JP2017227462A - Inspection equipment and inspection kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide inspection equipment capable of obtaining a color development of high concentration even in a state with a less detection target substance and capable of performing an inspection with a high degree of accuracy.SOLUTION: Inspection equipment for a lateral flow assay includes: a porous channel member with a flow channel where a detection target substance flows; and a detection part including a material that can be bonded to the detection target substance and arranged to abut on the channel member. If an area of a thickness direction in the channel member abutting on the detection part is set as a detection area, a width in the thickness direction of the detection area is narrower than a width in the thickness direction of the channel member other than in the detection area.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an inspection apparatus and an inspection kit.

  Conventionally, a method using a lateral flow assay test apparatus is known as one of immunoassays. In the test apparatus for lateral flow assay, the introduced test liquid flows through the porous flow path member in which the flow path is formed, and the detected substance contained in the test liquid is detected by, for example, a biochemical reaction by the detection unit. It is what is done.

Immunochromatography and nucleic acid chromatography are known as examples of methods using a lateral flow assay testing apparatus.
In an immunochromatographic test, as a method for detecting and quantifying an antigen or antibody using a reaction between an antigen and an antibody, for example, a flow path for flowing a sample for the purpose of testing a sample such as blood, food, or beverage An inspection apparatus in which is formed is used. The test apparatus includes, for example, a sample pad as a test solution receiver, a conjugate pad that reacts the test solution supplied from the sample pad, and a membrane film that flows the test solution supplied from the conjugate pad. It is configured.
As an inspection apparatus using an immunochromatography method, for example, an apparatus in which a fine structure is formed on the surface of an inspection part has been proposed for the purpose of increasing the contact opportunity between an antigen and an antibody and performing an inspection with a small amount of antigen (see Patent Document 1). .

In the nucleic acid chromatography that can detect the target nucleic acid, the presence or absence of nucleic acids (DNA, RNA) derived from viruses and bacteria, and the presence or absence of nucleic acids derived from mutated genes related to specific diseases and constitutions are examined. Therefore, it is possible to accurately diagnose genetic diseases such as infections, tumors, and constitutions. It is also applied to food inspection for detecting allergens and specific foods and environmental inspection for detecting microorganisms present in the environment.
As an inspection apparatus using a nucleic acid chromatography method, for example, a nucleic acid chromatography apparatus capable of detecting a target nucleic acid by applying a capture nucleic acid having a sequence complementary to the target nucleic acid to a detection unit and using a label capable of binding to the target nucleic acid. Has been proposed (see Patent Documents 2 and 3).

  However, there is a need for further technical proposals, and there is a need for an inspection apparatus that can provide high-concentration color development even when there are few substances to be detected.

  SUMMARY OF THE INVENTION An object of the present invention is to provide an inspection apparatus capable of obtaining a high-density color even in a state where the amount of a substance to be detected is small and performing a highly accurate inspection.

  In order to solve the above problems, an inspection apparatus according to the present invention includes a porous flow path member in which a flow path through which a detected substance and a detected substance that can be combined with the detected substance flow is formed, and the flow path member. And a detection portion containing the detection substance, and a width in the thickness direction of the detection region when a region in the thickness direction of the flow path member in contact with the detection portion is defined as a detection region Is smaller than the width in the thickness direction of the region of the flow path member other than the detection region.

  According to the present invention, it is possible to provide an inspection apparatus capable of obtaining high-concentration color development even in a state where the amount of a substance to be detected is small and performing inspection with high accuracy.

It is sectional drawing which shows an example of a part of test | inspection apparatus of this invention. It is a top view which shows an example of the inspection apparatus of this invention. It is sectional drawing in B-B 'of the inspection apparatus of FIG. It is a top view which shows an example of the test | inspection apparatus which is not contained in this invention. It is sectional drawing in C-C 'of the inspection apparatus of FIG. It is sectional drawing which shows an example of the inspection apparatus in the opposing part of a flow-path member and a resin layer. It is sectional drawing which shows another example of the inspection apparatus in the opposing part of a flow-path member and a resin layer. It is sectional drawing which shows another example of the inspection apparatus in the opposing part of a flow-path member and a resin layer. It is sectional drawing which shows another example of the inspection apparatus in the opposing part of a flow-path member and a resin layer. It is sectional drawing which shows another example of the inspection apparatus in the opposing part of a flow-path member and a resin layer. It is a conceptual diagram of the conjugate pad in the conventional inspection apparatus. It is a conceptual diagram of the membrane in the conventional inspection apparatus. It is a schematic sectional drawing which shows an example of the transfer medium for test | inspection apparatuses. It is a schematic sectional drawing which shows another example of the transfer medium for test | inspection apparatuses. It is the schematic which shows an example of the test | inspection kit of this invention. It is a top view which shows an example of the test | inspection apparatus used in the Example. It is sectional drawing in D-D 'of FIG. 13A. FIG. 13B is another example of a cross-sectional view taken along the line D-D ′ of FIG. 13A. It is a top view which shows another example of the inspection apparatus used in the Example. It is sectional drawing in E-E 'of FIG. 14A.

  Hereinafter, an inspection apparatus and an inspection kit according to the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, and other embodiments, additions, modifications, deletions, and the like can be changed within a range that can be conceived by those skilled in the art, and any aspect is possible. As long as the functions and effects of the present invention are exhibited, the scope of the present invention is included.

(Inspection equipment)
The inspection apparatus of the present invention is formed so as to be in contact with the flow path member, a porous flow path member in which a flow path through which a substance to be detected (hereinafter sometimes referred to as a specimen) flows is formed. And a detection part containing a substance that can bind to the substance (hereinafter also referred to as a detection substance), and when the area in the thickness direction of the flow path member in contact with the detection part is a detection area, the detection The width in the thickness direction of the region is smaller than the width in the thickness direction of the flow path member other than the detection region. The inspection apparatus of the present invention is particularly used as a lateral flow assay inspection apparatus.

  An embodiment of the inspection apparatus of the present invention will be described with reference to FIGS. 1 is a cross-sectional view showing a part of the inspection apparatus of the present embodiment, FIG. 2 is a top view of the inspection apparatus of the present embodiment, and FIG. 3 is a cross-sectional view of the inspection apparatus of FIG. It is. Hereinafter, in this embodiment, an example of an inspection apparatus for inspecting the presence or absence of an antigen contained in a test solution will be described. However, the inspection apparatus of the present invention is not limited to an apparatus using an antigen-antibody reaction. In addition, those using a nucleic acid reaction may be mentioned.

  In the inspection apparatus shown in FIG. 1, the base material 11, the porous flow path member 12, the absorption member 14, the sample pad 19, and the detection unit 15 b (details will be described later, but in the present embodiment, both the second resin layer 15 b and Is referred to). In this embodiment, the substance to be detected is supplied from the sample pad 19, flows through the flow path member 12, and is detected by the detection unit 15b.

  2 and 3, the first resin layer 15a, the second resin layer 15b, and the third resin layer 15c are illustrated, and the detection unit of the present embodiment is denoted by the same reference numerals as those of the second resin layer. Has been. A plurality of detection units may be provided. Moreover, when showing arbitrary resin layers among resin layers (15a, 15b, 15c), it represents with the resin layer 15. FIG.

  In the present invention, when a region in the thickness direction of the flow channel member in contact with the detection unit is set as a detection region, the width in the thickness direction of the detection region is larger than the width in the thickness direction of the flow channel member other than the detection region. Is also small. Here, an example of an inspection apparatus not included in the present invention is shown in FIGS. 4 is a top view, and FIG. 5 is a cross-sectional view taken along the line C-C ′ of FIG. 4. As shown in FIG. 5, the width in the thickness direction of the flow path member is constant.

  In the present embodiment, a region in the thickness direction in the flow path member in contact with the detection unit is defined as a detection region (second portion 12e), and a region in the flow channel member other than the detection region is defined as the first portion 12d. (See FIGS. 1 and 3). At this time, when the width in the thickness direction of the first portion 12d is d1, and the width in the thickness direction of the second portion 12e is d2, d2 is smaller than d1.

  For example, in the inspection apparatus of the present invention shown in FIG. 3, the inventor flows a specimen extract containing a substance to be detected such as an antigen from the sample pad 19, and d2 in the relationship between d1 and d2. It has been found that a color density higher than that of the prior art can be obtained when is less than d1.

The following two points can be cited as the reason why high color density can be obtained.
The first point is due to the fact that the member thickness of the detection unit is reduced. In general, the colored particles that can be detected with a porous material are about 5 μm deep from the surface. For this reason, color development in a portion deeper than 5 μm is not detected, and in order to combine a large number of substances to be detected and detection substances in the detectable part, it is preferable that the member thickness is thin. In addition, since the member thickness is small only in the detection part, more substance to be detected is supplied until the detection part is reached, so that a higher color density can be obtained.

  The second point is due to an increase in contact opportunities between the substance to be detected and the detection substance immobilized on the solid phase. In a lateral flow assay device typified by a general immunochromatography, a detection substance is uniformly permeated and fixed in the depth direction of the flow path member by, for example, a dispenser, so that the detected substance flowing through the flow path member Equally get the opportunity to come into contact with the detection substance.

  However, when the detection substance is solid-phased only on the surface facing the flow path member, in order to obtain a large number of detection substance-to-detection bonds, the detection substance in the sample extract is more It is necessary to pass or contact many opposing surfaces. Therefore, it is preferable that the width (height) d2 in the thickness direction of the region in the thickness direction (detection region, second portion 12e) in the flow path member in contact with the detection unit is as small as possible.

  In addition, by reducing d2 relative to the height d1 of the flow path member, the amount of liquid that can pass through the flow path member is reduced, and the specimen extract is temporarily decelerated near the detection unit, so that it is detected. The effect that the substance accumulates in the vicinity of the detection unit is obtained. Due to the effect of decelerating in the vicinity of the detection unit and the effect of the specimen extract being retained in the vicinity of the detection unit, the contact opportunity between the detection substance and the detection substance increases, and therefore, higher color development can be obtained.

  A method for reducing the thickness d2 of the second portion 12e relative to the thickness d1 of the first portion 12d is not particularly limited. Roughly classifying in terms of changes in the pore size of the porous flow path member, a method of contracting and deforming the portion corresponding to the detection site with respect to the flow path member, such as crushing the porous flow path member dynamically such as pressurization In addition, there is a method of selectively removing a specific part, such as cutting, peeling or mold-making at the time of preparing a substrate.

In both cases, there is a difference in the pore diameter of the porous flow path member in the second part, and in the former method of deforming the detection part in the flow path member, the second part 12e with respect to the first part 12d. The hole diameter becomes smaller.
It is preferable that an average pore diameter of the flow path member in the detection region (second portion 12e) is smaller than an average pore diameter of the flow channel member (first portion 12d) other than the detection region.
From the examination in the present invention, it has been found that color development at a higher density can be obtained by making the pore diameter of the second portion 12e smaller than that of the first portion 12d. The reason for this is that by reducing the pore diameter, it is easier to obtain the effect that the specimen extract is decelerated and stays in the vicinity of the detection section.

  Further, regarding the relationship between the height (width in the thickness direction) d1 of the flow path member and the height (width in the thickness direction) d2 of the flow path member including the detection unit, 0.4 ≦ d2 / d1 ≦ 0.8. It is preferable. If d2 / d1 is less than 0.4, the specimen extract is greatly decelerated, and it becomes difficult for the specimen extract to pass through the flow path member, or a part of the sample flows backward. It is not preferable. When d2 / d1 is greater than 0.8, there is almost no need for work in the present invention because the effect obtained when d1 and d2 are equal to each other is hardly obtained.

  As for the shape for reducing the thickness d2 of the second portion 12e relative to the thickness d1 of the first portion 12d, the cross-sectional view of FIG. 3 shows a rectangle, but there is no particular limitation. It can be appropriately selected according to the purpose. For example, a shape connected by straight lines such as a triangle, a quadrangle, a rectangle, and a polygon, and a shape in which a part or all of a semicircle, an ellipse, a U-character, etc. are connected by a curve are included. Alternatively, the surface may be intentionally roughened to increase the specific surface area. In the drawing, the upper side of the second portion 12e is recessed, but the present invention is not limited to this, and the lower side may be convex and d2 may be smaller than d1.

  Although there is no restriction | limiting in particular about the magnitude | size of the said 2nd site | part 12e, The structure where all the 1 or several detection substances are contained in the 2nd site | part 12e is preferable. In addition, if the second portion 12e is large and occupies a majority with respect to the length of the flow path member, the specimen extract is greatly decelerated, and the specimen extract becomes difficult to pass through the flow path member, and the time required for the test is prolonged. It is not preferable.

  In the inspection apparatus according to the present embodiment, it is preferable that the detection unit is formed by solidifying a substance capable of binding to the detection target substance on a surface facing the flow path member. In addition, it is preferable that a detection substance or a substance that reacts with the detection substance is solid-phased on the surface of the resin layer 15 facing the flow path member 12. A labeled antibody, a capture antibody, and the like will be described later, but the first resin layer 15a is solid-phased with a labeled antibody 16 that can bind to the substance to be detected, and the second resin layer 15b and the third resin layer. For 15c, it is preferable that a capture antibody that reacts with the labeled antibody 16 is immobilized.

  Further, as shown in the figure, it is preferable to provide a plurality of resin layers. When a plurality of resin layers are provided, for example, it is easy for the third resin layer 15c to develop the sample in the flow path member 12 without any problem. This is advantageous because it can be confirmed.

  In the test apparatus, the capture antibody is solid-phased on the surface of the resin layer facing the flow path member as in the conventional ELISA (Enzyme Linked Immuno Solvent Assay) method, whereby the specimen is placed on the resin layer side. However, since the capture antibody can be solid-phased at high density, the specimen can be detected with high sensitivity. Further, in the inspection apparatus, as the conventional immunochromatography method, the reaction proceeds while the test solution containing the sample is developed in the flow path member (flow path) by using the capillary phenomenon as a driving force. Sensitivity can be measured and a clear judgment line can be obtained.

The density of the antibody (the amount of immobilized antibody) is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 500 ng / cm ² or more. When the density of the antibody is 500 ng / cm ² or more, it is advantageous in that the amount of the immobilized antibody is appropriate, and the color development intensity of the line is sufficiently obtained.

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).
As a method for analyzing the amount of the immobilized antibody by the X-ray photoelectron spectroscopy (XPS), the binding energy of the element present on the surface of the resin layer is measured by the X-ray photoelectron spectroscopy (XPS), and C1s The element composition (atom%) is calculated by converting from the area of the peak corresponding to N1s, O1s, and the like.

  The amount of antibody present on the surface of the resin layer can be calculated from elemental components derived only from the antibody. At this time, about 5 samples with different amounts of immobilized antibody are prepared, The amount of antibody on the surface of the resin layer can be measured by creating a calibration curve from the difference in the ratio of the derived elemental composition.

  In addition, when the antibody is immobilized, the antibody solution applied to the surface of the resin layer is dried and solidified as it is, so the amount of the immobilized antibody is estimated from the amount of the antibody solution applied. Is also possible.

  There is no restriction | limiting in particular as the length in the flow path direction of the said whole resin layer, Although it can select suitably according to the objective, 0.5 mm or more and 2.0 mm or less are preferable. When the length of the entire resin layer in the flow path direction is 0.5 mm or more, visibility can be ensured without being too thin as a line, and a shape that can be recognized as a line when 2.0 mm or less can be maintained. This is advantageous.

  Next, details of the inspection apparatus of the present embodiment will be described with reference to FIGS. 6, 7A, 7B, 8A, and 8B. 6, 7, and 8 are cross-sectional views schematically showing an example of an inspection device at a facing portion of the flow path member and the resin layer.

  As shown in the figure, the test apparatus 10 includes a hydrophilic test liquid 30 (such as blood, spinal fluid, urine, or sample extract (for example, a liquid containing a sample collected by a sample collection means such as a stick)). It has a porous flow path member 12 in which a flow path for flowing an example of a specimen is formed, and resin layers (15a, 15b, 15c) provided on the flow path member 12. In addition, any or all of the detection units including the resin layer are processed with respect to the flow path member, and the height is small.

  On the surface of the resin layer (15a, 15b, 15c) facing the flow path member 12, the labeled antibody 16 that reacts with the antigen 31 contained in the test solution 30, the capture antibody 17 that captures the antigen 31, and the labeled antibody 16 are placed. 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, the case where the flow path member 12 is provided on the base material 11 and the absorption member 14 and the sample pad 19 are provided on the base material 11 and the flow path member 12 in the inspection apparatus 10 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. 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.

  In the inspection apparatus of the present embodiment, a plurality of resin layers (first resin layer 15a, second resin layer 15b, and third resin layer 15c) are provided, and the plurality of resin layers are labeled antibodies or It includes a plurality of capture antibodies that can bind to a substance to be detected or a labeled antibody. The first resin layer 15a includes the labeled antibody 16, and releases the labeled antibody 16 into the flow path. The second resin layer 15b includes a capture antibody 17 that captures the detection target substance bound to the labeled antibody 16 in the flow path. The third resin layer 15c includes a capture antibody 18 that captures the labeled antibody 16 in the flow path that is not bound to the substance to be detected. The second resin layer 15b serves as the detection unit.

As shown in FIG. 6, the first resin layer 15 a of the inspection apparatus 10 contains an amphiphilic resin 151 having many hydrophilic groups 152.
As content of the hydrophilic group 152, 50 mass% or more is preferable with respect to the 1st resin layer 15a whole quantity.
Here, the hydrophilic group is an atomic group that forms a weak bond such as a hydrogen bond with a water molecule, and means that there is an affinity for water. The amphiphilic means that both water and the organic solvent have affinity.

  The labeled antibody 16 is solid-phased on the surface of the first resin layer 15a facing the flow path member 12 by having the hydrophilic portion 16g. On the other hand, when the test liquid 30 is filled in the gap formed between the flow path member 12 and the first resin layer 15a, the hydrophilic portion 16g of the labeled antibody 16 and the hydrophilic test liquid 30 are compatible. Then, the labeled antibody 16 is released from the amphiphilic resin 151. Further, when the test solution 30 contains the antigen 31, the released labeled antibody 16 and the antigen 31 react and bind by the antigen-antibody reaction.

In order to prevent the binding between the labeled antibody 16 and the antigen 31 from being inhibited, the amphiphilic resin 151 is preferably a water-insoluble resin.
Here, the water 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. The reason why the mass of the resin changes is that by-products (such as monomer components) contained in the resin product are dissolved in water and the mass is reduced.

As shown in FIGS. 7A and 7B, the second resin layer 15b is preferably a resin having a hydrophobic group 153. Specifically, the second resin layer 15b contains an amphiphilic resin 154 having many hydrophobic resins 155 or hydrophobic groups 153, and the hydrophobic resin 155 or the amphiphilic resin 154 is a main component ( The content is preferably 50% by mass or more).
Here, the hydrophobic group is an atomic group that is not easily compatible with water, and means that it has a low affinity for water and is hardly soluble in water or mixed with water.

  The capture antibody 17 has a hydrophobic portion 17g, and the hydrophobic portion 17g is bonded to the surface of the second resin layer 15b facing the flow path member 12 by binding with an intermolecular force. Is done. 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. Accordingly, since the antigen 31 and the labeled antibody 16 are immobilized and colored, the second resin layer 15b can be used as a line (test line) for determining the presence or absence of the antigen 31.

  There is no restriction | limiting in particular as hydrophobic resin 155 and amphiphilic resin 154, Although it can select suitably according to the objective, It is preferable that it is the said water-insoluble resin, respectively. When the hydrophobic resin 155 and the amphiphilic resin 154 are each the water-insoluble resin, it is advantageous in that line bleeding can be prevented.

  As shown in FIGS. 8A and 8B, the third resin layer 15c 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 contained in the third resin layer 15c. It is preferable to be constituted as a main component (content is 50% by mass or more).

The capture antibody 18 is solid-phased on the surface of the third 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 and an antigen that specifically bind to the labeled antibody 16. . Accordingly, since the labeled antibody 16 is immobilized and colored, the third resin layer 15c can be used as a control line indicating that the labeled antibody 16 has reached.

  There is no restriction | limiting in particular as hydrophobic resin 155 and amphiphilic resin 154, Although it can select suitably according to the objective, It is preferable that it is the said water-insoluble resin, respectively. When the hydrophobic resin 155 and the amphiphilic resin 154 are each the water-insoluble resin, it is advantageous in that bleeding of the control line can be prevented.

  The resin layer 15 is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably a non-porous material. The non-porous body is a non-porous structure that does not substantially include voids, and is a structure that is contrary to a porous material including voids provided to promote absorption of liquid such as a membrane. 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.

  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.

  In general, the colored 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 the 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 average thickness of the porous material.

  In the inspection apparatus of the present embodiment, since the resin layer made of the non-porous material containing a large amount of hydrophobic groups is used for immobilizing the capture antibody, the capture antibody enters the resin layer. It is fixed only on the surface of the resin layer. The labeling particles are bonded to the capture antibody immobilized on the surface of the resin layer, and the color is developed. However, since the light can be detected through the resin layer made of the non-porous material, the label is not detected. The utilization efficiency of coloring by the particles for use can be greatly improved.

  Further, since there is no excess labeling particles (coloring 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 channel member made of a hydrophilic porous material has an average thickness of 100 μm, it is assumed that only a color having a thickness of 5 μm from the surface can be used, and the capturing used to obtain the same color intensity The amount of antibody applied can be reduced to 1/20.

  That is, since the resin layer made of the non-porous material containing a large amount of hydrophobic groups is used for immobilizing the capture antibody, the utilization efficiency of color development by the labeling particles can be greatly improved, and the excess in the thickness direction. Since no such colored particles are present, the amount of the capture antibody applied 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 is described. However, the inspection apparatus of the present invention is not limited to the apparatus using the 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.

  Moreover, as an embodiment other than the one using an antigen-antibody reaction, one using a nucleic acid reaction can be mentioned. In this embodiment, the substance to be detected is a nucleic acid, and the substance capable of binding to the substance to be detected is a capture nucleic acid having a sequence complementary to the nucleic acid. Further, similarly to the above-described embodiment, it is preferable that the detection unit is formed by solidifying a substance capable of binding to the detection target substance on a surface facing the flow path member. That is, it is preferable that a supplemental nucleic acid having a sequence complementary to the nucleic acid that is the substance to be detected is solid-phased on the resin on the surface facing the flow path member.

  Hereinafter, each member which comprises the inspection apparatus 10 is demonstrated in detail.

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

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

There is no restriction | limiting in particular as a shape of the base material 11, Although it can select suitably according to the objective, A sheet form is preferable.
There is no restriction | limiting in particular in the average thickness of the base material 11, Although it can select suitably according to the objective, 0.01 mm or more and 0.5 mm or less are preferable. When the average thickness is 0.01 mm or more, the strength as the substrate 11 is good, and when it is 0.5 mm or less, the flexibility of the substrate is good and can be suitably used as a sensor. .

  Here, the average thickness means, for example, that the thickness of the measurement object is 5 in the longitudinal direction (length direction), 3 in the width direction, and 15 in total so that the measurement locations are substantially evenly spaced. It can be set as the average value of thickness when measured with a meter (MDH-25M, manufactured by Mitutoyo Corporation). In the present embodiment, the thickness may be the length of the object in the direction perpendicular to the contact surface between the base material 11 and the flow path member 12.

<Flow channel member>
The flow path member 12 of the inspection apparatus 10 is not particularly limited as long as it is a member capable of flowing the inspection liquid 30, and can be appropriately selected according to the purpose. Examples thereof include a 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).

6 to 8B, the gap 12a is a gap formed in each cross section, and the gap 12b is a gap on the back side of the cross section. It is preferable that bubbles are present inside the hydrophilic porous material and the bubbles are connected to form an open cell.
The open cell is distinguished from the closed cell in which the bubbles are not connected to each other. In the continuous bubble, since a small hole is opened in the wall between the bubbles, it has a function of sucking liquid or allowing gas to pass therethrough by a capillary phenomenon. Since the flow path member 12 uses the capillary phenomenon to transfer the test solution 30 in the gaps (12a, 12b), an external driving device such as a pump is not necessary.

There is no restriction | limiting in particular as said hydrophilic porous material, Although it can select suitably according to the objective, The material which shows hydrophilic property and has a high porosity is used suitably.
The hydrophilic porous material means a porous material that can be easily penetrated by an aqueous solution. “Easy to penetrate” refers to a water permeability evaluation test in which 0.01 mL of pure water is dropped onto the surface of a plate-like test piece dried at 120 ° C. for 1 hour. All of 0.01 mL of pure water penetrates within 10 minutes. To do.

The porosity of the hydrophilic porous material is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 40% or more and 90% or less, and more preferably 65% or more and 80% or less. When the porosity is 90% or less, the 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 >), average thickness (micrometer), and composition specific gravity of a hydrophilic porous material, for example.

[Calculation Formula 1]
Porosity (%) = {1- [basis weight (g / m ² ) / average 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. When the average thickness is 0.01 mm or more, it is advantageous in that the strength of the substrate is good. Moreover, when the average thickness is 0.3 mm or less, it is advantageous in that the required amount of the test solution can be optimized.

  In addition, regarding the liquid velocity at which the liquid travels in the flow path member, the time for the liquid to move to the other end when the liquid is added to one end of the flow path member is generally represented by a capillary flow time. The capillary flow time can be obtained by the following calculation formula 2.

[Calculation formula 2]
Capillary flow time (second / cm) = time taken for liquid to reach one end from the other end (second) / length of channel member through which liquid passes (length from one end to the other end) (cm )

  The capillary flow time in the flow path member used in a lateral flow apparatus typified by immunochromatography is not particularly limited, but is generally 120 or more and 240 or less (seconds / 4 cm). It is selected at any time in consideration of the detection purpose such as the type of specimen extract, required detection accuracy, and measurement time.

  In general, if the capillary flow time is too large, it takes time for the liquid to pass through the flow path member, so that the inspection takes time. If the capillary flow time is too small, the detection unit before the detection substance and the detection substance are combined. It is known that the necessary color development cannot be obtained.

  In addition, it is known that the capillary flow time is affected by the hole diameter in the flow path member. As in the present invention, by pressing the flow path member to reduce the hole diameter or crush the hole, It is possible to slow down the liquid speed. From this, it is possible to select a capillary member having a small capillary flow time for the flow path member itself, to widen the selection range of the flow path member, and to further increase the passage speed of the sample extract until reaching the detection unit. Thus, the present invention is characterized in that the inspection time is shortened.

<Resin layer>
The function of the resin layer 15 in the present embodiment will be described in comparison with the conventional inspection apparatus shown in FIGS. FIG. 9 is a conceptual diagram of a conjugate pad in a conventional inspection apparatus. FIG. 10 is a conceptual diagram of a membrane in a conventional inspection apparatus.

  In the conventional inspection apparatus, if the hydrophilicity of the conjugate pad is too high, the inspection liquid tends to remain on the conjugate pad, and it is difficult to transfer to the membrane. Conversely, if the hydrophobicity of the conjugate pad is too high, the transfer of the test solution to the membrane will be faster, but the water absorption of the test solution from the sample pad will drop, so the test time will be longer or a large amount of test solution will be used. It was supposed to take.

  For this reason, the fiber F1 which can be used as a conjugate pad was limited. Furthermore, in the conventional inspection apparatus, since the labeled antibody 16 is solid-phased on the fiber F1 constituting the conjugate pad (see FIG. 9), as the labeled antibody 16 that can be released from the conjugate pad, It will be limited to the thing with a weak bond strength with the fiber F1. That is, the conventional inspection apparatus has a limited number of usable fibers F1 and labeled antibodies 16 in design.

  In the conventional inspection apparatus, since the capture antibody 17 is solid-phased on the fiber F2 constituting the membrane (see FIG. 10), the capture antibody 17 that can be immobilized on the membrane is the fiber F2. It will be limited to those with strong binding power. That is, the conventional inspection apparatus has a limited number of usable fibers F2 and capture antibodies 17 in design.

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

  Examples of a method for adjusting the strength of interaction between the resin layer 15 and the capture antibody 17 and the affinity with the test solution 30 include, for example, the capture antibody corresponding to the type of resin constituting the resin layer 15 and the resin composition ratio. The method of changing according to 17 is mentioned. 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 the capture antibody 17 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, the hydrophobic molecular species come to gather close to each other, and this is a phenomenon that appears to have a binding force acting between the molecules.

  When the hydrophilic ratio in the resin constituting the resin layer 15 is large, the interaction between the resin layer 15 and the hydrophilic capture antibody becomes strong, but when the binding portion comes into contact with the hydrophilic test solution 30, It is estimated that the reagent is likely to be released into the test liquid 30 in affinity with the test liquid 30.

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

  There is no restriction | limiting in particular as amphiphilic resin which comprises the 1st resin layer 15a, According to the objective, it can select suitably, For example, polyvinyl alcohols, polyvinyl acetal resin, polyacrylic acid, acrylic acid-acrylic Nitrile copolymer, 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 Polymer, styrene-α-methylstyrene-acrylic acid copolymer, styrene-α-methylstyrene-acrylic acid-acrylic acid ester copolymer, styrene-maleic acid copolymer, styrene-maleic anhydride copolymer, Vinyl naphthalene-acrylic acid copolymer, vinyl naphthalene-maleic acid copolymer, Examples thereof include vinyl acetate-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 second resin layer 15b and the third resin layer 15c include polystyrene resins such as polystyrene and acrylonitrile-butadiene-styrene copolymers, polypropylene resins, polyethylene resins, and ethylene-propylene copolymers. Polyolefin resins such as polymers or cyclic polyolefin resins, polycarbonate resins, polyethylene terephthalate resins, methacrylic resins such as polymethyl methacrylate resins, vinyl chloride resins, polybutylene terephthalate resins, polyarylate resins, polysulfone resins, polyether sulfones Resin, polyetheretherketone resin, polyetherimide resin, fluorine resin such as polytetrafluoroethylene, polymethylpentene resin, acrylic resin such as polyacrylonitrile, Such as cellulose resins such as onato resins. These may be used alone or in combination of two or more.

Examples of compounds other than the hydrophobic resin constituting the second resin layer 15b and the third resin layer 15c include beeswax, carnauba wax, whale wax, wood wax, candelilla wax, rice bran wax, and montan wax. Natural wax: synthetic wax such as paraffin wax, microcrystalline wax, oxidized wax, ozokerite, ceresin, ester wax, polyethylene wax, oxidized polyethylene wax; margaric acid, lauric acid, myristic acid, palmitic acid, stearic acid, furonic acid, behenine Higher fatty acids such as 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 the compounds constituting the second resin layer 15b and the third resin layer 15c, polystyrene resin, polyolefin resin, carnauba wax, and polyethylene wax are preferable from the viewpoint 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 first resin layer 15a is more hydrophilic than the resins constituting the second and third resin layers 15b and 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 first resin layer 15a is not particularly limited as long as it has a hydrophilic site and reacts with the antigen 31, and may be appropriately selected according to the purpose. Examples thereof include 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 second resin layer 15b is not particularly limited as long as it has a hydrophobic site and reacts with the antigen 31, and may be appropriately selected according to the purpose. Examples thereof include 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 third 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. For example, antibodies against Human IgG or the like, or the antibodies listed above can be mentioned. Moreover, the antigen itself which reacts with the labeled antibody 16 may be sufficient.

  The method for immobilizing the reagent such as the labeled antibody 16 and the capture antibody (17, 18) on the resin layer 15 is not particularly limited and may be appropriately selected depending on the intended purpose. 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.

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

(Transfer media for manufacturing inspection equipment)
In the first embodiment of the transfer medium for manufacturing an inspection apparatus according to the present embodiment, the transfer medium includes a support, a release layer provided on the support, and a reagent-immobilized layer provided on the release layer. . The surface of the reagent-immobilized layer has a reagent that reacts with the specimen.

In the second embodiment of the transfer medium for manufacturing an inspection apparatus according to this embodiment, in the first embodiment, the reagent-immobilized layer is a release layer / reagent-immobilized layer that also serves as the release layer. The surface of the release layer / reagent-immobilized layer has a reagent that reacts with the specimen.
The reagent-immobilized layer and the release layer / reagent-immobilized layer may be referred to as an upper layer.

  Here, with reference to the drawings, a transfer medium for manufacturing an inspection apparatus used when a resin layer is provided on a flow path member will be described. FIG. 11A is a schematic cross-sectional view showing an example of a transfer medium for an inspection apparatus according to the present invention. FIG. 11B is a schematic cross-sectional view showing another example of the transfer medium for an inspection apparatus of the present invention.

  When the thermal transfer method is used, the transfer medium 100 for an inspection apparatus on 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 is reduced. In addition, when the capture antibody is applied and arranged by a conventional method, it is necessary to dilute the capture antibody with a solvent until the viscosity is such that it can be applied (for example, can be ejected by an inkjet printer). When the capture antibody is disposed by thermal transfer, a high concentration capture antibody can be disposed in the flow path by using a transfer medium for an inspection apparatus to which a high concentration capture antibody is attached in advance.

As shown in FIG. 11A, the transfer medium 100 for an inspection apparatus includes a support 101, a release layer 102 provided on the support 101, and a reagent-immobilized layer 103 provided on the release layer 102. The reagent is immobilized on the surface of the reagent-immobilized layer 103. Further, the inspection apparatus transfer medium 100 further includes other layers such as a back layer 104 as necessary.
As shown in the inspection apparatus transfer medium 110 in FIG. 11B, 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 peeling layer / reagent solid phase layer 105 is used, the peeling layer / reagent solid phase layer 105 is completely transferred to the flow path member 12 or, among the test peeling layer / reagent solid phase layer 105, The portion including the surface on which the antibody is immobilized is transferred, but a part of the release layer / reagent-immobilized layer 105 may remain on the support 101 side.

  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.

<Reagent solid phase layer>
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 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 15 is improved, and the resin layer 15 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.

<Peeling layer and reagent solid phase layer>
The release layer / reagent-immobilized layer 105 has both functions of the release layer 102 and the reagent-immobilized layer 103, and improves the releasability between the support 101 and the reagent-immobilized layer 103 during transfer. In addition, 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.

  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.

  There is no restriction | limiting in particular as average thickness of the peeling layer and reagent solid-phased layer 105, Although it can select suitably according to the objective, 0.5 micrometer or more and 50 micrometers or less are preferable. When the average thickness is 0.5 μm or more, the durability of the release layer / reagent-immobilized layer 105 (resin layer 15) is improved, and the resin layer can be prevented from being damaged by friction or impact. Further, when the average thickness is 50 μm or less, heat from the thermal head can be uniformly transmitted, and the sharpness is improved.

The coating weight of the release layer and the reagent immobilization layer 105 is not particularly limited and may be appropriately selected depending on the purpose, 0.5 g / ^{m 2} or more 50 g / ^{m 2} or less. As a method for evaluating the adhesion amount, there is generally a method for evaluating a film thickness and a weight per unit area. However, regarding the film thickness, it is difficult to measure accurately and simply because the film thickness is thin, and the release layer / reagent-immobilized layer has a structure having voids, depending on the variation in thickness depending on the location and the external environment. Since variations in thickness are likely to occur, the present invention evaluates by weight per unit area.
When the adhesion amount is 0.5 g / m ² or more, the application amount is appropriate, and the release layer / reagent solid phase layer 105 (resin layer 15) is not damaged. Further, when the adhesion amount is 50 g / m ² or less, the drying time becomes appropriate, and the peeling layer / reagent solid phase layer 105 is less likely to be 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. 16 or a solution containing the capture antibody (17, 18) is applied to form a coating film. Subsequently, the labeled antibody 16 or the capture antibody (17, 18) can be immobilized on the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 by drying the coating film.

  When a test line and a control line are formed by transferring a plurality of transfer media for an inspection apparatus without gaps, the coating film is preferably formed so that the antibody density is uniform.

-Immobilization of labeled antibody-
The method for immobilizing the labeled antibody is not particularly limited and may be appropriately selected depending on the purpose. For example, the labeled antibody may be applied to the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105. Examples thereof include a method in which a coating liquid is applied to form a water film, which is then dried up by natural drying, reduced pressure drying, freeze drying, or the like to form a solid phase.
The water film is preferably applied so as to have a uniform thickness.
The 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.

  There is no restriction | limiting in particular as a drying method of the said coating liquid, According to the objective, it can select suitably, For example, ventilation drying, vacuum drying, natural drying, freeze-drying etc. are mentioned. Among these, natural drying under low humidity or drying under reduced pressure is preferable.

  There is no restriction | limiting in particular as humidity at the time of drying, Although it can select suitably according to the objective, 30% or less is preferable at a relative humidity. When the humidity during drying is 30% or less in terms of the relative humidity, it is advantageous in that drying is appropriate and the antibody can be sufficiently solid-phased.

  There is no restriction | limiting in particular as drying temperature of the said coating liquid, Although it can select suitably according to the objective, Room temperature or more and 50 degrees C or less are preferable. When the drying temperature is 20 ° C. or higher, the coating liquid can be appropriately dried and the productivity is improved, and when it is 50 ° C. or lower, the reagent can be prevented from being denatured by heat. Is advantageous.

  There is no restriction | limiting in particular as drying time of the said coating liquid, Although it can select suitably according to the objective, 30 minutes or more and 24 hours or less are preferable. When the drying time is 30 minutes or longer, the coating liquid can be appropriately dried. When the drying time is 24 hours or shorter, productivity is improved and discoloration can be prevented.

-Immobilization of capture antibody-
The method for immobilizing the capture antibody is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the capture antibody is formed on the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105. Apply a coating solution to form a water film and dry it by natural drying, vacuum drying, freeze drying, etc. (dry-up method), or in a humid environment so that the coating solution does not dry Examples of the method include a method in which the surface of the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 is washed with distilled water and dried to be immobilized (method of drying after adsorption). In any case, the coating film is preferably applied so as to have a uniform thickness.

  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. Be mentioned. Among these, natural drying under low humidity or drying under reduced pressure is preferable.

  There is no restriction | limiting in particular as humidity at the time of drying of the said coating liquid, Although it can select suitably according to the objective, 30% or less is preferable at relative humidity. When the humidity during drying is 30% or less in terms of the relative humidity, it is advantageous in that drying is appropriate and the antibody can be sufficiently solid-phased.

  There is no restriction | limiting in particular as drying temperature of the said coating liquid, Although it can select suitably according to the objective, Room temperature or more and 50 degrees C or less are preferable. When the drying temperature is 20 ° C. or higher, the coating liquid can be appropriately dried and the productivity is improved, and when it is 50 ° C. or lower, the reagent can be prevented from being denatured by heat. Is advantageous.

  There is no restriction | limiting in particular as drying time of the said coating liquid, Although it can select suitably according to the objective, 30 minutes or more and 24 hours or less are preferable. When the drying time is 30 minutes or longer, the coating liquid can be appropriately dried. When the drying time is 24 hours or shorter, productivity is improved and discoloration can be prevented.

  As a stationary condition when the capture antibody is solid-phased by a method of drying after adsorption, the temperature is preferably 0 ° C. or higher and 40 ° C. or lower. When the temperature of the stationary condition is 0 ° C. or higher, the capture antibody can be appropriately immobilized. Moreover, when the temperature is 40 ° C. or less, it is advantageous in that the capture antibody is hardly denatured.

  The relative humidity in the stationary condition is preferably 30% or more. If the relative humidity of the standing condition is 30% or more, it is advantageous in that a large amount of unnecessary components other than the antibody is not solid-phased because there is little volatilization of moisture during standing. is there.

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. An example is a method in which 100 μL or less of distilled water or the like is poured and then gently shaken at room temperature 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.

  There is no restriction | limiting in particular as drying temperature, Although it can select suitably according to the objective, Room temperature (20 degreeC) or more and 50 degrees C or less are preferable. 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.

  There is no restriction | limiting in particular as drying time, Although it can select suitably according to the objective, It is preferable to dry for 30 minutes or more and 24 hours or less. When the drying time is 30 minutes or longer, drying can be performed appropriately, and when it is 24 hours or shorter, productivity is improved and discoloration of the resin can be prevented.

The amount of immobilized capture antibody is preferably 500 ng / cm ² or more. When the immobilized amount of the capture antibody is 500 ng / cm ² or more, the immobilized amount is appropriate and 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).

  In addition, both the labeled antibody and the capture antibody may be formed so as to have coating solutions having different densities of the antibody on the surface of the same resin layer by using masking or the like.

<Back layer>
The transfer medium 100 for an inspection apparatus 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 method for manufacturing an inspection apparatus according to the present invention, a transfer medium having a support and an upper layer including at least a resin constituting the resin layer provided on the support is brought into contact with a porous flow path member, A step of transferring the upper layer, and a step of making the width in the thickness direction of the detection region smaller than the width in the thickness direction of the flow path member other than the detection region, and further performing other steps as necessary. Have. The upper layer corresponds to the reagent-immobilized layer and the release layer / reagent-immobilized layer.

  In the first embodiment of the method for producing an inspection apparatus according to the present invention, the reagent-immobilized layer of the inspection apparatus transfer medium is brought into contact with a porous flow path member so that the reagent-immobilized layer is A step of transferring to the flow path member (hereinafter, also referred to as “reagent-immobilized layer transfer step”).

  In the second embodiment of the method for manufacturing an inspection apparatus according to the present invention, the release layer / reagent is brought into contact with the release layer / reagent solid phase layer of the transfer medium for the inspection apparatus and a porous flow path member. There is a step of transferring the solid phase layer to the flow path member (hereinafter also referred to as “transfer step of the release layer / reagent solid phase layer”).

<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 of the transfer medium 100 for an inspection apparatus can be used. 105 and the flow path member 12 are brought into contact with each other, and the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 is transferred to the flow path 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 repeated desorption, the slower the rate of permeation through the flow path. On the other hand, a highly hydrophobic component penetrates without adsorbing to the stationary phase, and thus moves quickly in the flow path. By selectively extracting and reacting the target component of the test liquid 30 using the difference in moving speed in the test liquid, the test apparatus 10 can be used as a highly functional sensor for chemical or biochemical applications. .

<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. A labeled antibody 16 (an example of a reagent) that is released from the resin layer 15a by bringing it into contact with the test solution 30, and further includes other steps as necessary.

  As a method for testing using the test apparatus 10, when the test liquid 30 is supplied to the flow path member 12 of the test apparatus 10 and when the test liquid 30 contains the antigen 31, the antigen 31 (part of the sample) May include a step of capturing with a 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 sample pad 19 (see FIG. 2) provided in the flow path member 12 of the test apparatus 10. Next, the supplied test solution 30 and the labeled antibody 16 immobilized on the first resin layer 15a are brought into contact with each other, and the labeled antibody 16 is released from the first resin layer 15a. When the test solution 30 contains the antigen 31, the labeled antibody 16 released from the first resin layer 15a reacts with and binds to the antigen 31 (see FIG. 6).

  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 second resin layer 15b is disposed. The capture antibody 17 immobilized on the surface of the second 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 second resin layer 15b by the hydrophobic group 17g, the capture antibody 17 does not have affinity with the test solution 30 and is not easily released even when it comes into contact with the test solution 30. 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 second resin layer 15b, so that the test line is clearly colored (see FIGS. 7A and 7B).

  The labeled antibody 16 that has passed without being captured in the second resin layer 15b is developed along the flow path member 12, and reaches the region where the third resin layer 15c is disposed. In the present embodiment, a capture antibody 18 having a hydrophobic group is immobilized on the surface of the third resin layer 15c that faces 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 third resin layer 15 c by a hydrophobic group, even if it contacts the test solution 30, it does not have affinity with the test solution 30 and is not easily released. Even if a part of the capture antibody 18 is released into the test solution 30, it immediately binds to the fibers constituting the flow path member 12. As a result, the labeled antibody 16 is immobilized in the vicinity of the third resin layer 15c, so that the control line is clearly colored (see FIGS. 8A and 8B).

(Inspection kit)
The test kit of the present invention includes the test apparatus of the present invention and a collecting means for collecting a substance to be detected (specimen). Furthermore, it is preferable to have a liquid for extracting the collected substance to be detected, and other members are provided as necessary.

As an example shown in FIG. 12, the test kit includes the test apparatus 10 of the present invention, an instrument for collecting a sample (an example of a collection unit), and a liquid for extracting the sample.
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 extracting 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 above-described embodiment, an example has been described in which the support 101 and the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 in the transfer medium 100 for inspection apparatus are peeled off by heat during transfer. The invention is not limited to this embodiment. For example, the support 101 and the reagent-immobilized layer 103 or the separation layer / reagent-immobilized layer 105 may be separated by light. In this case, the release layer 102 or the release layer / reagent-immobilized layer 105 is mixed with a light absorber such as carbon black, and the light is absorbed thereby to generate heat, whereby the release layer 102 or the release layer. Alternatively, the reagent-immobilized layer 105 may be melted, and the reagent-immobilized layer 103 or the release layer / reagent-immobilized layer 105 may be peeled off. 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”.

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

(Preparation Example 1)
-Preparation of back layer coating solution-
16.8 parts by mass of silicone rubber emulsion (manufactured by Shin-Etsu Chemical Co., Ltd., KS779H, solid content 30% by mass), 0.2 part by mass of chloroplatinic acid catalyst, and 83 parts by mass of toluene were mixed. A back layer coating solution was prepared.

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

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

(Preparation Example 4)
-Preparation of reagent coating solution for test line-
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 concentration of the antibody was 100 μg / mL, and the test line reagent coating solution of Preparation Example 4 was prepared.

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

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

(Preparation Example 7)
-Preparation of reagent coating solution for test line-
Anti-hCG monoclonal antibody (Medix Biochemica, Anti-Alpha subunit 6601 SPR-5) as an antibody diluent, Dulbecco's phosphate buffered saline (Ca, Mg-free, D-PBS (-), Nacalai Tesque, Inc. Manufactured, 14249-95) to 100 μg / mL, and the test line reagent coating solution of Preparation Example 7 was prepared.

(Preparation Example 8)
-Preparation of reagent coating solution for control line-
The above-mentioned D-PBS (−) was added to an anti-mouse immunoglobulin antibody (Z0259 antibody manufactured by Dako Japan Co., Ltd.) as an antibody diluent to give 100 μg / mL, thereby preparing a control line reagent coating solution of Preparation Example 8.

(Preparation Example 9)
-Preparation of reagent coating solution for labeled antibody-
1 mL of KH2PO4 buffer (pH 7.0) adjusted to 50 mM was added to 9 mL of colloidal gold solution (BBI, EMGC50), and then anti-hCG monoclonal antibody (Medix Biochemica, Anti-hCG, adjusted to 50 μg / mL). 1 mL of 5008 SP-5) was added and stirred. After allowing this to stand for 10 minutes, 550 μL of a 1% by weight polyethylene glycol aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd., 168-11285) 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, leaving about 1 mL, and the gold colloid was redispersed with an ultrasonic cleaner. Centrifugation was carried out using a centrifuge (manufactured by Hitachi Koki Co., Ltd., himacCF16RN) under conditions of a centrifugal acceleration of 8,000 × g and 4 ° C. Then, it was dispersed in 20 mL of colloidal gold stock solution (20 mM Tris-HCl buffer (pH 8.2), 0.05 mass% polyethylene glycol, 150 mM NaCl, 1 mass% BSA aqueous solution, 0.1 mass% NaN3 aqueous solution), and again the above After centrifuging under the same conditions as above, the supernatant was removed, leaving about 1 mL, and the gold colloid was redispersed with an ultrasonic cleaner. These operations were repeated to obtain a reagent solution for a labeled antibody so that the colloidal gold preservation solution had an OD of 8.0.

  The reagent solution for the antibody described above was diluted 4-fold with a colloidal gold coating solution (20 mM Tris-HCl buffer (pH 8.2), 0.05 mass% polyethylene glycol, 150 mM, 3.5% sucrose), and the OD was 2.0. Thus, a labeled antibody reagent coating solution of Preparation Example 9 was obtained.

Example 1
<Preparation of thermal transfer media for test lines>
-Formation of back layer-
The back layer coating solution of Preparation Example 1 was applied to one side of a polyethylene terephthalate (PET) film (Toray Co., Ltd., Lumirror F57) having an average thickness of 4.5 μm as a support, and dried at 80 ° C. for 10 seconds. A back layer having an average thickness of 20 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 / reagent solid phase layer 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 coated with the test line reagent coating solution was placed in a container maintained at a relative humidity of 80% so that the water film did not dry, and was allowed to stand at 25 ° C. for 10 minutes. After allowing to stand, a thermal transfer medium is 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). Distilled water was poured onto the surface 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 the shaking, the supernatant liquid after washing was thoroughly cut from the surface of the thermal transfer medium and washed. After washing, the thermal transfer medium was dried as it was in a desiccator at a temperature of 25 ° C. and a relative humidity of 20% for 15 minutes to immobilize the reagent on the release layer / reagent immobilization layer (for immobilization). Thus, a test line thermal transfer medium was obtained.

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

<Preparation of thermal transfer medium for labeled antibody>
The back layer and release layer / reagent layer are fixed 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 production of the thermal transfer medium for test lines. After the formation of the phase-immobilizing layer (for immobilization), the reagent-immobilized layer (for release) coating solution of Preparation Example 3 is applied onto the release layer / reagent-immobilized layer (for immobilization), at 40 ° C. It was dried for 10 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 onto the reagent-immobilized layer (for release) so as to be 14 μL / cm ^2, and dried in a vacuum dryer at 25 ° C. for 5 hours. Thus, the reagent was immobilized on the reagent-immobilized layer (for release). Thus, a thermal transfer medium for labeled antibody of Example 1 was obtained.

<Production of inspection device>
The inspection apparatus shown in FIGS. 13A and 13B was produced as follows. FIG. 13A is a top view illustrating an example of the inspection apparatus used in the example. 13B is a cross-sectional view taken along the line DD ′ of FIG. 13A.

-Fabrication of paper substrate-
A polyester hot melt adhesive (Aron Melt PES375S40, manufactured by Toa Gosei Co., Ltd.) is used as a thermoplastic resin on top of a PET film (Lumilar S10, manufactured by Toray Industries, Inc., average thickness 50 μm) 11 cut to a width of 40 mm × length of 80 mm. Using an roll coater, the film was heated to 190 ° C. and coated to form an adhesive layer so that the average thickness was 50 μm on the PET film.

After the PET film 11 on which the adhesive layer was formed was allowed to stand for 2 hours or more, a nitrocellulose membrane (Merck Millipore, HF75, porosity 70%, cut into a width of 40 mm and a length of 65 mm on the surface of the adhesive layer) The thickness of 140 μm) is overlaid so that one end on the major axis side of each member is aligned 5 cm away from one end on the major axis side of the adhesive layer surface, and a load of 1 kgf / cm ² is applied at a temperature of 150 ° C. for 10 seconds. I took it. Finally, the paper substrate 12 (flow channel member 12) was obtained by cutting along the long axis direction so as to be 4 mm wide × 80 mm long.

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

[Calculation Formula 1]
Porosity (%) = {1− [basis weight (g / m ² ) / average thickness (μm) / specific gravity of composition]} × 100

  Further, in the obtained paper substrate 12, the average hole diameter of the detection region (second portion 12e) is smaller than the average hole diameter of the region (first portion 12d) in the flow path member other than the detection region. .

-Transcription of labeled antibody-
After the paper substrate 12 and the side of the labeled antibody thermal transfer medium on which the reagent is solid-phased are faced and overlapped, using a thermal transfer printer, as shown in FIGS. 13A and 13B, the paper substrate 12 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.

-Processing of detection area-
75 kg using a hand press machine (manufactured by CG Corporation) in a line shape of 4 mm wide and 0.9 mm long from the surface opposite to the surface to which the labeled antibody line is transferred 44.8 mm away from the upstream end of the paper substrate 12 Weight / cm ² Pressed for 1 second. At this time, the thickness d1 of the detection region was 100 μm while the thickness d1 of the membrane was 140 μm.

-Transfer of test line and control line-
As shown in FIG. 13A and FIG. 13B, the test line thermal transfer medium was first transferred in a line shape of 4 mm width × 0.5 mm length to a position 15 mm away from the transfer position of the labeled antibody thermal transfer medium. . Further, the control line thermal transfer medium was transferred in a line shape having a width of 4 mm and a length of 0.5 mm at a position 5 mm away from the transfer position of the test line thermal transfer medium. Each line was formed under the same printing conditions as the transfer of the labeled antibody.

-Preparation of sample pad and absorbent member-
As shown in FIG. 13A and FIG. 13B, the immunochromatographic assay of Example 1 was performed by providing the sample pad 19 (Bencott M-3II manufactured by Asahi Kasei Corporation) and the absorbing member 14 (Surewick C248 manufactured by Merck Millipore). An inspection device 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 obtain a test solution in which the concentration of the antigen was adjusted to 500 μg / mL.

-Reaction-
100 μL of the test solution was dropped on the sample pad 19 of the immunochromatographic assay shown in FIGS. 13A and 13B, and the state of development was observed.

-Color evaluation of lines-
Immunochromatography reader C10066-10 (manufactured by Hamamatsu Photonics Co., Ltd.) was used for evaluation of the color density of the line. The reflectance in the vicinity of the test line was measured using the same product, and the absorption intensity was digitized using analysis software to calculate the peak area. The evaluation of the color density was carried out 15 minutes after the sample extraction droplet and 1 day after the sample extract was sufficiently dried.

(Example 2)
It was produced in the same manner as in Example 1 except that the pressure by the hand press machine was changed from 75 kg weight / cm ² to 200 kg weight / cm ² in the processing area of the detection region. At this time, the thickness d2 of the detection region was 70 μm.

(Example 3)
It was produced in the same manner as in Example 1 except that the pressure by the hand press machine was changed from 75 kg weight / cm ² to 350 kg weight / cm ² in the processing area of the detection region. At this time, the thickness d2 of the detection region was 40 μm.

Example 4
Instead of pressing with a hand press machine at the processing portion of the detection area, a method of peeling the surface with cello tape (manufactured by Nichiban Co., Ltd., registered trademark) was used. Cut the cello tape into a width of 10 mm and a length of 0.9 mm, paste it in parallel on the transfer surface of the membrane so that it overlaps with a width of 4 mm and a length of 0.9 mm, leave it for 10 seconds, and then peel off the cello tape vertically. Then, the surface of the membrane was peeled off. At this time, the thickness d2 of the detection region was 100 μm. Other than that was produced by the same method as in Example 1.

(Example 5)
Stripping with the cellophane described in Example 4 was repeated twice at the processed portion of the detection region. At this time, the thickness d2 of the detection region was 70 μm. Other than that was produced by the same method as in Example 1.

(Example 6)
Stripping with the cellophane described in Example 4 was repeated three times at the processed portion of the detection region. At this time, the thickness d2 of the detection region was 40 μm. Other than that was produced by the same method as in Example 1.

(Comparative Example 1)
It was produced in the same manner as in Example 1 except that the detection area was not processed.

(Example 7)
-Formation of test lines and control lines-
In Example 1, the test line and the control line were transferred by thermal transfer. In Example 7, the test line and the control line were formed using a positive pressure spray apparatus (Biojet, Biojet).
At a position 15 mm away from the transfer position of the labeled antibody thermal transfer medium, the test line reagent coating solution of Preparation Example 4 is 50 μL per unit area (cm ² ) in a line shape of 4 mm width × 0.5 mm length. It was applied to. Furthermore, the control line reagent coating solution of Preparation Example 5 was placed at a distance of 5 mm from the transfer position of the test line thermal transfer medium in a line form of 4 mm width × 0.5 mm length to 50 μL per unit area (cm ² ). It was applied so that it was dried at 20 ° C. in an environment with a relative humidity of 20% for 1 hour. It was produced by the same method as in Example 1 except that the formation method of the test line and the control line was changed. The inspection apparatus shown in FIGS. 13A and 13C was manufactured as described above.

(Example 8)
It was produced in the same manner as in Example 7 except that the pressure by the hand press machine was changed from 75 kg weight / cm ² to 200 kg weight / cm ² in the processing portion of the detection region. At this time, the thickness d2 of the detection region was 70 μm.

  In FIG. 13C, the flow path member 12 in the vicinity of the second resin layer 15b is not interrupted, but is locally recessed, and the speed at which the test solution flows is slow.

Example 9
It was produced in the same manner as in Example 7 except that the pressure by the hand press machine was changed from 75 kg weight / cm ² to 350 kg weight / cm ² in the processing area of the detection region. At this time, the thickness d2 of the detection region was 40 μm.

(Comparative Example 2)
It was produced in the same manner as in Example 7 except that the detection area was not processed.

(Example 10)
<Production of inspection device>
The inspection apparatus shown in FIGS. 14A and 14B was produced as follows. FIG. 14A is a top view illustrating an example of the inspection apparatus used in the example. 14B is a cross-sectional view taken along line EE ′ of FIG. 14A.

-Preparation of labeled antibody holding pad-
The reagent solution for labeled antibody of Preparation Example 9 was applied to a glass fiber pad (GFCP203000, manufactured by Merck Millipore) cut to a width of 40 mm × 12 mm so as to be 1.5 μL / cm ² , dried under reduced pressure for 5 hours, and labeled. An antibody holding pad 15a ′ was prepared.

-Fabrication of paper substrate-
A polyester hot melt adhesive (Aronmelt PES375S40, manufactured by Toa Gosei Co., Ltd.) is used as a thermoplastic resin on the upper part of a PET film (Lumirror S10, manufactured by Toray Industries, Inc., average thickness 50 μm) 11 cut to a width of 40 mm × length of 75 mm. Using an roll coater, the film was heated to 190 ° C. and coated to form an adhesive layer so that the average thickness was 50 μm on the PET film.
After the PET film 11 having the adhesive layer formed thereon was allowed to stand for 2 hours or longer, a nitrocellulose membrane (manufactured by Merck Millipore, HF180, porosity 70%, cut to a width of 40 mm × length of 30 mm on the surface of the adhesive layer) 140 μm in thickness) are stacked so that one end on the long axis side of each member is aligned at a position 25 mm away from one end on the long axis side of the adhesive layer surface, and a load of 1 kgf / cm ² is applied for 10 seconds at a temperature of 150 ° C. I took it.

-Processing of detection area-
Using a hand press machine (manufactured by CG Corporation) in a line shape of width 40 mm × length 2.0 mm from the surface opposite to the surface to which the labeled antibody line is transferred at a position 39 mm away from the upstream end of the paper substrate 12, 75 kg weight / cm ² Pressed for 1 second. At this time, the thickness d1 of the detection region was 100 μm while the thickness d1 of the membrane was 140 μm.

-Formation of test lines and control lines-
Using a positive pressure spray device (Biojet, Biojet), the reagent coating solution for test line of Preparation Example 7 was formed in a unit area (cm) in a line shape of width 4 mm × length 1.0 mm at a position 40 mm away from the upper end side. ² ) It was applied so as to be 50 μL per unit. Furthermore, the reagent coating solution for the control line of Preparation Example 8 was added to a 50 μL per unit area (cm ² ) in the form of a line 4 mm wide × 1.0 mm long at a position 45 mm away from the upper end using a positive pressure spray device. It was applied so that it was dried at 20 ° C. in an environment with a relative humidity of 20% for 1 hour.

-Cutting paper substrates-
A paper substrate 12 was obtained by cutting so as to be 4 mm wide × 75 mm long along the long axis direction. In addition, the direction in which 25 mm of the adhesive layer remains is the upper end side, and the direction in which the adhesive layer remains 10 mm is the lower end side.

-Bonding each component to paper substrate-
The labeled antibody holding pad 15a ′ containing the labeled antibody on a paper substrate is cut so as to have a width of 4 mm × a length of 12 mm, and one end on the long axis side is aligned from the upper end side and bonded so as to overlap the 2 mm membrane. Next, as shown in FIGS. 14A and 14B, the immunochromatography of Example 10 was performed by providing a sample pad 19 (Bencott M-3II manufactured by Asahi Kasei Corporation) and an absorbing member 14 (Surewick C248 manufactured by Merck Millipore). An assay (test device 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 hCG antigen (Fitzgerald 30-1132) to obtain a test solution in which the concentration of the antigen was adjusted to 500 mIU / mL.

-Reaction-
100 μL of the test solution was dropped on the sample pad 19 of the immunochromatographic assay shown in FIGS. 14A and 14B, and the state of development was observed. The labeled antibody holding pad 15 a ′ in FIG. 14B is porous, and the test solution passes through the labeled antibody holding pad 15 a ′ to reach the flow path member 12.

-Color evaluation of lines-
Immunochromatography reader C10066-10 (manufactured by Hamamatsu Photonics Co., Ltd.) was used for evaluation of the color density of the line. The reflectance in the vicinity of the test line was measured using the same product, and the absorption intensity was digitized using analysis software to calculate the peak area. The evaluation of the color density was carried out 15 minutes after the sample extraction droplet and 1 day after the sample extract was sufficiently dried.

(Example 11)
It was produced in the same manner as in Example 10 except that the pressing by the hand press machine was changed from 75 kg weight / cm ² to 200 kg weight / cm ² in the processing portion of the detection area. At this time, the thickness d2 of the detection region was 70 μm.

(Example 12)
It was produced in the same manner as in Example 10 except that the pressing by the hand press machine was changed from 75 kg weight / cm ² to 350 kg weight / cm ² in the processing portion of the detection region. At this time, the thickness d2 of the detection region was 40 μm.

(Comparative Example 3)
It was produced by the same method as in Example 10 except that the processing operation of the detection unit was not performed.

(Example 13)
In the processing portion of the paper substrate of Example 1, the flow path member to be superimposed on the adhesive layer was changed from a nitrocellulose membrane (manufactured by Merck Millipore, HF75, porosity 70%, thickness 140 μm) to hydrophilic PTFE (Merck Millipore). JMWP14225, porosity 80%, thickness 85 μm). Further, regarding the processing method by pressurizing the detection region, the hand is formed in a line shape of 4 mm wide and 0.9 mm long from the surface opposite to the surface to which the labeled antibody line is transferred at a position 44.8 mm away from the upstream end of the paper substrate 12. By pressing using a press machine (manufactured by CG Corporation) at 150 kg weight / cm ^{2 for} 1 second, the thickness d1 of the hydrophilic PTFE was 85 μm, and the thickness d2 of the detection region was 45 μm.

(Comparative Example 4)
The detection region was fabricated in the same manner as in Example 13 except that the processing of the detection area was not performed.

(Example 14)
In the processing portion of the paper substrate of Example 1, the flow path member to be superimposed on the adhesive layer was changed from a nitrocellulose membrane (manufactured by Merck Millipore, HF75, porosity 70%, thickness 140 μm) to qualitative filter paper No4 (ADVANTEC). Manufactured, porosity 48%, thickness 120 μm). Further, regarding the processing method by pressurizing the detection region, the hand is formed in a line shape of 4 mm wide and 0.9 mm long from the surface opposite to the surface to which the labeled antibody line is transferred at a position 44.8 mm away from the upstream end of the paper substrate 12. By pressing with a press machine (manufactured by CG Corporation) at 150 kg weight / cm ^{2 for} 1 second, the thickness d1 of the qualitative filter paper No4 was 120 μm, and the thickness d2 of the detection region was 60 μm.

(Comparative Example 5)
It was produced by the same method as in Example 14 except that the detection area was not processed.

The results obtained are shown in the table below.
Note that, in the example in which the detection area is pressed by a hand press machine, the average pore diameter of the detection area (second part 12e) is the area (first part 12d) in the flow path member other than the detection area. ) Smaller than the average pore diameter.

  The contents of Examples 1 to 6 and Comparative Example 1 are summarized in Table 1.

[Evaluation criteria]
A: A sufficient color density was obtained after 15 minutes.
(After 15 minutes: 10000 or more, and 1 day later: 18000 or more)
○: A sufficient color density is obtained after drying, but the color density is low after 15 minutes.
(One day later: over 18000)
Δ: Visible, but sufficient color density is not obtained even after drying.
(One day later: 10000 or more and less than 18000)
X: The color density is low even after drying and is difficult to visually recognize.
(One day later: less than 10,000)

  From the results in Table 1, it was confirmed that the coloring density was higher in Examples 1 to 6 than in Comparative Example 1. Particularly in Examples 1, 2, and 5, the color density increased 15 minutes after the sample extraction droplet, indicating that it is useful in actual diagnosis. In Examples 3 and 6, it took time until the color density was obtained because the thickness of the detection region was too small and the progress of the specimen extract in the detection region was hindered. Further, although it was shown that the color density of Example 4 was higher than that of Comparative Example 1, since the effect was limited, it is larger when the hole diameter is reduced by pressing as in Example 1. An effect can be obtained.

  Next, the contents of Examples 1 to 3, Examples 7 to 9, and Comparative Examples 1 to 2 are summarized in Table 2.

[Evaluation criteria]
A: A sufficient color density was obtained after 15 minutes.
(After 15 minutes: 10000 or more, and 1 day later: 18000 or more)
○: A sufficient color density is obtained after drying, but the color density is low after 15 minutes.
(One day later: over 18000)
Δ: Visible, but sufficient color density is not obtained even after drying.
(One day later: 10000 or more and less than 18000)
X: The color density is low even after drying and is difficult to visually recognize.
(One day later: less than 10,000)

  From the results of Table 2, it was confirmed that Examples 7 to 9 had higher color density than Comparative Example 2. This indicates that the color density is increased by processing the detection region and making d1 larger than d2, regardless of the test line formation method.

  Next, the contents of Examples 10 to 12 and Comparative Example 3 are shown together in Table 3.

[Evaluation criteria]
A: A sufficient color density was obtained after 15 minutes.
(After 15 minutes: 20000 or more, and 1 day later: 36000 or more)
○: A sufficient color density is obtained after drying, but the color density is low after 15 minutes.
(One day later: over 36000)
Δ: Visible, but sufficient color density is not obtained even after drying.
(One day later: 20000 or more and less than 36000)
X: The color density is low even after drying and is difficult to visually recognize.
(One day later: less than 20000)

  From the results in Table 3, it was confirmed that the color density was higher in Examples 10 to 12 than in Comparative Example 3. This indicates that the effects of the present invention are widely applied to general antigen antibodies.

  Next, Table 4 summarizes the contents of Examples 2, 13, and 14 and Comparative Examples 1, 4, and 5.

[Evaluation criteria]
A: A sufficient color density was obtained after 15 minutes.
(After 15 minutes: 10000 or more, and 1 day later: 18000 or more)
○: A sufficient color density is obtained after drying, but the color density is low after 15 minutes.
(One day later: over 18000)
Δ: Visible, but sufficient color density is not obtained even after drying.
(One day later: 10000 or more and less than 18000)
X: The color density is low even after drying and is difficult to visually recognize.
(One day later: less than 10,000)

  From the results of Table 4, in Examples 2, 13, and 14, it was confirmed that the color density increased 15 minutes after the sample extraction droplet by the processing by pressing. From this, it was shown that the effect can be obtained by processing if the flow path member is generally used in a lateral flow assay inspection apparatus.

DESCRIPTION OF SYMBOLS 10 Inspection apparatus 10 'Inspection apparatus 11 Base material 12 Flow path member 12a Space | gap 12b Space | gap 12d 1st site | part 12e 2nd site | part (detection area | region)
14 Absorbing member 15 Resin layer 15a First resin layer 15b Second resin layer (test line)
15c Third resin layer (control line)
16 Labeled antibody (example of reagent)
17 Capture antibody (example of reagent)
18 Capture antibody (example of reagent)
19 Sample pad 30 Test solution (example of specimen)
31 Antigen 50 Test Kit 51 Sterile Cotton Swab 52 Diluent 100 Transfer Medium for Inspection Device 110 Transfer Medium for Inspection Device 101 Support 102 Release Layer 103 Reagent Immobilization Layer 104 Back Layer 105 Release Layer / Reagent Immobilization Layer

JP 2013-113633 A JP 2001-157598 A JP 2005-503556 A

Claims (7)

A porous flow path member in which a flow path for the substance to be detected is formed;
A detection unit that is formed so as to be in contact with the flow path member and includes a substance that can bind to the detection target substance;
When the area in the thickness direction of the flow path member in contact with the detection unit is set as a detection area, the width in the thickness direction of the detection area is smaller than the width in the thickness direction of the flow path member other than the detection area. Characteristic lateral flow assay testing device.   The inspection apparatus according to claim 1, wherein a substance that can bind to the substance to be detected is solidified in a resin on a surface that faces the flow path member.   The inspection apparatus according to claim 1 or 2, wherein the substance to be detected is an antigen, and the substance capable of binding to the substance to be detected is an antibody.   The inspection apparatus according to claim 1 or 2, wherein the substance to be detected is a nucleic acid, and the substance capable of binding to the substance to be detected is a capture nucleic acid having a sequence complementary to the nucleic acid.   The inspection apparatus according to claim 1, wherein an average hole diameter of the flow path member in the detection area is smaller than an average hole diameter of the flow path member other than the detection area.   When the width in the thickness direction of the flow path member other than the detection region is d1, and the width in the thickness direction of the detection region is d2, 0.4 ≦ d2 / d1 ≦ 0.8. The inspection apparatus according to claim 1. The inspection apparatus according to any one of claims 1 to 6,
An inspection kit comprising: a collecting means for collecting the substance to be detected; and a liquid for extracting the collected substance to be detected.