilized layer having a labeled antibody and polyethylene glycol on the surface is provided at one location of the support, and further includes other members as necessary.

Here, a transfer medium for an inspection apparatus used when a resin layer is provided on a flow path member will be described with reference to the drawings. 8A and 8B are schematic cross-sectional views showing an example of a transfer medium for an inspection apparatus according to the present invention.
When the thermal transfer method is used, the transfer medium 100 to which the capture antibody is uniformly attached in advance can be used, so that the concentration difference of the capture antibody (17, 18) in the test line or the control line becomes small. 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, of the release layer / reagent solid phase layer 105, the antibody Although the portion including the surface on which the solid phase is immobilized is transferred, a part of the release layer / reagent solid phase layer 105 may remain on the support 101 side.

There is no restriction | limiting in particular in the support body 101, what was synthesize | combined suitably may be used and a commercial item 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 contains a wax and a binder resin, and further contains 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, furoic acid, behenic acid Fatty acids; higher alcohols such as stearic alcohol and behenyl alcohol; esters such as fatty acid esters of sorbitan; amides such as stearamide and oleinamide. These may be used alone or in combination of two or more. Among these, carnauba wax and polyethylene wax are preferable from the viewpoint of excellent peelability.

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

There is no restriction | limiting in particular as a formation method of the 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 in the 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.
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.

<First and second reagent-immobilized layers>
Of the first and second reagent-immobilized layers, an arbitrary reagent-immobilized layer is indicated as a reagent-immobilized layer 103.
The reagent-immobilized layer 103 only needs to contain a resin constituting the resin layer 15 in the inspection apparatus 10, and the material thereof is not particularly limited and can be appropriately selected according to the purpose.
The first reagent-immobilized layer corresponds to the resin layers 15b and 15c.
The second reagent-immobilized layer corresponds to the resin layer 15a.
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 the sharpness 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.

<First and second release layer / reagent-immobilized layer>
Of the first and second release layer / reagent-immobilized layer, an arbitrary release layer / reagent-immobilized layer is indicated as a release layer / reagent-immobilized layer 105.
The release layer / reagent-immobilized layer 105 has both functions of a release layer and a reagent-immobilized layer, improves the releasability between the support 101 and the reagent-immobilized layer 103 during transfer, and By including the resin constituting 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.
The first release layer reagent-immobilized layer corresponds to the resin layers 15b and 15c.
The second release layer reagent-immobilized layer corresponds to the resin layer 15a.

  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 phase 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.
The average thickness of the release layer / reagent-immobilized layer 105 is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.5 μm or more and 50 μm or less. When the average thickness is 0.5 μm or more, the durability of the release layer / reagent solid-phased 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 the sharpness is improved.
The adhesion amount of the release layer / reagent solid phase layer 105 is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.5 g / m ² or more and 50 g / m ² or less. When the adhesion amount is 0.5 g / m ² or more, the coating amount is appropriate, and the release layer / reagent solid phase layer 105 (resin layer 15) is not damaged. Moreover, when the adhesion amount is 50 g / m ² or less, the drying time is appropriate, and the release layer / reagent solid phase layer 105 is not uneven.

-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. A solution containing 16 and polyethylene glycol or a capture antibody (17, 18) is applied to form a uniform coating film. Subsequently, by drying the coating film, the labeled antibody 16 and polyethylene glycol, or the capture antibody (17, 18) are immobilized on the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105. be able to.

-Immobilization of labeled antibody and polyethylene glycol-
The method for immobilizing the labeled antibody is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the surface of the second reagent-immobilized layer as the reagent-immobilized layer 103 or the release layer The second release layer as the reagent-immobilized layer 105 is coated with a coating solution containing a labeled antibody and polyethylene glycol on the surface of the reagent-immobilized layer to form a water film, naturally dried, dried under reduced pressure, freeze-dried, etc. For example, a method of drying up and immobilizing the solution.
The water film is preferably applied so as to have a uniform thickness.
The application amount of the labeled antibody is not particularly limited and may be appropriately selected depending on the purpose. When a colloidal gold labeled antibody is used as the labeled antibody, the OD (optical density) is 1.0 to 20. It is preferable to apply 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. Further, when the coating amount is 600 μL or less, the amount of colloidal gold labeled antibody is appropriate, and the color of the line is good.
The coating amount of the polyethylene glycol is preferably 0.15 times or more and 1.5 times or less, more preferably 0.03 times or more and 0.3 times or less by mass ratio with respect to the labeled antibody.
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.) to 50 ° C., and the drying time is preferably 30 minutes to 24 hours. When the drying temperature is 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 more, drying can be performed appropriately, and when it is 24 hours or less, productivity is improved and discoloration can be prevented.

-Immobilization of capture antibody-
The method for immobilizing the capture antibody is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the surface of the first reagent-immobilized layer as the reagent-immobilized layer 103 or peeling The first release layer as the layer and reagent-immobilized layer 105 is coated with a capture antibody coating solution on the surface of the reagent-immobilized layer to form a water film, and dried up by natural drying, vacuum drying, freeze drying, etc. And then leaving the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 in distilled water or the like after leaving it in a humid environment so that the coating solution does not dry. And a method of washing and drying to solid phase (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 applied per unit area (cm ² ) to the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 is appropriate. Components such as inorganic salts contained are reduced, and the antibody can be sufficiently immobilized. In addition, when the coating concentration is 5,000 μg / mL or less, the wettability and viscosity of the coating solution are appropriate, so that 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 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 is not denatured. When the relative humidity is 30% or more, moisture volatilization is small during standing, so that unnecessary components other than antibodies are not solidified in a large amount.
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 (25 ° 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 room temperature (20 ° C.) to 50 ° C., and the drying time is preferably 30 minutes to 24 hours.
When the drying temperature is 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 immobilized amount of the 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 sufficient color development intensity can be obtained.
Here, examples of the method for analyzing the amount of the immobilized antibody present on the surface of the resin layer 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. For this reason, 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 may be used. 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 in the average thickness of the said protective film, Although it can select suitably according to the objective, 5 micrometers or more and 100 micrometers or less are preferable, and 10 micrometers or more and 30 micrometers or less are more preferable.

(Inspection device manufacturing method)
In the first embodiment of the method for manufacturing an inspection apparatus of the present invention, the first and second reagent-immobilized layers of the transfer medium for the inspection apparatus of the first embodiment of the present invention, and a porous flow Including a step of bringing the reagent-immobilized layer into contact with the channel member and transferring the reagent-immobilized layer to the channel member (hereinafter, also referred to as “reagent-immobilized layer transfer step”), and if necessary It includes other steps.

  In the second embodiment of the method for producing an inspection apparatus of the present invention, the first and second release layer / reagent-immobilized layers of the transfer medium for the inspection apparatus of the second embodiment of the present invention, and a porous A step of transferring the release layer / reagent-immobilized layer to the flow-path member by contacting with a quality channel member (hereinafter also referred to as “transfer step of release layer / reagent-immobilized layer”) ) And other steps as necessary.

<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 higher the hydrophilic component, the easier it is to adsorb to the porous part, which is the stationary phase, and the greater the number of times of desorption, the slower the rate of permeation through the flow path. On the other hand, a highly hydrophobic component penetrates without adsorbing to the stationary phase, and thus moves quickly in the flow path. By using the difference in the moving speed in the test liquid, the target component of the test liquid 30 is selectively extracted and reacted, so that the test apparatus 10 can be used as a highly functional chemical or biochemical sensor. 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 and polyethylene glycol (an example of a reagent) that have been converted from the resin layer 15a by bringing them 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 and polyethylene glycol immobilized on the resin layer 15a to release the labeled antibody 16 from the resin layer 15a. When the test solution 30 contains the antigen 31, the labeled antibody 16 released from the resin layer 15a reacts with and binds to the antigen 31 (see FIG. 3).

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

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

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

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

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

In the 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. The test solution 30 is a solvophilic one containing an organic solvent such as alcohols such as methyl alcohol, ethyl alcohol, 1-propyl alcohol and 2-propyl alcohol, and ketones such as acetone and MEK (methyl ethyl ketone). There may be. In this case, “hydrophilicity” in the above embodiment is replaced with “hydrophobic”, and “hydrophobic” is replaced with “hydrophilic”.

Hereinafter, although experimental examples of the present invention will be described, the present invention is not limited to these experimental examples.
In the following experimental examples, the weight average molecular weight of polyethylene glycol was measured as follows.

<Weight average molecular weight of polyethylene glycol>
The weight average molecular weight of polyethylene glycol (PEG) was measured by gel permeation chromatography (GPC) method. Specifically, the sample was dissolved in tetrahydrofuran (THF), filtered through a membrane filter, and the obtained filtrate was injected into the column. The column was a Shodex GPC KF-801 (8.0 mm ID × 300 mm) connected in a four-sided row. The carrier was THF, and the detector was a differential refractometer under the condition of a flow rate of 1.0 mL / min.
Using a commercially available PEG molecular weight standard, the weight average molecular weight of PEG in the sample was determined by comparing the retention time of the sample with the retention time of the standard.

(Preparation Example 1)
-Preparation of back layer coating solution-
Silicone rubber emulsion (manufactured by Shin-Etsu Chemical Co., Ltd., KS779H, 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 obtained.

(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 (manufactured by Ricoh Co., Ltd., B110AX stripping solution) was used as a stripping 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 (Sekisui Chemical Co., Ltd., BL-1, 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 (manufactured by Sigma Aldrich, I1886) was diluted 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 above-mentioned D-PBS (−) was added as an antibody diluent to Human IgG (Sigma Aldrich, I2511-10MG) to make 100 μg / mL, and a control line reagent coating solution was prepared.

(Preparation Example 6)
-Preparation of reagent coating solution for labeled antibody-
After adding 1 mL of KH ₂ PO ₄ buffer (pH 7.0) adjusted to 50 mM to 9 mL of colloidal gold solution (BBI, EMGC50), anti-hCG monoclonal antibody (Medix Biochemica, Anti-HCG, prepared to 50 μg / mL) was added. 1 mL of -hCG 5008 SP-5) was added and stirred. After standing for 10 minutes, 550 μL of a 1% by weight polyethylene glycol aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd., 168-1285) was added and stirred, and then a 10% by weight BSA aqueous solution (manufactured by Sigma Aldrich, A- 7906) was added and stirred.

Next, after centrifuging this solution for 30 minutes, the supernatant was removed, and the gold colloid was redispersed with an ultrasonic cleaner. Centrifugation was performed using a centrifuge (HimacCF16RN, manufactured by Hitachi Koki Co., Ltd.) under conditions of a centrifugal acceleration of 8,000 × g and 4 ° C. Thereafter, a gold colloid preservation solution [20 mM Tris-HCl buffer (pH 8.2), 0.05 mass% polyethylene glycol (weight average molecular weight 2,000), 150 mM NaCl, 1 mass% BSA aqueous solution, 0.1 mass% NaN ₃ aqueous solution] Dispersed in 20 mL and centrifuged again under the same conditions as described above. After removing about 1 mL of the supernatant, the supernatant was removed, and the gold colloid was redispersed with an ultrasonic cleaner. These operations were repeated, and the mixture was prepared so that OD = 15 with a colloidal gold preservation solution, and used as a labeled antibody reagent coating solution.

(Preparation Example 7)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 0.1 mass% polyethylene glycol (weight average molecular weight 2,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 7 was prepared.

(Preparation Example 8)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, preparation was performed except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 0.5 mass% polyethylene glycol (weight average molecular weight 2,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 8 was prepared.

(Preparation Example 9)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, Preparation Example 6 except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 1 mass% polyethylene glycol (weight average molecular weight 2,000). In the same manner as described above, a reagent coating solution for labeled antibody of Preparation Example 9 was prepared.

(Preparation Example 10)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 2.5 mass% polyethylene glycol (weight average molecular weight 2,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 10 was prepared.

(Preparation Example 11)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, Preparation Example 6 except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 5 mass% polyethylene glycol (weight average molecular weight 2,000). In the same manner as described above, a reagent coating solution for labeled antibody of Preparation Example 11 was prepared.

(Preparation Example 12)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 0.05 mass% polyethylene glycol (weight average molecular weight 8,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 12 was prepared.

(Preparation Example 13)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 0.1 mass% polyethylene glycol (weight average molecular weight 8,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 13 was prepared.

(Preparation Example 14)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 0.5 mass% polyethylene glycol (weight average molecular weight 8,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 14 was prepared.

(Preparation Example 15)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, Preparation Example 6 except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the colloidal gold preservation solution was changed to 1 mass% polyethylene glycol (weight average molecular weight 8,000). In the same manner as described above, a reagent coating solution for labeled antibody of Preparation Example 15 was prepared.

(Preparation Example 16)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 2.5 mass% polyethylene glycol (weight average molecular weight 8,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 16 was prepared.

(Preparation Example 17)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, Preparation Example 6 except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 5 mass% polyethylene glycol (weight average molecular weight 8,000). In the same manner as described above, a reagent coating solution for labeled antibody of Preparation Example 17 was prepared.

(Preparation Example 18)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 0.05 mass% polyethylene glycol (weight average molecular weight 20,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 18 was prepared.

(Preparation Example 19)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 0.1 mass% polyethylene glycol (weight average molecular weight 20,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 19 was prepared.

(Preparation Example 20)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 0.5 mass% polyethylene glycol (weight average molecular weight 20,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 20 was prepared.

(Preparation Example 21)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, Preparation Example 6 except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 1 mass% polyethylene glycol (weight average molecular weight 20,000). In the same manner as described above, a reagent coating solution for labeled antibody of Preparation Example 21 was prepared.

(Preparation Example 22)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 2.5 mass% polyethylene glycol (weight average molecular weight 20,000). In the same manner as in Example 6, the reagent coating solution for labeled antibody of Preparation Example 22 was prepared.

(Preparation Example 23)
-Preparation of reagent coating solution for labeled antibody-
In Preparation Example 6, Preparation Example 6 except that 0.05 mass% polyethylene glycol (weight average molecular weight 2,000) in the gold colloid preservation solution was changed to 5 mass% polyethylene glycol (weight average molecular weight 20,000). In the same manner as described above, a reagent coating solution for labeled antibody of Preparation Example 23 was prepared.

(Experimental example 1)
<No. 1>
<< Preparation of thermal transfer medium for test line >>
-Back layer formation-
The back layer coating solution of Preparation Example 1 was applied to one side of a polyethylene terephthalate (PET) film (Toray Co., Ltd., Lumirror F57) having an average thickness of 4.5 μm as a support, and dried at 80 ° C. for 10 seconds. A back layer having an average thickness of 0.02 μm was formed.

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

-Production of thermal transfer media for test lines-
Next, after the reagent coating solution for test line of Preparation Example 4 is applied on the release layer and reagent-immobilized layer (for fixation) so as to be 12 μL per unit area (cm ² ), a water film is formed. The thermal transfer medium was placed in a container maintained at a relative humidity of 80% so that the water film was not dried, and left at 25 ° C. for 10 minutes. After standing, the thermal 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.

-Washing-
The thermal transfer medium peeled 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 r / min 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 off from the surface of the thermal transfer medium and washed (5 washings). 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). As described above, No. of Experimental Example 1 A thermal transfer medium for test line 1 was obtained.
Here, when the surface of the release layer / reagent-immobilized layer was analyzed by XPS (AXIS ULTRA, manufactured by Shimadzu Kratos Co., Ltd.), it was confirmed that the capture antibody was immobilized at 728 ng / cm ² .

<< Preparation of thermal transfer medium for control line >>
In the production of the test line thermal transfer medium, except that the test line reagent coating solution of Preparation Example 4 was changed to the control line reagent coating solution of Preparation Example 5, No. of Experimental Example 1 A thermal transfer medium for control line 1 was obtained.

<< Preparation of thermal transfer medium for labeled antibody >>
A back layer and a release layer / reagent solid phase are formed on one side of a polyethylene terephthalate (PET) film (Lumirror F57, manufactured by Toray Industries, Inc.) having an average thickness of 4.5 μm as a support in the same manner as in the preparation of the test line thermal transfer medium. After the formation of the 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), and 10 ° C. at 40 ° C. It was dried for 5 minutes to form a reagent solid phase layer (for release) having an average thickness of 5 μm.
Next, the reagent coating solution for labeled antibody of Preparation Example 6 is applied to the reagent-immobilized layer (for release) so as to be 8 μL / cm ² and dried at 25 ° C. for 5 hours in a vacuum dryer. Thus, the reagent was immobilized on the reagent-immobilized layer (for release). As described above, No. of Experimental Example 1 No. 1 thermal transfer medium for labeled antibody was obtained.
In 1 mL of the reagent coating solution for labeled antibody, 0.5 μg of polyethylene glycol (weight average molecular weight 2,000) is contained per 33 μg of labeled antibody, and the labeled antibody has a polyethylene glycol with a mass ratio of 0.015 times. It is solid-phased with (weight average molecular weight 2,000).

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

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

Here, the porosity of the paper substrate 12 was determined by the following calculation formula 1 from the basis weight (g / m ² ), thickness (μm), and composition specific gravity of the paper substrate. Was 70%. In addition, it can be said that the said paper substrate is porous as the porosity of the said paper substrate is 40% or more and 90% or less.
[Calculation Formula 1]
Porosity (%) = {1- [basis weight (g / m ² ) / thickness (μm) / specific gravity of composition]} × 100

-Transcription of labeled antibody-
After the paper substrate 12 and the side of the labeled antibody thermal transfer medium on which the reagent is solid-phased are overlapped with each other, the paper substrate 12 is used as shown in FIGS. 10A and 10B by using a thermal transfer printer. The labeled antibody thermal transfer medium was transferred in a pattern of 3 mm width × 10 mm length (resin layer 15 a) at a position 20 mm away from the upstream end.
The thermal transfer printer has a thermal head with a dot density of 300 dpi (manufactured by TDK Corporation), and has built an evaluation system with a printing speed of 8.7 mm / sec and 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 thermal transfer medium was transferred in a line of 4 mm height × 1 mm length to a position 15 mm away from the transfer position of the labeled antibody thermal transfer medium (test line). 15b). Further, the control line thermal transfer medium was transferred in a line shape of 4 mm height × 1 mm length at a position 5 mm away from the transfer position of the test line thermal transfer medium (control line 15c). Each line was formed under the same printing conditions as the transfer of the labeled antibody.

-Production of absorbent member-
As shown in FIGS. 10A and 10B, by providing the absorbing member 14 (manufactured by Merck Millipore, Surewick C248), No. 1 of Experimental Example 1 was obtained. 1 immunochromatographic assay (test apparatus 10) was obtained.

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

-Reaction-
100 μL of the test solution was dropped on the upstream end of the immunochromatographic assay shown in FIGS. 10A and 10B, and after 30 minutes, the test line concentration was measured.

-Measurement of line density-
The immunochromatographic assay after completion of the reaction was placed in a measurement housing case, measured using a chromatographic reader (Otsuka Electronics Co., Ltd., DiaScan 10), the color density of the test line was determined from the readings, and evaluated according to the following criteria: . The results are shown in Table 1. The larger the reading value, the deeper the color of the test line is preferable.
[Evaluation criteria]
◎: Reading value is 120 or more ○: Reading value is less than 120 and 100 or more Δ: Reading value is less than 100 and 75 or more ×: Reading value is less than 75

-Observation of release during deployment-
The test solution was dropped and the time required for the labeled antibody to finish developing was observed. In addition, in order to perform sufficiently efficient detection and quick diagnosis, “◯” which can be said to be that the labeled antibody has been released slowly is preferable.
[Evaluation criteria]
○: Time required to complete deployment is 15 minutes or more and less than 30 minutes Δ: Time required to complete deployment is 5 minutes or more and less than 15 minutes ×: Time required to complete deployment is less than 5 minutes Yes, or longer than 30 minutes

(Experimental example 1)
<No. 2-18>
No. of Experimental Example 1 1 except that the labeled antibody reagent coating solution of Preparation Example 6 was replaced with the labeled antibody reagent coating solution of Preparation Examples 7 to 23 shown in Table 1. In the same manner as in No. 1, 2 to 18 immunochromatographic assays (testing apparatus 10) were prepared and evaluated in the same manner. The results are shown in Table 1.

(Experimental example 1)
<No. 19>
No. of Experimental Example 1 No. 1 in Experimental Example 1 in <Manufacturing Inspection Apparatus>. Instead of producing one paper substrate, a test strip was produced by the following procedure.

<Preparation of labeled antibody holding pad>
The labeled antibody reagent coating solution of Preparation Example 13, the width 3.5 mm, glass fiber pad was cut to a length 16 mm (Merck Millipore, GFCP203000) was coated to a 8 [mu] L / cm ² to 5 hours drying under reduced pressure Thus, a labeled antibody holding pad was prepared.

-Formation of test lines and control lines-
Next, on the position shown in FIG. 11A and FIG. 11B on the nitrocellulose membrane filter (HF180, manufactured by Merck Millipore Corporation, porosity 70%) cut to a width of 3.5 mm and a length of 38 mm, No. The test line 15b and the control line 15c were formed in the same manner as in FIG. No. No. 19 test transfer thermal transfer medium is No. 1 in Experimental Example 1. This is the same as the thermal transfer medium for test line 1 and the analysis result of the antibody amount by XPS is also the same.

−Assembly−
As shown in FIGS. 11A and 11B, a PET film obtained by cutting the nitrocellulose membrane filter 12 forming the test line 15b and the control line 15c into a width of 3.5 mm and a length of 76 mm (manufactured by Toray Industries, Inc., Lumirror S10, thickness) 100 μm) 11 was bonded to a position 32 mm away from one end on the long axis side so that the side opposite to the reagent application surface and the PET film 11 faced each other. Next, the labeled antibody holding pad 15a produced as described above was attached to the upper surface of the nitrocellulose membrane filter 12 so that the upstream end of the nitrocellulose membrane filter overlapped by 2 mm. Furthermore, a sample pad having a width of 3.5 mm and a length of 34 mm (Bencott M-3II, manufactured by Asahi Kasei Fibers Co., Ltd.) was disposed and attached so as to overlap the upper surface of the labeled antibody holding pad to obtain a sample dropping pad 20.
Next, an absorption pad (Surwick C248, manufactured by Merck Millipore) having a width of 3.5 mm and a length of 25 mm as the absorbing member 14 is overlapped with the downstream end of the PET film 11 on the upper surface of the nitrocellulose membrane filter 12 by 19 mm. The test strip was obtained.

-Housing-
The test strip is housed in a housing case for measurement. Nineteen immunochromatographic assays (test apparatus 10) were obtained.

-Evaluation-
No. produced For the 19 immunochromatographic assay, No. Evaluation similar to 1 was performed. The results are shown in Table 1.

(Experimental example 1)
<No. 20>
No. of Experimental Example 1 No. 19 in Experimental Example 1 except that the labeled antibody reagent coating solution of Preparation Example 14 was used. In the same manner as in No. 19, no. 20 immunochromatographic assays (inspection apparatus 10) were prepared. Evaluation similar to 1 was performed. The results are shown in Table 1.

(Experimental example 1)
<No. 21>
No. of Experimental Example 1 No. 19 in Experimental Example 1 except that the labeled antibody reagent coating solution of Preparation Example 24 was used. In the same manner as in No. 19, no. No. 21 immunochromatographic assay (inspection apparatus 10) was prepared. Evaluation similar to 1 was performed. The results are shown in Table 1.

From the results in Table 1, it was found that the labeled antibody was immobilized on a resin layer together with polyethylene glycol having a weight average molecular weight of 8,000. Nos. 8 and 9 are No. 8 in which a labeled antibody was immobilized on a glass fiber holding pad together with polyethylene glycol as a labeled antibody holding part. 19-No. Compared to 21, the color intensity was high, and a clear line could be confirmed.
In contrast, no. Nos. 19 and 20 are polyethylene glycol having a weight average molecular weight of 8,000 as a labeled antibody reagent. In spite of being equivalent to 8 and 9, since the glass fiber holding pad was used as the labeled antibody holding part, the color intensity was low. No. No. 21 was a polyethylene glycol having a weight average molecular weight of 20,000 as a labeled antibody reagent. Despite being included in the same amount as No. 13, the glass fiber holding pad was used as the labeled antibody holding portion, so the color intensity was low.

Regarding the release property at the time of development, No. 1 containing polyethylene glycol having a weight average molecular weight of 8,000 and 20,000 in a mass ratio of 0.03 to 1.5 with respect to the labeled antibody. 8-12, no. From 14 to 18, the labeled antibody was gradually released, and good results were obtained.
Further, No. 1 containing polyethylene glycol having a weight average molecular weight of 2,000. 2 to 6 also showed sustained release of the labeled antibody, but even after 30 minutes or more, the labeled antibody that was not released from the resin layer and remained was observed. This is because the hydrophilicity of the labeled antibody-immobilized resin layer is increased by the addition of polyethylene glycol, which is a hydrophilic polymer. However, when the weight average molecular weight is 2,000, the resin layer is hydrophilic to the developing solution. This is considered to be because the labeled antibody remains in the resin layer because of the excessively high value.
In addition, when the weight average molecular weight of polyethylene glycol is 20,000, the improvement of the coloring intensity of about 8,000 is not observed because of the inhibition of antibody antigen reaction by polyethylene glycol. This is probably because of this.
From the above, no. As shown in 8 and 9, when the labeled antibody is immobilized on the resin layer, the polyethylene glycol having a weight average molecular weight of 8,000 is in a solid phase together with the labeled antibody at a mass ratio of 0.03 to 0.15 with respect to the labeled antibody. As a result, it was found that an inspection apparatus with improved color intensity and a clear line can be obtained.

Aspects of the present invention are as follows, for example.
<1> a porous flow path member for flowing a specimen;
A resin layer in at least one place on the flow path member,
The test apparatus is characterized in that an antibody that reacts with the specimen and polyethylene glycol are immobilized between the resin layer and the flow path member.
<2> The inspection apparatus according to <1>, wherein the antibody that reacts with the specimen is a capture antibody or a labeled antibody.
<3> a porous flow path member having a flow path for flowing a specimen;
A first resin layer provided in at least one location on the flow path member and having a capture antibody immobilized on a surface facing the flow path member;
A second resin layer provided on the flow path member and having a polyethylene glycol and a labeled antibody immobilized on a surface facing the flow path member;
The inspection apparatus according to any one of <1> to <2>.
<4> The inspection apparatus according to any one of <1> to <3>, wherein the polyethylene glycol has a weight average molecular weight of 5,000 to 10,000.
<5> The test according to any one of <1> to <4>, wherein a content of the polyethylene glycol is 0.015 times or more and 1.5 times or less by mass ratio with respect to the antibody or the labeled antibody. Device.
<6> The weight average molecular weight of the polyethylene glycol is 5,000 or more and 20,000 or less, and the content of the polyethylene glycol is 0.03 times or more and 1. The inspection apparatus according to any one of <1> to <3>, which is 5 times or less.
<7> The weight average molecular weight of the polyethylene glycol is 5,000 or more and 10,000 or less, and the content of the polyethylene glycol is 0.03 times or more and 0.0. The inspection apparatus according to any one of <1> to <6>, which is 15 times or less.
<8> The weight average molecular weight of the polyethylene glycol is 8,000, and the content of the polyethylene glycol is 0.03 times or more and 0.15 times or less by mass ratio with respect to the antibody or labeled antibody. The inspection apparatus according to any one of <1> to <7>.
<9> The inspection apparatus according to any one of <1> to <8>, wherein an amount of the capture antibody to be immobilized is 500 ng / cm ² or more.
<10> The inspection apparatus according to any one of <3> to <9>, including a plurality of the first resin layers.
<11> The inspection apparatus according to any one of <1> to <10>, wherein a porosity of the flow path member is 40% or more and 90% or less.
<12> The inspection apparatus according to any one of <1> to <11>, wherein an average thickness of the flow path member is 0.01 mm or more and 0.3 mm or less.
<13> The inspection apparatus according to any one of <1> to <12>,
A test kit comprising: a collecting means for collecting a sample; and at least one selected from a liquid for processing the sample.
<14> a support;
A release layer provided on the support;
A first reagent-immobilized layer provided on at least one location of the release layer and having a capture antibody on the surface;
A second reagent-immobilized layer provided on the release layer and having a labeled antibody and polyethylene glycol on the surface;
It is a transfer medium for inspection devices characterized by having.
<15> The transfer medium for an inspection apparatus according to <12>, wherein the polyethylene glycol has a weight average molecular weight of 5,000 or more and 10,000 or less.
<16> The inspection apparatus according to any one of <14> to <15>, wherein the polyethylene glycol content is 0.015 times or more and 1.5 times or less by mass ratio with respect to the labeled antibody. It is a transfer medium.
<17> The polyethylene glycol has a weight average molecular weight of 5,000 or more and 20,000 or less, and the polyethylene glycol content is 0.03 times or more and 1. The transfer medium for an inspection apparatus according to <14>, wherein the transfer medium is 5 times or less.
<18> The weight average molecular weight of the polyethylene glycol is 5,000 or more and 10,000 or less, and the content of the polyethylene glycol is 0.03 times or more and 0.0. The transfer medium for an inspection apparatus according to any one of <14> to <17>, wherein the transfer medium is 15 times or less.
<19> The weight average molecular weight of the polyethylene glycol is 8,000, and the content of the polyethylene glycol is 0.03 times or more and 0.15 times or less in terms of mass ratio with respect to the labeled antibody. 14> to <18>. The inspection medium transfer medium according to any one of <14> to <18>.
<20> The above-described <14> to <19>, wherein the first and second reagent-immobilized layers are first and second release layers / reagent-immobilized layers that also function as release layers. The transfer medium for an inspection apparatus described.
<21> The first and second reagent-immobilized layers of the transfer medium for an inspection apparatus according to any one of <14> to <19>, and the first and second described in <20>. Any one of the release layer and reagent-immobilized layer is brought into contact with a porous flow path member, and the first and second reagent-immobilized layers, and the first and second release layers and reagents are contacted. It is a manufacturing method of the inspection apparatus characterized by including the process of transcribe | transferring any one of a solid-phased layer to the said flow-path member.
<22> supplying a sample to the flow path member of the inspection apparatus according to any one of <1> to <12>;
Releasing the reagent immobilized on the resin layer from the resin layer by bringing the reagent into contact with the specimen;
It is an inspection method characterized by including.
<23> supplying a sample to the flow path member of the inspection apparatus according to any one of <1> to <12>;
And a step of capturing a part of the specimen by a reagent solid-phased on the resin layer.
<24> The first resin layer, wherein the resin layer is provided in at least one place on the flow path member, and a capture antibody is solid-phased on a surface facing the flow path member;
<22> to <23>, including a second resin layer provided at one location on the flow path member and having a saccharide and a labeled antibody immobilized on a surface facing the flow path member The inspection method according to any one of the above.

  The inspection device according to any one of <1> to <12>, the inspection kit according to <13>, the transfer medium for an inspection device according to any one of <14> to <20>, <21 > And the inspection method according to any one of <22> to <24> can solve the conventional problems and achieve the object of the present invention.

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

JP 2009-264879 A JP 2002-148266 A Japanese Patent Laid-Open No. 05-133958

Claims (14)

A porous channel member for flowing a specimen;
A resin layer in at least one place on the flow path member,
An inspection apparatus, wherein an antibody that reacts with the specimen and polyethylene glycol are immobilized between the resin layer and the flow path member. A porous flow path member having a flow path for flowing a specimen;
A first resin layer provided in at least one location on the flow path member and having a capture antibody immobilized on a surface facing the flow path member;
A second resin layer provided on the flow path member and having a polyethylene glycol and a labeled antibody immobilized on a surface facing the flow path member;
The inspection apparatus according to claim 1.   The inspection apparatus according to claim 1, wherein the polyethylene glycol has a weight average molecular weight of 5,000 or more and 10,000 or less.   The inspection apparatus according to any one of claims 1 to 3, wherein a content of the polyethylene glycol is 0.015 times or more and 1.5 times or less by mass ratio with respect to the antibody or the labeled antibody.   The weight average molecular weight of the polyethylene glycol is 5,000 or more and 20,000 or less, and the content of the polyethylene glycol is 0.03 times or more and 1.5 times or less by mass ratio with respect to the antibody or labeled antibody. The inspection apparatus according to any one of claims 1 to 2.   The weight average molecular weight of the polyethylene glycol is 5,000 or more and 10,000 or less, and the content of the polyethylene glycol is 0.03 or more and 0.15 or less by mass ratio with respect to the antibody or labeled antibody. The inspection apparatus according to any one of claims 1 to 5. An inspection apparatus according to any one of claims 1 to 6;
A test kit, comprising: a collecting means for collecting a specimen; and at least one selected from a liquid for processing the specimen. A support;
A release layer provided on the support;
A first reagent-immobilized layer provided on at least one location of the release layer and having a capture antibody on the surface;
A second reagent-immobilized layer provided on the release layer and having a labeled antibody and polyethylene glycol on the surface;
A transfer medium for an inspection apparatus, comprising:   The transfer medium for an inspection apparatus according to claim 8, wherein the polyethylene glycol has a weight average molecular weight of 5,000 or more and 10,000 or less.   The transfer medium for an inspection apparatus according to any one of claims 8 to 9, wherein a content of the polyethylene glycol is 0.015 times or more and 1.5 times or less by mass ratio with respect to the labeled antibody.   The weight average molecular weight of the polyethylene glycol is 5,000 or more and 20,000 or less, and the content of the polyethylene glycol is 0.03 times or more and 1.5 times or less by mass ratio with respect to the antibody or labeled antibody. The transfer medium for an inspection apparatus according to claim 8.   The weight average molecular weight of the polyethylene glycol is 5,000 or more and 10,000 or less, and the content of the polyethylene glycol is 0.03 or more and 0.15 or less by mass ratio with respect to the antibody or labeled antibody. The transfer medium for an inspection apparatus according to any one of claims 8 to 11.   The transfer for an inspection apparatus according to claim 8, wherein the first and second reagent-immobilized layers are first and second release-layer / reagent-immobilized layers that also function as release layers. Medium.   The said 1st and 2nd reagent solid-phase layer of the transfer medium for test | inspection apparatuses in any one of Claim 8 to 12, and the said 1st and 2nd peeling layer and reagent solid-phase of Claim 13 Any one of the first and second reagent-immobilized layers and the first and second release layers / reagent-immobilized layers by bringing any one of the immobilized layers into contact with the porous flow path member. The manufacturing method of the test | inspection apparatus characterized by including the process of transcribe | transferring to a said flow-path member.