JP6876328B2 - Container and its manufacturing method and detection method of test substance - Google Patents

Description

The present invention relates to a container used for detecting a test substance by an enzyme-linked immunosorbent assay (ELISA method), a method for producing the same, and a method for detecting the test substance using the container.

The ELISA method is a kind of immunoassay that uses a combination of a specific binding reaction between an antigenic determinant of an antigen and an antibody and a coloration reaction by an antibody or an enzyme labeled with an antigen. In the ELISA method, since the antigen-antibody reaction having high specificity is used to convert the color development based on the enzymatic reaction into an electric signal for measurement, the detection can be performed with high sensitivity and the quantification is also excellent. In addition, it is safer, cheaper and simpler than radioimmunoassay (Radioimmunoassay, RIA) using a radioactive substance as a labeling substance. Therefore, the ELISA method is used for the diagnosis of bio-related substances such as antibodies, influenza viruses, plasma proteins, cytokines, DNA, peptides and ligands; chemical substances such as residual pesticides and environmental hormones contained in foods; diabetes and cancer. It is widely used for the detection and quantification of various test substances such as blood glucose and diagnostic substances such as tumor markers.

In the ELISA method, a microplate is used as a container. The microplate is a flat plate provided with a plurality of dents (wells), and can perform antigen-antibody reaction and washing at once. In the microplate manufacturing process and the test substance detection process, a cleaning operation for washing away unreacted antibodies and the like is performed manually or by a dedicated plate washer. Manual cleaning is not easy for unskilled personnel due to the complexity of the work, and if a microplate with insufficient cleaning is used, unreacted antibodies remain, resulting in reduced sensitivity and measurement error. May cause an increase in. On the other hand, since the plate washer is equipped with peripheral devices such as pumps and valves, it is expensive and large in size, and can be used only by inspection institutions and research institutes, which is not convenient. Therefore, a container that can be washed by a simpler method has been developed. For example, Patent Document 1 discloses a disk-shaped analysis chip using centrifugal force. The analysis chip is provided with a plurality of tanks, and the reaction liquid and the washing liquid are injected into each tank, and the washing and the reaction are carried out by changing the magnitude and direction of the centrifugal force. Since centrifugal force is used and peripheral devices such as pumps and valves are not required, the washer can be inexpensive and miniaturized.

U.S. Patent Application Publication No. 2015/226730

However, since the analysis chip described in Patent Document 1 has a complicated tank structure, the antigen-antibody reaction and washing can be switched by changing the magnitude and direction of the centrifugal force, or a plurality of tanks are filled with a washing liquid in advance. There was room for improvement in that it lacked convenience. Therefore, an object of the present invention is to provide a container capable of easily performing a washing operation in an enzyme immunoassay.

The container of the present invention that has achieved the above object is used for detecting a test substance by an enzyme immunoassay, and has a base material having one main surface and the other main surface, and has a base material. On the other hand, the gist is that it has a central opening that is on the main surface side and includes the center of gravity, and a plurality of internal flow paths that communicate with the central opening and extend in the radial direction of the base material. Have. According to the container of the present invention, it is easy to move the cleaning liquid to a plurality of internal flow paths communicating with the central opening all at once by a simple method of putting the cleaning liquid in the central opening and applying centrifugal force to the container. And it can be washed sufficiently. Therefore, the detection accuracy of the test substance can be ensured regardless of the proficiency level of the operator. In addition, a small and inexpensive centrifuge that does not require peripheral devices such as pumps and valves can be used as a washer.

In the above container, the base material has an upper base material and a lower base material laminated with each other, and the lower base material has a first opening including the center of gravity of the lower base material on one main surface side and a first opening. It has a second opening that communicates with one opening and extends in the radial direction, the upper substrate has a first through opening that overlaps the first opening, and the central opening has a central opening. It is preferably formed from the first opening and the first through opening, and the internal flow path is formed from the second opening.

In the above container, the lower base material is composed of a lower first base material and a lower second base material laminated with each other, and the first opening and the second opening form the lower first base material. It is preferably provided and penetrates from the upper base material side toward the lower second base material side.

In the above container, it is preferable that the outer end of the first opening is formed inward of the outer end of the first through port in the radial direction.

In the above container, it is preferable that the base material has a plurality of outer openings that are outward in the radial direction from the central opening and communicate with the inner flow path.

In the above container, it is preferable that a filter for absorbing liquid is provided around the internal flow path.

In the above container, it is preferable that an antibody for detecting a test substance is held on a wall surface forming an internal flow path.

The present invention also provides a method for producing a container used for detecting a test substance by an enzyme immunoassay. The method for manufacturing a container of the present invention is a lower base material having one main surface and the other main surface, and communicates with a first opening including the center of gravity of the lower base material on one main surface side and the first opening. A step of preparing a lower base material having a plurality of second openings extending in the radial direction, and an upper base material having one main surface and the other main surface, including the center of gravity of the upper base material. A step of preparing an upper base material having a first through-hole and a plurality of second through-holes arranged around the first through-hole, and a second opening by overlapping the first opening and the first through-hole. A step of joining one main surface of the lower base material and the other main surface of the upper base material in a state where the second through-holes are overlapped, and a step of putting a liquid containing an antibody that detects a test substance from the second through-holes. The main point is that it has a step of putting a cleaning liquid through the first through port and a step of rotating the container and discharging the residual liquid of the liquid containing the antibody and the cleaning liquid by centrifugal force.

The method for manufacturing the container further includes a step of pouring a liquid containing a blocking agent through the second through-hole, a step of pouring a cleaning liquid through the first penetrating port, and a step of rotating the container to prepare the liquid containing the blocking agent by centrifugal force. It is preferable to have a step of discharging the residual liquid and the cleaning liquid.

Furthermore, the present invention also provides a method for detecting a test substance by an enzyme immunoassay method using the above container. The detection method of the present invention has a gist in that it has a step of putting a cleaning liquid through a central opening and a step of rotating a container to move the cleaning liquid to a plurality of internal flow paths by centrifugal force.

According to the container of the present invention, it is easy to move the cleaning liquid to a plurality of internal flow paths communicating with the central opening all at once by a simple method of putting the cleaning liquid in the central opening and applying centrifugal force to the container. And it can be washed sufficiently. Therefore, the detection accuracy of the test substance can be ensured regardless of the proficiency level of the operator. In addition, a small and inexpensive centrifuge that does not require peripheral devices such as pumps and valves can be used as a washer.

The perspective view of the container which concerns on embodiment of this invention is shown. The front view of the container which concerns on embodiment of this invention is shown. A cross-sectional view taken along the line III-III of FIG. 2 is shown. The plan view of the upper base material of the container shown in FIG. 1 is shown. The plan view of the lower first base material of the container shown in FIG. 1 is shown. The plan view of the lower second base material of the container shown in FIG. 1 is shown. A front view showing a modified example of the container shown in FIG. 2 is shown. A cross-sectional view taken along the line VIII-VIII of FIG. 7 is shown. A front view showing another modification of the container shown in FIG. 2 is shown. A cross-sectional view taken along the line XX of FIG. 9 is shown. The plan view of the upper base material of the container shown in FIG. 9 is shown. The plan view of the lower first base material of the container shown in FIG. 9 is shown. The plan view of the filter of the container shown in FIG. 9 is shown. The plan view of the lower second base material of the container shown in FIG. 9 is shown. The schematic diagram which shows the manufacturing method of a container is shown. The schematic diagram which shows the manufacturing method of a container is shown. The schematic diagram which shows the manufacturing method of a container is shown. The schematic diagram which shows the manufacturing method of a container is shown.

Hereinafter, the present invention will be described in more detail based on the following embodiments, but the present invention is not limited by the following embodiments as well as the present invention, and appropriate changes are made to the extent that it can be adapted to the purpose of the above and the following. In addition, it is of course possible to carry out, and all of them are included in the technical scope of the present invention.

1. 1. Container The container of the present invention is a container for containing a sample or a washing solution when a test substance is detected by an enzyme-linked immunosorbent assay (ELISA method), and is preferably used in place of a conventional microplate. The container of the present invention can detect, measure, and analyze a test substance or an interaction specific to the test substance (antigen-antibody reaction, etc.), as well as to the human body based on these detection results, measurement results, or analysis results. It can also be used for various evaluations such as the degree of influence and diagnosis of illnesses.

Examples of the test substance include biological substances such as antibodies, influenza viruses, C-reactive proteins, plasma proteins, cytokines, DNAs, peptides and ligands; chemical substances such as residual pesticides and environmental hormones contained in foods; diabetes. , Blood plasma used for diagnosing cancer, diagnostic substances such as tumor markers, and the like.

The container of the present invention will be described with reference to FIGS. 1 to 6. 1 to 3 show a perspective view, a front view, and a cross-sectional view taken along the line III-III of FIG. 2, respectively, showing a perspective view and a front view of the container 1 (1A) according to the embodiment of the present invention. 4 to 6 show a plan view of the upper base material 20, the lower first base material 37, and the lower second base material 38 of the container 1A shown in FIG. 1, respectively.

The container 1 has a base material 10 having one main surface 11 and the other main surface 12, and the base material 10 has a central opening 13 on the one side of the main surface 11 and including the center of gravity C, and a central opening 13. It has a plurality of internal flow paths 15 that communicate with the base material 10 and extend in the radial direction of the base material 10.

In the present invention, the radial direction refers to the direction away from the center of gravity C of the base material 10, and the thickness direction refers to the direction from one main surface 11 of the base material 10 toward the other main surface 12.

The base material 10 constitutes the overall shape of the container 1, and is preferably formed in a flat plate shape. The shape of the base material 10 when viewed from the main surface 11 side can be circular, elliptical, polygonal, or a combination thereof, but is preferably circular.

The material constituting the base material 10 is, for example, polymethyl methacrylate resin (PMMA), polydimethylsiloxane (PDMS), glass, cycloolefin polymer (COP), cycloolefin copolymer (COC), polyethylene terephthalate (PET), polystyrene. (PS) and polypropylene (PP) can be mentioned. When the base material 10 is composed of a plurality of base materials, the material of the base material (for example, the lower second base material 38 described later) arranged on the other side of the main surface 12 has high transparency and a refractive index. It is preferable to use PMMA having characteristics such as small spatial distribution and anisotropy and small surface roughness. This makes it easier to detect the scattered light obtained from the test substance by irradiating the lower second base material 38 with the laser light.

In order to make the liquid supplied to the internal flow path 15 visible, the base material 10 is preferably made of a transparent or translucent material.

When the concentration of the test substance is measured by irradiating the test substance with laser light and detecting the scattered light obtained, it is preferable that the base material 10 has light transmittance. Specifically, the base material 10 preferably transmits light having a wavelength of 532 nm by 85% or more, more preferably 90% or more, still more preferably 95% or more.

The thickness of the base material 10 is not particularly limited, but may be, for example, 0.1 mm or more, 0.2 mm or more, 0.5 mm or more, or 1 mm or more, and is 10 mm or less, 8 mm or less, or 5 mm or less. Is also acceptable.

From the viewpoint of reducing the amount of the cleaning liquid and the sample supplied to the container 1, the outer diameter of the base material 10 as viewed from the main surface 11 side is preferably 150 mm or less, 100 mm or less, or 50 mm or less. From the viewpoint of improving the handleability of the container 1, the size of the base material 10 as viewed from the main surface 11 side is preferably 10 mm or more, 15 mm or more, or 20 mm or more.

A central opening 13 including the center of gravity C is provided on one main surface 11 side of the base material 10. The central opening 13 functions as a supply port for the cleaning liquid. Further, the base material 10 is also provided with a plurality of internal flow paths 15 that communicate with the central opening 13 and extend in the radial direction of the base material 10. The container 1 of the present invention communicates with the central opening 13 by a simple method of pouring a cleaning liquid through the central opening 13 and applying centrifugal force to the container 1, for example, rotating the base material 10 around the center of gravity C. The cleaning liquid can be moved to the plurality of internal flow paths 15 all at once. Therefore, the container can be easily and sufficiently washed, and the detection accuracy of the test substance can be ensured regardless of the proficiency level of the operator. In addition, a small and inexpensive centrifuge that does not require peripheral devices such as pumps and valves can be used as a washer.

The number of central openings 13 provided in the base material 10 is not particularly limited, but it is preferable that only one is provided. Since the cleaning liquid can be sent to the plurality of internal flow paths 15 by one operation of filling the cleaning liquid through the central opening 13, the operation can be simplified.

The shape of the central opening 13 when viewed from the one main surface 11 side of the base material 10 can be a circular shape, an elliptical shape, a polygonal shape, or a combination thereof, but it is easy to drop the cleaning liquid into the central opening 13. From the viewpoint of this, it is preferable to have a circular shape. The center of the circular central opening 13 may overlap with the center of gravity of the base material 10.

The internal flow path 15 is a flow path provided inside the base material 10, and corresponds to a well of a conventional microplate.

The internal flow path 15 may extend along the radial direction, that is, it may be formed radially, may be formed linearly, or may be formed non-linearly, for example, curvedly. You may. The internal flow path 15 may extend along the rotation direction of the container 1, or may extend along the direction opposite to the rotation direction of the container 1. Further, the internal flow path 15 may have a curved portion or a bent portion.

The number of internal flow paths 15 in one container 1 may be odd or even, and is not particularly limited, but can be set to, for example, 4 or more, 6 or more, 8 or more, or 10 or more, and 30 or less, 25 or less, It is also permissible to set it to 20 or less, or 15 or less.

The height of the internal flow path 15 in the thickness direction of the base material 10 may be, for example, 0.1 mm or more, 0.2 mm or more, 0.3 mm or more, or 0.5 mm or more, and 3 mm or less and 2 mm or less. , Or may be 1 mm or less.

It is preferable that the plurality of internal flow paths 15 are formed in the same shape. In order to make the amount of the cleaning liquid flowing into each internal flow path 15 uniform, it is preferable that the plurality of internal flow paths 15 are formed rotationally symmetrically with the center of gravity C as the center.

The base material 10 may be composed of a single member, or may be composed of a combination of a plurality of members. An example in which a plurality of members are combined to form the base material 10 will be described with reference to FIGS. 1 to 6. The base material 10 of the container 1 may have an upper base material 20 and a lower base material 30 that are laminated with each other. In that case, the lower base material 30 communicates with the first opening 35 including the center of gravity of the lower base material 30 on the main surface 11 side, and extends in the radial direction. It is preferable to have two openings 36. Further, the upper base material 20 has a first through opening 25 that overlaps with the first opening 35, and the central opening 13 is formed from the first opening 35 and the first through opening 25, and the internal flow. It is preferable that the road 15 is formed from the second opening 36. By forming the base material 10 from a plurality of members in this way, it is possible to change the material and thickness of each member and to impart different functions to each member. For example, it is preferable that the wall surface of the upper base material 20 facing the lower base material 30 is coated with a material that easily modifies an antibody, for example, polystyrene resin.

The upper base material 20 constitutes the upper wall surface (lid) of the inner flow path 15, and when centrifugal force is applied to the base material 10, liquid is unintentionally discharged from the inner flow path 15 or the container 1 is left in a predetermined time. Prevents the liquid from evaporating when left unattended. As the upper base material 20, as shown in FIG. 4, a flat plate having one first through hole 25 for forming the central opening 13 can be used. Although details will be described later, the upper base material 20 may be provided with a second through hole 26 for forming a plurality of outer openings 18.

Since the first opening 35 of the lower base material 30 constitutes the lower side of the central opening 13 and the second opening 36 constitutes the lateral side wall surface and the bottom wall surface of the internal flow path 15, the first opening 35 and the second opening 36 is preferably a non-penetrating groove in the thickness direction. In the thickness direction, the first opening 35 and the second opening 36 of the lower base material 30 may have the same height or may have different heights.

The plurality of second openings 36 are preferably formed in the same shape. In order to make the amount of the cleaning liquid flowing into each internal flow path 15 uniform, it is preferable that the plurality of second openings 36 are formed rotationally symmetrically about the center of gravity of the lower base material 30.

In FIGS. 1 and 3, when the base material 10 is viewed from the main surface 11 side, the upper base material 20 and the lower base material 30 are formed in the same circular outer shape. The outer end may extend outward from the outer end of the upper base material 20. The upper base material 20 and the lower base material 30 can be made of the same material or different materials.

Examples of the method of joining the upper base material 20 and the lower base material 30 include adhesion by an adhesive such as double-sided tape, heat welding such as laser welding and ultrasonic welding, and a convex portion provided on one member and another member. Examples thereof include engagement of the recess provided.

Further, the lower base material 30 may be composed of a plurality of members. The lower base material 30 is preferably composed of a lower first base material 37 and a lower second base material 38 that are laminated with each other. In that case, the first opening 35 and the second opening 36 are provided in the lower first base material 37 and penetrate from the upper base material 20 side toward the lower second base material 38 side. preferable. By composing the lower base material 30 from the lower first base material 37 and the lower second base material 38, it is possible to change the material and thickness of these base materials and to impart different functions. Further, since the first opening 35 and the second opening 36 penetrate, the cleaning liquid is referred to from the first opening 35, and the liquid containing a test substance such as an antigen is referred to from the second opening 36 (hereinafter, "sample liquid"). (Sometimes referred to as) can be inserted into the internal flow path 15.

In FIGS. 1 and 3, the lower first base material 37 is provided on one main surface 11 side of the base material 10, and the lower second base material 38 is provided on the other main surface 12 side of the base material 10. As shown in FIG. 5, the lower first base material 37 communicates with the first opening 35 for forming the central opening 13 and the first opening 35 for forming the internal flow path 15. A flat plate having a second opening 36 can be used. As the lower second base material 38, as shown in FIG. 6, a flat plate having no grooves or through holes can be used. The lower base material 30 can be formed by joining the lower first base material 37 and the lower second base material 38. The lower first base material 37 and the lower second base material 38 can be joined in the same manner as the upper base material 20 and the lower base material 30.

In the thickness direction, the lower base material 30 may be thicker than the upper base material 20, and the lower base material 30 may be thinner than the upper base material 20. Similarly, the magnitude relationship between the thicknesses of the lower first base material 37 and the lower second base material 38 is not particularly limited. For example, the upper base material 20 is a plate made of PMMA having a thickness of 0.5 mm, and the lower first base material 37 and the lower second base material 38 are made of PMMA having a thickness of 0.2 mm, respectively. It may be an acrylic plate.

The first opening 35 of the lower base material 30 and the first through port 25 of the upper base material 20 may have the same shape or different shapes. As shown in FIG. 3, in the present embodiment, the first opening 35 and the first through port 25 are formed in a circular shape having the same area.

It is preferable that the base material 10 has a plurality of outer openings 18 that are outward in the radial direction from the central opening 13 and communicate with the internal flow path 15, respectively. The sample liquid can be supplied from the outer opening 18 to the inner flow path 15. Further, the outer opening 18 can also be used as a drainage port for drainage.

The opening area of the outer opening 18 may be smaller than the opening area of the central opening 13 as long as the liquid supplied to the inner flow path 15 can be dropped or the liquid can be discharged from the inner flow path 15.

The position where the outer opening 18 is provided is not particularly limited, but when the outer opening 18 is used as at least one of a cleaning liquid discharge port and a sample liquid supply port, the outer opening 18 is an internal flow path 15 in the radial direction. It is preferable that it overlaps with the outer end of. Further, when the outer opening 18 is used only as a supply port for the sample liquid, the outer opening 18 may exist inward of the outer end of the inner flow path 15.

The outer opening 18 is preferably provided inward of the midpoint between the inner and outer ends of one internal flow path 15 in the radial direction. This prevents the liquid supplied to the internal flow path 15 through the outer opening 18 from being unintentionally discharged.

When the base materials 10 have an upper base material 20 and a lower base material 30 laminated with each other, it is preferable that the outer opening 18 is formed from the second through hole 26 of the upper base material 20. The outer opening 18 can be formed by a simple method of providing the second through hole 26 in the upper base material 20.

The internal flow path 15 preferably has a narrow portion 16 whose width narrows toward the central opening 13. By providing the narrow portion 16 in the internal flow path 15, it is possible to prevent the washing liquid from unintentionally moving to the internal flow path 15 when centrifugal force is not applied, for example, when an antigen-antibody reaction is being carried out. The width of the narrow portion 16 is not particularly limited, but is preferably one-half or less, more preferably one-third or less, of the maximum width of the internal flow path 15.

The narrow portion 16 is preferably formed inward of the midpoint between the inner and outer ends of the internal flow path 15 in the radial direction. Further, the length of the narrow portion 16 in the radial direction is preferably one-fifth or less of the length of the internal flow path 15, and more preferably one-tenth or less. With these configurations, the effect of suppressing the movement of the cleaning liquid is effectively exhibited.

It is preferable that the container 1 holds an antibody for detecting a test substance on the wall surface forming the internal flow path 15. Specifically, it is more preferable that the antibody is immobilized on the inner wall surface of the base material 10 on the one main surface 11 side. Further, it is preferable that the antibody is retained on the inner wall surface on one main surface 11 side of the base material 10 and not on the inner wall surface on the other main surface 12 side. When the antibody is retained in this way, it becomes easy to measure the scattered light intensity from the test substance by irradiating the laser from the other main surface 12 side of the base material 10.

Further, it is preferable that the blocking agent adheres to the wall surface forming the internal flow path 15. The blocking agent suppresses the adhesion of unreacted antibody to the wall surface of the internal flow path 15. The blocking agent is preferably adhered to the inner wall surface on one main surface 11 side of the base material 10, and more preferably adhered to the entire inner wall surface constituting the internal flow path 15. As the blocking agent, a commercially available blocking reagent (for example, ELISA ULTRABLOCK AbD serotec) can be used.

Next, a container 1 (1B) having a mode different from that of the container 1A shown in FIGS. 1 to 6 will be described with reference to FIGS. 7 to 8. In the description of FIGS. 7 to 8, the description of the part overlapping with the above description will be omitted. FIG. 7 is a plan view of the container 1B provided with the convex portion 17, and FIG. 8 is a cross-sectional view of the container 1B shown in FIG. 7 along the line XIII-XIII.

As shown in FIG. 7, on the one main surface 11 side of the base material 10, inside the central opening 13, it is lower than the one main surface 11 of the base material 10 in the thickness direction and higher than the bottom surface of the central opening 13. It is preferable that the convex portion 17 formed in a convex shape is provided. The convex portion 17 prevents the sample liquid in the internal flow path 15 from reaching the central opening 13 due to the capillary phenomenon and mixing with the sample liquid in another internal flow path 15 adjacent to the internal flow path 15. It is provided to do. Therefore, it is preferable that the convex portion 17 is provided between two internal flow paths 15 adjacent to each other in the circumferential direction of the base material 10.

In order to form the convex portion 17, as shown in FIGS. 7 to 8, when viewed from one main surface 11 side of the base material 10, the lower first through port 25 of the upper base material 20 is formed. It is preferable that the first opening 35 of the base material 37 is arranged. That is, in the radial direction, it is preferable that the outer end of the first opening 35 is formed inward of the outer end of the first through opening 25. As a result, the central opening 13 is formed so as to narrow toward the other main surface 12 side of the base material 10. In the thickness direction of the base material 10, the central opening 13 is wider on one main surface 11 side of the base material 10, so that the cleaning liquid can be easily dropped.

The convex portion 17 is preferably provided between all the internal flow paths 15. As a result, the effect of suppressing the sample liquids existing in the internal flow path 15 from being mixed with each other is preferably exhibited.

In the circumferential direction of the base material 10, the minimum separation distance between adjacent convex portions 17 is preferably formed to be narrower than the maximum width of the internal flow path 15. Further, it is preferable that the convex portion 17 has a portion whose width narrows toward the center of gravity C of the base material 10. By forming the convex portion 17 in this way, it is possible to prevent the movement of the cleaning liquid to the internal flow path 15 from being hindered when centrifugal force is applied to the container 1.

Although not shown, the first through hole 25 of the upper base material 20 may be arranged in the first opening 35 of the lower base material 30 when viewed from the one main surface 11 side of the base material 10. As a result, the central opening 13 is formed in a shape that is recessed toward one main surface 11 side of the base material 10. In the thickness direction of the base material 10, the central opening 13 is wider on the other main surface 12 side of the base material 10, so that the cleaning liquid tends to diffuse outward in the radial direction.

Next, the container 1 (1C) having a mode further different from the containers 1A and 1B shown in FIGS. 1 to 8 will be described with reference to FIGS. 9 to 14. In the description of FIGS. 9 to 14, the description of the part overlapping with the above description will be omitted. FIG. 9 is a plan view of the container 1C in which the filter 40 for absorbing the drainage liquid is provided in the base material 10, and FIG. 10 is a cross-sectional view of the container 1C shown in FIG. 9 along the XX line. 11A is a plan view of the upper base material 20, FIG. 12 is a plan view of the lower first base material 37, FIG. 13 is a plan view of the filter 40, and FIG. 14 is a lower side view. It is a top view of the 2nd base material 38.

The container 1 (1C) is composed of the members shown in FIGS. 11 to 14. As shown in FIG. 10, it is preferable that the container 1C is provided with a filter 40 that absorbs a liquid around the internal flow path 15. By providing the filter 40 in this way, it is possible to absorb the liquid discharged from the internal flow path 15 when centrifugal force is applied.

In order to prevent unintentional absorption of the liquid in the internal flow path 15, the filter 40 preferably extends outward from the outer end of the internal flow path 15 in the radial direction. Further, from the viewpoint of securing the absorption capacity, the filter 40 may extend inward from the outer end of the internal flow path 15.

The filter 40 may be provided inside the base material 10 as shown in FIGS. 9 to 10. Specifically, it is preferably provided between the upper base material 20 and the lower base material 30 in the thickness direction. Further, in order to prevent the thickness of the base material 10 from increasing, the filter 40 may be provided outside the outer end of the base material 10 in the radial direction. The size and arrangement position of the filter 40 may be set according to the amount of liquid added to the internal flow path 15. For example, the filter 40 may be provided only in a part of the base material 10 in the circumferential direction, or may be provided over the entire circumferential direction of the base material 10 as shown in FIG.

The shape of the filter 40 is not particularly limited, but can be formed into, for example, a circular shape, a polygonal shape, an annular shape, or the like. In particular, since the annular filter 40 as shown in FIG. 13 can be arranged so as to surround the internal flow path 15, the container 1 is compared with the configuration in which the filter 40 is provided under the internal flow path 15. Can be prevented from increasing in thickness.

From the viewpoint of suppressing the thickness of the container 1, the filter 40 is preferably in the form of a film. Specifically, in the thickness direction, the filter 40 preferably has a thickness of 37 or less of the lower first base material. The filter 40 is preferably made of a hydrophilic porous material, and for example, paper such as filter paper or plain paper, or a water-absorbent synthetic resin film can be used.

As shown in FIG. 14, it is preferable that an auxiliary opening 19 is provided on one main surface 11 side of the lower second base material 38. The auxiliary opening 19 functions as a tank for temporarily storing the liquid discharged from the internal flow path 15 by centrifugal force until it is absorbed by the filter 40. The auxiliary opening 19 of the lower second base material 38 preferably communicates with the second through port 26 of the lower first base material 37. The auxiliary opening 19 of the lower second base material 38 is preferably provided inward in the radial direction with respect to the filter 40 (preferably the outer end of the filter 40). The auxiliary opening 19 provided around the internal flow path 15 is preferably a groove that does not penetrate the lower second base material 38 in the thickness direction. The auxiliary opening 19 may be formed in the same shape as the central opening 13 or the outer opening 18.

2. Method for Manufacturing a Container Next, a method for manufacturing a container 1 used for detecting a test substance by an enzyme immunoassay will be described with reference to FIGS. 15 to 18. The container 1 manufactured here has a lower base material 30 having a first opening 35 and a second opening 36, and an upper base material 20 having a first through port 25 and a second through port 26. Have. The manufacturing method of the container 1 of the present invention is a lower base material 30 having one main surface and the other main surface, and communicates with a first opening 35 including a center of gravity on one main surface side and the first opening 35. A step (step 1) of preparing a lower base material 30 having a plurality of second openings 36 extending in the radial direction of the cage, and an upper base material 20 having one main surface and the other main surface, the upper side. A step (step 2) of preparing a first through-hole 25 including the center of gravity of the base material 20 and an upper base material 20 having a plurality of second through-holes 26 arranged around the first through-hole 25, and a first step. Step of joining one main surface of the lower base material 30 and the other main surface of the upper base material 20 in a state where the first opening 35 and the first through port 25 are overlapped and the second opening 36 and the second through port 26 are overlapped. (Step 3), a step of putting a liquid containing an antibody that detects a test substance from the second through hole 26 (step 4), a step of putting a cleaning liquid from the first through port 25 (step 5), and rotating the container 1. It has a step (step 6) of discharging the residual liquid of the liquid containing the antibody and the cleaning liquid by centrifugal force. Hereinafter, each step will be described.

(Step 1)
First, the lower base material 30 for forming the central opening 13 and the internal flow path 15 is prepared. The lower base material 30 has one main surface and the other main surface, and communicates with the first opening 35 including the center of gravity on one main surface side and the first opening 35 and extends in the radial direction. It has a plurality of second openings 36. Specifically, the lower base material 30 described in "1. Container" can be used.

(Step 2)
The upper base material 20 for forming the central opening 13 and the outer opening 18 is prepared. The upper base material 20 has one main surface and the other main surface, and has a first through port 25 including the center of gravity of the upper base material 20 and a plurality of second through ports 25 arranged around the first through port 25. It has a through port 26. Specifically, the upper base material 20 described in "1. Container" can be used.

Examples of the method for forming the internal flow path 15 and the opening (groove) for the central opening 13 in the steps 1 and 2 include laser machining, machining, sandblasting, injection molding, and the like. Among them, a flat plate. Is preferably formed by laser processing.

(Step 3)
As shown in FIG. 3, one main surface of the lower base material 30 and the upper base material 20 in a state where the first opening 35 and the first through port 25 are overlapped and the second opening 36 and the second through port 26 are overlapped with each other. The other main surface of is joined. As a result, the central opening 13 is formed by the first opening 35 and the first through opening 25, the internal flow path 15 is formed by the other main surface of the second opening 36 and the upper base material 20, and the outer flow path 15 is formed by the second through opening 26. The opening 18 is formed. As a method for joining the upper base material 20 and the lower base material 30, the method described in "1. Container" can be used.

In step 3, it is preferable to overlap the first opening 35 and the first through port 25 so that the center of gravity of the upper base material 20 and the center of gravity of the lower base material 30 coincide with each other. As a result, when centrifugal force is applied to the container 1, it becomes easy to uniformly supply the cleaning liquid to the plurality of internal flow paths 15.

(Step 4)
As shown in FIG. 15, the liquid 50 containing the antibody 45 that detects the test substance is put into the second through hole 26 (outer opening 18). As a result, the liquid 50 containing the antibody 45 is supplied to the internal flow path 15, so that it faces the wall surface of the internal flow path 15, particularly the lower base material 30 of the upper base material 20, as shown in FIG. The surface can carry the antibody 45 that detects the test substance. Examples of the method of inserting the liquid 50 containing the antibody 45 from the second through-hole 26 include a method of dropping the liquid 50 little by little with a burette or a micropipette. The amount of the liquid 50 supplied to each second through hole 26 can be, for example, 1 μL or more, 3 μL or more, 5 μL or more, or 7 μL or more, and can be 30 μL or less, 20 μL or less, or 10 μL or less. ..

In order to ensure that the antibody 45 is retained on the wall surface of the internal flow path 15, it is preferable to leave the liquid 50 in the internal flow path 15 at a constant temperature after step 4. The leaving time may be, for example, 30 minutes or more, 60 minutes or more, 90 minutes or more, or 120 minutes or more.

(Step 5)
In order to wash away the unreacted antibody 45, that is, the antibody 45 that was not retained on the wall surface of the internal flow path 15, a washing liquid is put into the first through port 25 as shown in FIG. As a result, the cleaning liquid can be supplied to each of the internal flow paths 15 formed by the upper base material 20 and the lower base material 30 all at once. The method of pouring the cleaning liquid through the first through port 25 can be performed in the same manner as the method of pouring the liquid 50 through the second through port 26. The amount of the cleaning liquid supplied to the first through hole 25 can be, for example, 1 μL or more, 5 μL or more, 10 μL or more, or 15 μL or more, and 100 μL or less, 80 μL or less, 50 μL or less, 40 μL or less, or 30 μL. It can also be as follows. As the cleaning solution, for example, phosphate buffered saline (PBS) can be used.

(Step 6)
As shown in FIG. 18, the container 1 is rotated, and the residual liquid of the liquid 50 containing the antibody 45 and the cleaning liquid 51 are discharged by centrifugal force. By applying a centrifugal force to the container 1, the liquid existing in the internal flow path 15 moves outward in the radial direction by the centrifugal force, so that the internal flow path 15 is washed. As the centrifuge, a commercially available centrifuge (for example, a desktop microcentrifuge mini-centrifuge manufactured by Nippon Genetics Co., Ltd. (model number: NE-NG002B)) can be used. The magnitude of the centrifugal force applied to the container 1 may be constant or may be changed with time. Although FIG. 18 shows an example in which the liquid is discharged from the second through-hole 26 forming the outer opening 18, it may be discharged from an opening other than the second through-hole 26.

In step 6, the rotation direction of the container 1 is not particularly limited. It may be rotated in only one direction, or it may be rotated in one direction and then in the other direction. Further, one direction and the other direction may be rotated alternately.

Steps 5 to 6 may be repeated, for example, 2 times or more and 3 times or more, but may be 15 times or less, 10 times or less, or 5 times or less.

Before the step 5 of adding the cleaning liquid, the step of rotating the container 1 and discharging only the residual liquid of the liquid 50 containing the antibody 45 by centrifugal force may be performed. Since the residual liquid of the liquid 50 containing the antibody 45 and the cleaning liquid 51 can be discharged in different steps, it is difficult for unnecessary liquid to remain in the internal flow path 15.

The method for producing the container 1 of the present invention further includes a step of pouring a liquid containing a blocking agent from the second penetrating port 26 (step 7), a step of pouring a cleaning liquid 51 from the first penetrating port 25 (step 8), and a container. It is preferable to have a step (step 9) of rotating 1 and discharging the residual liquid of the liquid containing the blocking agent and the cleaning liquid 51 by centrifugal force. By these steps 7 to 9, since the blocking agent can be held on the other main surface side of the upper base material 20, it is possible to suppress the unreacted antibody from adhering to the other main surface side of the upper base material 20. A container 1 capable of accurately detecting the test substance can be obtained.

In step 7, the blocking agent described in “1. Container” can be used. In step 7, the method of adding the liquid containing the blocking agent can be carried out in the same manner as in step 4.

The amount and type of the cleaning liquid in step 8 and the method of applying centrifugal force in step 9 can be carried out in the same manner as in steps 5 and 6, respectively.

Steps 8 to 9 may be repeated, for example, 2 times or more and 3 times or more, but may be 15 times or less, 10 times or less, or 5 times or less.

After the step 7, before the step 8, it is preferable to leave the liquid in the internal flow path 15 at a constant temperature. The leaving time may be, for example, 30 minutes or more, 60 minutes or more, 90 minutes or more, or 120 minutes or more. Further, the leaving time of the liquid containing the blocking agent may be shorter than the leaving time of the liquid containing the antibody, for example, the former may be 2 hours and the latter may be 1 hour.

3. 3. Method for Detecting Test Substance The present invention also provides a method for detecting a test substance by an ELISA method using a container 1 having a central opening 13 and a plurality of internal flow paths 15 described in “1. Container”. .. The ELISA method is roughly classified into a direct method, an indirect method, a competitive method, and a sandwich method according to the difference in measurement principle.

In the direct method, the test substance is immobilized on the inner wall surface constituting the internal flow path 15, and then an enzyme-labeled antibody or an antigen to which the antibody in the test substance specifically binds is added, and the antigen or antibody in the test substance is added. To react with. Next, the contaminants are removed by washing, a color-developing substrate is added to the labeled enzyme to react, and the absorbance and scattered light intensity of the colored dye are measured to measure the amount of antigen or antibody in the test substance. To do. For the method of measuring the scattered light intensity of the developed dye, International Publication No. 2015/060269 can be referred to.

In the indirect method, a test substance is immobilized on the inner wall surface constituting the internal flow path 15, and then an enzyme-labeled antibody (primary antibody) and then an enzyme-labeled antibody (secondary antibody) against the antibody are reacted. After that, the operation can be performed in the same manner as the operation after cleaning the contaminants by the direct method.

In the competitive method, the test substance and the enzyme-labeled antibody are placed in the internal flow path 15 of the container 1 in which the antibody is immobilized, and an antigen-antibody reaction is carried out. After that, the operation can be performed in the same manner as the operation after cleaning the contaminants by the direct method.

In the sandwich method, the antibody (primary antibody) immobilized on the internal flow path 15 of the container 1 is reacted with the antigen in the test substance, and then the internal flow path 15 is washed to obtain an enzyme-labeled antibody (secondary antibody). ) Is added to cause an antigen-antibody reaction at yet another site. After that, the operation can be performed in the same manner as the operation after cleaning the contaminants by the direct method.

The detection method includes a step (step A) of feeding a liquid (sample solution) containing the test substance through an opening other than the central opening 13 when the test substance is supplied to the internal flow path 15 of the container 1. preferable. For example, when the container 1 is provided with the outer opening 18, it is preferable to have a step of putting a liquid containing the test substance through the outer opening 18.

The detection method includes a step of pouring the cleaning liquid through the central opening 13 (step B) and a step of rotating the container 1 and moving the cleaning liquid to a plurality of internal flow paths 15 by centrifugal force (step C). It is something that is. By simply pouring the cleaning liquid through the central opening 13 and applying centrifugal force to the container 1 in this way, the cleaning liquid can be easily moved to the plurality of internal flow paths 15 communicating with the central opening 13 all at once. The container 1 can be sufficiently washed. Therefore, according to the test substance detection method of the present invention, the detection accuracy of the test substance can be ensured regardless of the proficiency level of the operator. In addition, a small and inexpensive centrifuge that does not require peripheral devices such as pumps and valves can be used as a washer.

The method of pouring the cleaning liquid through the central opening 13 in step B and the method of applying centrifugal force to the container 1 in step C can be performed in the same manner as in steps 5 and 6 of "2. Container manufacturing method", respectively. it can.

1, 1A, 1B, 1C: Container 10: Base material 11: One main surface 12: The other main surface 13: Central opening 15: Internal flow path 16: Narrow portion 17: Convex portion 18: Outer opening 19: Auxiliary opening 20 : Upper base material 25: First through port 26: Second through port 30: Lower base material 35: First opening 36: Second opening 37: Lower first base material 38: Lower second base material 40: Filter 45: Antibody 50: Liquid containing an antibody that detects the test substance 51: Washing liquid

Claims (7)

A container used to detect a test substance by enzyme immunoassay.
It has a base material having one main surface and the other main surface,
The base material has a central opening on one main surface side including the center of gravity, and a plurality of internal flow paths communicating with the central opening and extending in the radial direction of the base material. ,
The base material has an upper base material and a lower base material that are laminated with each other.
The lower base material has a first opening including the center of gravity of the lower base material on one main surface side and a second opening communicating with the first opening and extending in the radial direction. Have and
The upper base material has a first through hole that overlaps with the first opening.
The central opening is formed from the first opening and the first through port.
The internal flow path is formed from the second opening, and the internal flow path is formed from the second opening.
A container characterized in that, in the radial direction, the outer end of the first opening is formed inward with respect to the outer end of the first through port. The lower base material is composed of a lower first base material and a lower second base material that are laminated with each other.
The first aspect according to claim 1, wherein the first opening and the second opening are provided in the lower first base material and penetrate from the upper base material side toward the lower second base material side. Container. The container according to claim 1 or 2, wherein the base material is outward from the central opening in the radial direction and has a plurality of outer openings communicating with the internal flow path, respectively. The container according to any one of claims 1 to 3, wherein a filter for absorbing a liquid is provided around the internal flow path. The container according to any one of claims 1 to 4, wherein an antibody for detecting a test substance is held on a wall surface constituting the internal flow path. A method for manufacturing a container used for detecting a test substance by an enzyme immunoassay.
On the other hand a lower base member having a principal surface and the other main surface, a first opening including the center of gravity of the lower substrate to the one main surface side, wherein the first opening and the communication with your Riho morphism direction A step of preparing a lower substrate having a plurality of second openings extending in the
An upper base material having one main surface and the other main surface, which has a first through-hole including the center of gravity of the upper base material and a plurality of second through-holes arranged around the first through-hole. The process of preparing the upper base material and
One main surface of the lower base material and the other main surface of the upper base material are joined in a state where the first opening and the first through hole are overlapped and the second opening and the second through port are overlapped. Process and
The step of putting a liquid containing an antibody that detects a test substance from the second through-hole, and
The step of pouring the cleaning liquid through the first through port and
A step of rotating the container and discharging the residual liquid of the liquid containing the antibody and the cleaning liquid by centrifugal force.
The step of putting a liquid containing a blocking agent through the second through hole and
The step of pouring the cleaning liquid through the first through port and
A method characterized by having a step of rotating the container and discharging a residual liquid of a liquid containing the blocking agent and the cleaning liquid by centrifugal force. A method for detecting a test substance by an enzyme immunoassay using the container according to any one of claims 1 to 5.
The process of pouring the cleaning liquid through the central opening and
A detection method comprising a step of rotating the container and moving the cleaning liquid to each of the plurality of internal flow paths by centrifugal force.

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