JP4935424B2 - Immunoassay chip - Google Patents

Description

  The present invention relates to an immunoassay chip, and more particularly to an immunoassay chip and an immunoassay kit that can be easily applied to a centrifuge.

  In an immunoassay for analyzing a trace amount of a test substance in a specimen, use of a small chip called Lab-on-chip (μTAS) has been proposed. In recent years, a technique related to a chip for feeding liquid using centrifugal force due to such rotation has been developed.

  For example, Patent Document 1 discloses a microfluidic device having a first set of two or more microchannel structures, and a specific structural unit of each microchannel structure connected by a microconducting path. A technique is described in which a liquid is caused to flow by utilizing centrifugal force by rotating a device to perform synthesis, preparation for decomposition, and the like in the chemical and biological chemical fields. Further, Patent Document 2 describes a technique for rotating a micro system platform in which a fine channel is embedded and inducing fluid motion on the platform by using centripetal force generated thereby.

JP 2005-507762 A JP 2000-514928 gazette

However, the above-described conventional technique has a problem that a special liquid feeding device is required and it is difficult to proceed a series of analysis operations quickly. In addition, since the structures are complicated, there are problems that reaction conditions are limited and customization by the user is difficult.
In view of such a conventional problem, an object of the present invention is to provide an immunoassay chip capable of completing an operation in a short time and capable of efficient analysis.

The present invention provides the following [1] to [18].
[1] An immunoassay chip that has a reaction chamber containing a carrier to which an antigen and / or an antibody are bound, and detects a test substance in the reaction chamber. The chip is attached to an angle rotor and / or a swing rotor of a centrifuge. An immunoassay chip that can be worn.
[2] The immunoassay chip according to [1], wherein the carrier has a minor axis of 10 to 200 μm.
[3] The immunoassay chip according to [1] or [2], further comprising a reagent / sample reservoir that communicates with the reaction chamber and has a first opening on the opposite side of the reaction chamber.
[4] The immunoassay chip according to [3], wherein the reaction chamber includes a second opening on a side opposite to the reagent / sample reservoir, and has a damming means for the carrier in the second opening. .
[5] The immunoassay chip according to [3] or [4], wherein the volume of the reaction chamber is smaller than the volume of the reagent / sample reservoir.
[6] The immunoassay chip according to any one of [3] to [5], wherein a volume ratio of the reagent / sample reservoir to the reaction chamber is 100 or more.
[7] The immunoassay chip according to any one of [3] to [5], wherein a volume ratio of the reagent / sample reservoir to the reaction chamber is 100 to 5 × 10 ⁸ .
[8] The ratio of the projected sectional area of the reagent / specimen reservoir in the liquid feeding direction to the projected sectional area of the reaction chamber in the liquid feeding direction is 50 or more. [3] to [6] The immunoassay chip described.
[9] The ratio of the maximum cross-sectional area parallel to the liquid feeding direction of the reagent / sample reservoir to the maximum cross-sectional area parallel to the liquid feeding direction of the reaction chamber is 2 to 400. [7] The immunoassay chip according to any one of [7].
[10] The immunoassay chip according to any one of [1] to [9], wherein the reaction chamber has a volume of 1 nL to 100 μL.
[11] The immunoassay chip according to any one of [4] to [10], further including a waste tank on the opposite side of the reagent / sample reservoir through the second opening of the reaction chamber.
[12] The immunoassay chip according to [11], wherein the reaction chamber and the waste liquid tank are separable.
[13] The immunoassay chip according to any one of [1] to [12], wherein at least a part of the reaction chamber is transparent.
[14] The fitting according to any one of [1] to [13], which is fitted into the centrifuge tube and can be attached to the angle rotor and / or swing rotor of the centrifuge while being fitted to the centrifuge tube. The immunoassay chip described.
[15] The immunoassay chip according to [14], wherein a protrusion is further provided on the outer wall of the chip.
[16] The immunoassay chip according to any one of [1] to [15], wherein the test substance is a cytokine and / or a chemokine.
[17] It includes at least a carrier to which an antigen and / or an antibody are bound, and a chip body having a reaction chamber capable of accommodating the carrier and attachable to an angle rotor and / or a swing rotor of a centrifuge. Immunoassay kit.
[18] It includes at least a carrier capable of binding an antigen and / or an antibody, and a chip body having a reaction chamber capable of accommodating the carrier and attachable to an angle rotor and / or a swing rotor of a centrifuge. An immunoassay kit.

According to the immunoassay chip of the present invention, in a reaction chamber containing a carrier to which an antigen and / or an antibody are bound, a special liquid feeding device is not required, and a test substance in a sample can be highly sensitive in a short time. It can be conveniently contacted with an antibody and / or an antigen. In particular, since the immunoassay chip of the present invention can be attached to an angle rotor and / or a swing rotor of a centrifuge, the liquid discharged downstream from the reaction chamber enters the reaction chamber due to the action of gravity when the apparatus is stopped. There is no risk of backflow, and a mechanism such as a hydrophobic valve to prevent backflow is not required.
In addition, since the immunoassay kit of the present invention can select and use the user's antibody and / or antigen, it can be customized to meet the user's needs.

  The immunoassay chip of the present invention is a chip for performing immunoassay of a specimen. In the present invention, immunoassay means a technique for analyzing a test substance in a sample using an antigen-antibody reaction, and a representative example thereof is ELISA (Enzyme-Linked Immunosorbent Assay solid phase enzyme immunoassay). , RIA (Radioimmunoassay radioimmunoassay), FIA (Fluorescence immunoassay fluorescent immunoassay), FLISA (Fluorescence-Linked Immunosorbent solid phase fluorescent immunoassay).

The analysis method is as follows: 1) a direct method for directly recognizing and detecting the target substance with the labeled antibody;
2) An indirect method of recognizing a target substance with an antibody and recognizing and detecting an antibody bound to the target substance with a labeled antibody,
3) Competition method,
4) A two-antibody sandwich method in which a target substance is captured by an immobilized antibody (primary antibody) and further detected by another labeled antibody (secondary antibody),
5) A three-antibody sandwich method in which the target substance is captured by an immobilized antibody, the target substance is recognized by another antibody, and the target substance is detected by an antibody labeled with the recognized antibody.
Etc.
Further, a method of detecting a test substance using avidin, streptavidin or the like, such as ABC method, may be used.

  The test substance in the immunoassay may be any substance that specifically binds to an antigen or antibody, such as a protein, sugar, lipid, nucleic acid, glycoprotein, glycolipid, or cell. Examples include cytokines, chemokines, interleukins, allergens, DNA, RNA, antibodies, lipids, enzymes, and other chemical substances. In particular, IL-6, IL-8, and TNF are preferable. The organism from which the test substance is derived does not matter. The test substance may be one type or two or more types.

  The purpose of immunoassay is not particularly limited, and there is no particular limitation such as detection of the presence or absence of a test substance in a sample, quantification of a test substance. The immunoassay in the present invention can be used for analysis in clinical tests, food tests, environmental tests and the like.

  The specimen refers to a sample that may contain the test substance, and is preferably a liquid. For example, liquids collected from a living body including body fluids such as blood, urine, spinal fluid, saliva, sputum, cell suspension, cell disruption fluid, and soil extract can be exemplified.

  When performing immunoassay, the immunoassay chip of the present invention needs to be rotated so that the rotation axis outside the chip is revolved. In other words, the rotation axis is outside the immunoassay chip, and it is necessary to adjust so that at least the reaction chamber of the chip rotates (revolves) along a trajectory centered on the rotation axis. The trajectory may be substantially circular, and may be an elliptical trajectory.

  The immunoassay chip of the present invention can be attached to an angle rotor and / or a swing rotor of a centrifuge. There are two types of chips that can be mounted.

  As the first type of the immunoassay chip of the present invention, one that can be inserted and fitted into the centrifuge tube can be mentioned. That is, it may be a type that can be attached to the angle rotor and / or swing rotor of the centrifuge while being fitted inside the centrifuge tube. In the case of this type of tip, when the tip of the present invention is put in the centrifuge tube, the tip is fixed to the inner wall surface of the centrifuge tube. In other words, any point or surface on the outer surface of the immunoassay chip is fixed at an arbitrary point or surface on the inner wall surface of the centrifuge tube. Typical examples include immunoassay chips A, B, C, D, and F shown in FIGS. 1 to 4 and FIG. 7, respectively. These chips can be used by being inserted into and fitted into a centrifuge tube as shown in FIG.

  On the other hand, as a second type of the immunoassay chip of the present invention, there can be mentioned a type that can be directly mounted on a centrifuge tube mounting portion of an angle rotor or swing rotor of a centrifuge. A typical example is an immunoassay chip E shown in FIG. 5 or FIG.

  Here, the centrifuge tube in the present invention means a so-called test tube (usually a cap tube) that can be applied to a centrifuge, and includes all those called centrifuge tubes, conical tubes, Eppendorf tubes, and the like. The centrifuge is not particularly limited as long as it is compatible with a centrifuge tube, and a small desktop centrifuge may be used as long as the centrifuge tube can be attached.

In the immunoassay chip of the present invention, an immunoassay of a specimen is performed in a reaction chamber.
The shape and size of the reaction chamber need only be able to accommodate a carrier to which an antigen and / or antibody is bound. The shape is preferably tubular, and the cross section of the tube is not particularly limited, such as a circle or a polygon. The smaller the reaction chamber size, the smaller the amount of the carrier to which the antigen and / or antibody is bound, the cost can be reduced, the volume ratio with the reservoir can be easily increased, and it can be mounted on a commercially available centrifuge. A large concentration effect can be obtained by size. The volume of the reaction chamber is usually 1 nL to 100 μL, preferably 10 nL to 10 μL.

  The reaction chamber in the immunoassay chip of the present invention will be described based on the examples shown in FIGS. 1 has a reaction chamber 11 connected to a reagent / sample reservoir 12 described later, and has an opening 11A on the opposite side of the reagent / sample reservoir 12 from the opening 12A. The reaction chamber 11 contains a carrier 13 that binds an antigen and / or an antibody. The reaction chamber 11 of the immunoassay chip E shown in FIGS. 5 and 6 is provided adjacent to the central tube wall of the immunoassay chip E, and is opened on the opposite side to the opening 12A of the reagent / sample reservoir 12. Part 11A.

  In the immunoassay chip of the present invention, the reaction chamber can include an opening (second opening). When the later-described reagent / sample reservoir is provided, the position of the opening is desirably opposite to the reservoir. The opening can be further provided with a carrier damming means, whereby the carrier can be held so as not to leak from the chip. As the damming means, for example, a wire mesh or a filter can be used. In the case of a wire mesh, a wire mesh of an appropriate size (for example, an opening having a size of 20 μm × 20 μm) can be pressed into the opening to form a damming means. In the case of a filter, it can be obtained by press-fitting a cellulose acetate filter or the like into the opening. The damming means is not limited to a wire mesh or a filter, and a cap or the like can be used.

  The case where the opening of the reaction chamber is closed with a filter as a blocking means will be described with reference to the embodiment of FIG. FIG. 2 is a diagram schematically showing a longitudinal section of the immunoassay chip B of the embodiment of the present invention. The opening 11A ′ of the reaction chamber 11 is wider than the reaction chamber 11, and a filter is press-fitted into this portion 11A ′. Note that it is not necessary that the width of the opening is wide when providing the blocking means, and the opening 11A such as the immunoassay chip A of FIG. 1 or the opening of the immunoassay chip E shown in FIGS. In 11A, a wire mesh can be pressed into the opening to provide damming means (not shown).

  The reaction chamber contains a carrier to which an antigen and / or an antibody is bound. The number of carriers to be accommodated is one or more, and it is preferable to accommodate a plurality of carriers from the viewpoint of increasing the efficiency of immunoassay. In addition, since a plurality of carriers are accommodated in the reaction chamber, a pressure loss occurs with respect to the liquid. Therefore, even if the liquid is injected into the reagent / specimen reservoir, the liquid does not flow out through the reaction chamber only by the action of gravity. is there.

The shape of the carrier may be a so-called microrod such as a cylinder or a polygonal column, or a plate-like microplate, in addition to spherical or elliptical microbeads.
The size of the carrier depends on the size of the reaction chamber, but the minor axis is preferably in the range of 1 to 1000 μm, preferably 10 to 200 μm, regardless of the shape of the carrier.

The material of the carrier is not particularly limited, and glass, ceramic (eg, yttrium partially stabilized zirconia), metal (eg, gold, platinum, stainless steel), resin (eg, nylon, polystyrene, polyvinyl alcohol, polymethyl methacrylate, polyacrylamide), agarose Of these, resins, particularly polystyrene, are preferred.
When a plurality of carriers are stored in the reaction chamber, the shape, size, and material of each carrier may be uniform or varied. In addition, it is not necessary that antigens and / or antibodies are bound to all the carriers stored in the reaction chamber, and a part of the carriers that do not bind anything may be included.

  Antigens and / or antibodies to be bound to the carrier can be selected from various antibodies, antigen-binding fragments of antibodies such as Fab fragments and F (ab ′) 2 fragments, various antigens, and the like in samples in immunoassays. Antigens and antibodies that specifically bind to the test substance can be appropriately selected, and may be one kind or plural. There are no particular restrictions on the binding density, number of bindings, binding mode, etc. of the antigen or antibody to the carrier.

  The antigen and / or antibody can be bound to the carrier by, for example, a method in which the carrier and the antigen or antibody are mixed in a solution such as a buffer and contacted to bind. Bonding by contact can be carried out usually under conditions of 1 hour to 24 hours (day), low temperature, generally 4 to 37 ° C., with stirring as necessary. The obtained carrier may be washed with a buffer solution, a washing solution or the like before use. The binding method is not limited to this. For example, a method of chemically binding an antigen or antibody and a carrier using a crosslinking agent containing a hydrophilic polymer (polyethyleneimine, polyethylene glycol, polyvinyl alcohol, sodium polysulfonate, etc.). Etc. can also be used.

  The method for accommodating the carrier to which the antigen and / or antibody are bound in the reaction chamber of the chip body can be performed, for example, as follows. That is, the beads to which antigens and / or antibodies are bound are suspended in a buffer containing a surfactant and / or a blocking agent, and this suspension is poured into the specimen / reagent reservoir of the chip body. Is inserted into a centrifuge tube and centrifuged in a centrifuge for 10 seconds to 1 minute.

  Samples and reagents for immunoassay are sent into the reaction chamber. The specimen has already been described. A reagent means a chemical substance or a drug other than a specimen used in immunoassay. For example, drugs, substances, washing solutions, etc. for detection of test substances, and more specifically, labeled antibodies (secondary antibodies) labeled with fluorescence or enzymes, antigens, washing solutions, fluorescence or luminescent substrates, etc. Can be mentioned.

  The immunoassay chip of the present invention can comprise a reagent / sample reservoir. As a result, the operation is easier than in the case where the reagent or sample is directly fed into the reaction chamber. In other words, the reaction can be performed simply by holding the reagent or specimen in the reagent / specimen reservoir and then centrifuging, and the reaction chamber has a narrower tube shape, improving the storage density of the carrier and improving the efficiency of immunoassay Can be increased.

  1 is provided with a reagent / sample reservoir 12 connected to a reaction chamber 11, and the reagent / sample reservoir 12 has an opening 12A on the outside. Similarly, in the immunoassay chip E of FIGS. 5 and 6, the reagent / sample reservoir 12 is also connected to the reaction chamber 11.

The volume (size) of the reagent / sample reservoir is preferably larger than that of the reaction chamber. This is because the reaction chamber is sufficiently smaller than the reservoir to provide a concentration effect of the test substance, and it is easy to hold the reagent and specimen in the reservoir. Specifically, for example, the volume ratio of the reagent / sample reservoir to the reaction chamber is usually 100 or more, preferably in the range of 100 to 5 × 10 ⁸ , particularly preferably in the range of 100 to 3000. In the conventional technology in which the analysis mechanism is integrated on the disk, the limit is about 15 times. By setting the above range, the concentration effect of the test substance is very large compared to the conventional technology. It becomes.
The volume of the reagent / sample reservoir is generally 30 μL to 500 mL, and preferably 30 μL to 1000 μL (1 mL).

  On the other hand, the ratio of the projected sectional area of the reagent / sample reservoir in the liquid feeding direction (area when projected onto a plane perpendicular to the liquid feeding direction) to the projected sectional area of the reaction chamber in the liquid feeding direction is usually 50. Or more, preferably 100 or more. Moreover, generally an upper limit shall be 10,000 or less. In the case of the conventional technology in which the analysis mechanism is integrated on the disk, the entire apparatus has a two-dimensional structure, so that the projected cross-sectional area ratio in the liquid feeding direction between the reservoir and the reaction chamber can only differ by a factor of about 10 times at most. However, in the case of the chip of the present invention having a three-dimensional structure, the area ratio can be increased and the concentration efficiency of the test substance can be remarkably improved.

Further, the ratio of the maximum cross-sectional area parallel to the liquid feeding direction of the reagent / sample reservoir and the maximum cross-sectional area parallel to the liquid feeding direction of the reaction chamber is determined from the viewpoint of obtaining the concentration effect of the test substance. 2 to 400 is preferable. The maximum cross-sectional area parallel to the liquid feeding direction of the reagent-analyte reservoir is preferably a 10 mm ² to 200 mm ^2. The maximum cross-sectional area parallel to the liquid feeding direction of the reaction chamber is preferably set to 0.5 mm ² to 5 mm ^2.

  The shape of the reagent / sample reservoir is not particularly limited, and can be appropriately selected from various shapes such as a cylindrical shape and a polygonal shape. However, from the viewpoint of smoothly feeding the reagent or sample into the reaction chamber by centrifugal force, the area of the cross section However, it is preferable that the shape gradually narrows toward the side connected to the reaction chamber. For example, the shape of the reagent / sample reservoir 12 in the immunoassay chip A shown in FIG. 1 is basically a rectangular parallelepiped, with four corners on the reaction chamber 11 side being rounded. On the other hand, the shape of the reagent / sample reservoir 12 in each of the immunoassay chips B, C, and D shown in FIGS. 2, 3 and 4 is basically a rectangular parallelepiped, and the four side surfaces are the reaction chamber side. It is squeezed towards. The shape of the reagent / sample reservoir 12 of the immunoassay chip E in FIGS.

  The reagent / sample reservoir is directly connected to the reaction chamber, and has an opening (first opening) on the side opposite to the reaction chamber. Although the shape of the opening to the outside is not particularly limited, it can be appropriately determined in such a size that the injected reagent or specimen does not leak out. For example, the minor axis can be appropriately determined to be in the range of 1 mm to 100 mm, preferably 1 to 20 mm.

The immunoassay chip of the present invention may further comprise a waste liquid tank. By providing the waste liquid tank, it is possible to prevent the waste liquid from being scattered in the centrifuge. The waste liquid tank is connected to the reaction chamber at the second opening. As described above, when the damming means is provided in the second opening, the waste liquid tank is connected to the tip of the damming means.
In the present invention, the waste liquid tank may be a space where the waste liquid can be accumulated. For example, in the case of an immunoassay chip of the type that can be inserted and fitted into the centrifuge tube as shown in FIGS. 1 to 4, when the centrifuge tube is attached to the centrifuge tube as shown in FIG. The space formed in this can be the waste liquid tank 16. On the other hand, although not shown in FIGS. 1 to 4, it is possible to attach a bag or container to the second opening 11 </ b> A to form a waste liquid tank.
In the case of an immunoassay chip of the type that can be directly mounted on an angle rotor or a swing rotor as shown in FIGS. 5 and 6, a waste tank 16 can be provided in the chip.

  Moreover, in the case of the chip | tip which can be inserted and fitted in the centrifuge tube among the immunoassay chips of this invention, the protrusion part may be further provided in the outer wall. When the tip is fitted to the centrifuge tube by providing the protrusion, the tip is fitted with the protrusion in contact with the wall of the centrifuge tube, and a space as a waste liquid tank as described above is formed at the bottom of the centrifuge tube. Can be secured. In addition, the chip can be easily removed from the centrifuge tube. Furthermore, when the chip is taken out from the centrifuge tube, the chip can be taken out and analyzed in a state where the waste liquid remains in the centrifuge tube, so that the influence of measurement noise derived from the waste liquid can be eliminated.

  The protrusion may be provided on the outer wall of the chip, and its shape and position are not specified. A position and shape that can be fixed at an arbitrary point or surface on the inner wall surface of the centrifuge tube when the tip is placed in the centrifuge tube can be appropriately determined. Examples of the manner of fixing on the inner wall surface of the centrifuge tube include a mode in which the tip is suspended and fixed, and a mode in which the tip is raised to the bottom of the centrifuge tube via a scaffold.

  The protruding portion can be provided on the side surface of the tip, and the tip can be suspended and fixed by the contact or contact surface between the inner wall surface of the centrifuge tube and the protruding portion. Specifically, an ear protruding in the vertical direction can be provided around the opening of the reagent / sample reservoir. In the immunoassay chip D of FIG. 4, an ear 14 is provided around the opening 12A. The shape and size of the ear can be determined as appropriate in relation to the size of the centrifuge tube.

  Moreover, a protrusion can be provided on the bottom surface of the chip and extended to the bottom surface of the centrifuge tube to fix the chip. Specifically, a leg extending from the bottom surface of the reagent / sample reservoir can be provided. In the immunoassay chip F of FIG. 7, two legs 14B from which the chip extends from the bottom surface of the chip are provided. When the immunoassay chip F is attached to the centrifuge tube, the bottom is raised by the two legs 14B, and the space between the bottom surface of the centrifuge tube and the chip (mainly the space 14C between the legs 14B) is secured as a waste liquid tank.

  Among the immunoassay chips of the present invention, in the case of a type that fits inside the centrifuge tube, it is preferable that the chip is detachably fitted from the centrifuge tube for the convenience of subsequent operations.

The size and shape of the chip body of the immunoassay chip of the present invention can be determined appropriately depending on the type.
That is, in the case of a chip that is detachably fitted to the centrifuge tube, the size of the chip body can be determined in consideration of the fact that it can be inserted and fitted into the centrifuge tube as described above. The size of the centrifuge tube generally used is 8 to 40 mm in short diameter and 5 to 120 mm in height. Therefore, for example, the short diameter of the chip body is usually 6 to 40 mm and the height is usually 5 to 120 mm. It can be determined within the range. Since a centrifuge tube having a volume of 0.5 ml to 2.5 ml, such as an Eppendorf tube, which is preferable as a centrifuge tube because of its smaller size, its size is 8 to 10 mm. The size can be determined in the range of 6 to 10 mm for the minor axis, 5 to 30 mm for the height, and more preferably 5 to 15 mm for the height. On the other hand, in the case of a chip of a type that can be mounted as it is on the centrifuge tube's angle rotor or the centrifuge tube mounting portion of the swing rotor, it may be a size that matches the size of the rotor of the mounting destination centrifuge.

  Further, the shape of the chip body of the immunoassay chip of the present invention can be determined in consideration of the ease of molding. For example, in the case of a chip that is detachably fitted to a centrifuge tube, it can be selected from a polygonal column shape such as a triangular column or a quadrangular column, a cylindrical shape, a pyramid shape, a conical shape, or the like. In the embodiment shown in FIGS. 1 to 4, all are cubic. On the other hand, in the case of a type of chip that can be directly attached to the centrifuge tube's angle rotor or swing rotor's centrifuge tube mounting site, the shape is matched to the size of the rotor of the centrifuge at the mounting destination, that is, the same shape as the centrifuge tube If it is.

  The material of the immunoassay chip of the present invention is not particularly limited, and examples thereof include resin and glass. In particular, from the viewpoint of facilitating observation of the reaction chamber from the outside, it is preferable that at least a part of the reaction chamber is transparent. By making at least a part of the reaction chamber transparent, a more concentrated test substance can be easily detected. Therefore, it is preferable to use a transparent material for a part of the reaction chamber, and it is particularly preferable to form the whole from a transparent material. Further, the surface of the reaction chamber made of a transparent material may be a flat surface or a lens shape (concave surface).

  Examples of the transparent material include various organic materials and inorganic materials, such as polymethyl methyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, polydimethyl. Resins such as siloxane and silicon, copolymers or composites containing such polymer compounds; quartz glass, pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, borosilicate glass, and the like; The composite; a metal whose surface is coated with an insulating material, a composite thereof, a ceramic, a composite thereof, and the like are preferably used. Of these, polymethyl methyl methacrylate (PMMA) is particularly preferably used.

  In addition, in the immunoassay chip of the present invention, the surface of the reaction chamber, reagent / specimen reservoir, where the solution of the test substance comes into contact is subjected to an adsorption suppression process that suppresses nonspecific adsorption of the test substance. preferable. Adsorption suppression treatment methods include a coating treatment that electrostatically adsorbs hydrophilic polymer material to the surface, a method of irradiating high-energy rays and covalently bonding the hydrophilic polymer to the resin surface to immobilize it firmly. Is used.

  Hereinafter, the method for operating the immunoassay chip of the present invention will be described with reference to FIG. 5 taking as an example the case of analyzing cytokines by ELISA. In the reaction chamber of the immunoassay chip, primary antibody adsorption beads of cytokine are accommodated.

The operation of the immunoassay chip of the present invention can be performed by a method comprising at least the following steps.
Step (a): After introducing the sample / reagent into the reagent / sample reservoir of the immunoassay chip, the step of revolving the rotation axis outside the immune analysis chip with the immune analysis chip, and the step (b): The process of measuring the amount of the test substance in the reaction chamber of the immunoassay chip

The above process will be described as follows using the immunoassay chip C of FIG. 3 as an example.
For step (a), first, a specimen and a secondary antibody are injected into the specimen / reagent reservoir ((1) in FIG. 9), and then the specimen is transferred to the reaction chamber by centrifugal force ((2) in FIG. 9). The primary antibody on the bead carrier is reacted with an antigen antibody ((3) in FIG. 9). That is, the immunoassay chip after sample injection is inserted into a centrifuge tube, and this centrifuge tube is set in a centrifuge and rotated. By this process, the sample is transferred from the sample / reagent reservoir to the reaction chamber by centrifugal force, and the reaction is performed at the same time. The sample and the secondary antibody may not be injected simultaneously as described above, and the sample may be injected and centrifuged, and then the secondary antibody may be injected and centrifuged.

  Subsequently, after injecting the fluorescent substrate into the specimen / reagent reservoir ((1) in FIG. 9), the substrate is similarly transferred to the reaction chamber by centrifugal force ((2) in FIG. 9). It reacts with the next antibody ((3) in FIG. 9). That is, in the transfer of the sample, centrifugation is performed in the same manner except that the sample is replaced with a fluorescent substrate.

  The revolution speed and time of the immunoassay chip can be appropriately adjusted according to the type of the test substance and the measurement concentration range. About the speed of revolution, it is preferable to set it as 10-10000G, for example, and it is more preferable to set it as 100-5000G. The time is preferably 5 seconds to 120 minutes, more preferably 10 seconds to 60 minutes.

  Specific examples of the step (a) are as follows. First, an antigen and a secondary antibody are suspended in a buffer solution or the like as needed to make 150 μL, and then injected into the sample / reagent reservoir of the chip. Then, the chip is centrifuged at 500 G for 3 minutes. Subsequently, PBS-T is injected into the specimen / reagent reservoir, and then centrifuged again for 1 minute. Next, 100 μL of the substrate is injected into the specimen / reagent reservoir and then centrifuged again for 20 seconds, and then left to react for 5 minutes.

  Subsequently, in step (b), the fluorescence intensity in the reaction chamber is measured with a fluorescence detection device (such as a fluorescence microscope) while the chip is taken out from the centrifuge tube or the chip is inserted into the centrifuge tube. In the case of detecting the presence or absence of a cytokine in a sample as a test substance, if the fluorescence intensity can be measured, it is confirmed that the cytokine is present in the sample. On the other hand, in the case of immunoassay for the purpose of quantifying cytokines in a sample, the concentration of cytokines is specified in comparison with a calibration curve prepared by measuring the concentrations of cytokines in advance in the same manner.

  As described so far, the operation of the immunoassay chip of the present invention includes (1) introduction of a sample / reagent, (2) liquid feeding by centrifugal force, and (3) ELISA in a bead carrier part as shown in FIG. The three steps of each reaction can be repeated. In addition, after the sample injection and the substrate injection, a washing solution or a buffer solution may be injected into the reservoir as necessary for washing the reaction chamber, and the centrifugal treatment may be similarly performed. A plurality of test substances may be provided.

The immunoassay chip of the present invention can also be provided as a kit in which individual members are assembled when used on the user side.
That is, as a first form of the immunoassay kit of the present invention, an antigen and / or antibody-bound carrier and a reaction chamber containing the carrier are attached to an angle rotor and / or a swing rotor of a centrifuge. Examples include at least a possible chip body. That is, the kit includes a carrier to which an antigen and / or an antibody is bound in advance.

  In addition, as a second form of the immunoassay kit of the present invention, an angle rotor and / or a swing rotor of a centrifuge having a carrier capable of binding an antigen and / or an antibody and a reaction chamber capable of accommodating the carrier. And at least a chip body that can be mounted on the chip. That is, it is a kit containing a carrier to which no antigen and / or antibody is bound, and the user can select and bind the antigen and / or antibody corresponding to the detection target.

  The method for binding the antigen and / or antibody to the carrier and the method for inserting the carrier into the reaction chamber in the two immunoassay kits of the present invention are as already described with specific examples.

FIG. 1 is a perspective view schematically showing one embodiment of the immunoassay chip of the present invention. FIG. 2 is a longitudinal sectional view schematically showing one embodiment of the immunoassay chip of the present invention. FIG. 3 is a longitudinal sectional view schematically showing one embodiment of the immunoassay chip of the present invention. FIG. 4 is a longitudinal sectional view schematically showing an embodiment of the immunoassay chip of the present invention. FIG. 5 is a longitudinal sectional view schematically showing one embodiment of the immunoassay chip of the present invention. FIG. 6 is a longitudinal sectional view schematically showing an embodiment of the immunoassay chip of the present invention. FIG. 7 is a longitudinal sectional view schematically showing one embodiment of the immunoassay chip of the present invention. FIG. 8 is a perspective view schematically showing a state where the immunoassay chip of the present invention is inserted into a centrifuge tube. FIG. 9 is an explanatory diagram showing an example of the procedure of the method for operating the immunoassay chip of the present invention.

Explanation of symbols

11 Reaction chambers 11A, 11A ′ Opening 12 Specimen / reagent reservoir 12A Opening 13 Carrier 14A ear 14B Foot 14C Space 15 bound to antigen and / or antibody 16 Side corner 16 of immunological analysis chip Waste tanks A to D, E, F, H Immunoassay chip G Centrifuge tube

Claims (13)

An immunoassay chip that has a reaction chamber containing a carrier to which an antigen and / or an antibody are bound, and detects a test substance in the reaction chamber,
A reagent / specimen reservoir that communicates with the reaction chamber and has a first opening on the opposite side of the reaction chamber;
The minor axis of the carrier is 10 to 200 μm, and the shape of the carrier is a shape selected from microbeads, microrods, and microplates,
The volume ratio of the reagent / sample reservoir to the reaction chamber is 100 or more,
An immunoassay chip that can be attached to an angle rotor and / or a swing rotor of a centrifuge. 2. The immunoassay chip according to claim 1 , wherein the reaction chamber includes a second opening on a side opposite to the reagent / sample reservoir, and has a damming means for the carrier in the second opening. The immunoassay chip according to claim 1 or 2 , wherein a ratio of a projected sectional area of the reagent / sample reservoir in the liquid feeding direction and a projected sectional area of the reaction chamber in the liquid feeding direction is 50 or more. The maximum cross-sectional area parallel to the liquid feeding direction of the reagent-sample reservoir, the ratio of the maximum cross-sectional area parallel to the liquid feeding direction of the reaction chamber, more of claims 1 to 3 is 2 to 400 The immunoassay chip according to claim 1. The immunoassay chip according to any one of claims 2 to 4 , further comprising a waste liquid tank that communicates with the second opening of the reaction chamber and is opposite to the reagent / sample reservoir. The immunoassay chip according to claim 5 , wherein the reaction chamber and the waste liquid tank are separable. The immunoassay chip according to any one of claims 1 to 6 , wherein at least a part of the reaction chamber is transparent. The immunoassay chip according to any one of claims 1 to 7 , which is fitted into a centrifuge tube and can be attached to an angle rotor and / or a swing rotor of a centrifuge while being fitted to the centrifuge tube. . The immunoassay chip according to claim 8 , wherein a protrusion is further provided on the outer wall of the chip. The immunoassay chip according to any one of claims 1 to 9, wherein a volume of the reaction chamber is 1 nL to 100 µL. The immunoassay chip according to claim 1, wherein a volume of the reagent / sample reservoir is 30 μL to 500 mL. A carrier to which an antigen and / or an antibody binds and has a short diameter of 10 to 200 μm and is selected from microbeads, microrods and microplates; a reaction chamber capable of accommodating the carrier; and the reaction chamber And a reagent / sample reservoir having a first opening on the opposite side of the reaction chamber, the volume ratio of the reagent / sample reservoir to the reaction chamber being 100 or more, and an angle rotor and / or swing of the centrifuge Chip body that can be attached to the rotor,
An immunoassay kit comprising at least A carrier capable of binding an antigen and / or antibody and having a short diameter of 10 to 200 μm and a shape selected from microbeads, microrods and microplates; a reaction chamber capable of accommodating the carrier; and the reaction chamber A reagent / sample reservoir having a first opening on the opposite side of the reaction chamber, the volume ratio of the reagent / sample reservoir to the reaction chamber being 100 or more, and an angle rotor of the centrifuge and / or Chip body that can be attached to the swing rotor,
An immunoassay kit comprising at least

Images