JP4512840B2 - Sample reaction apparatus and sample reaction method - Google Patents

Description

  The present invention relates to an apparatus and method used for reacting a sample with a reaction solution.

  After the completion of the Human Genome Project, proteome research has been actively conducted. The term “proteome” is intended to mean all proteins that are translated and produced in specific cells, organs, and organs. Examples of such research include protein profiling.

  One of the most widely used methods for profiling proteins is protein electrophoresis, particularly two-dimensional electrophoresis. Because proteins have unique properties of charge and molecular weight, they combine a proteome, which is a mixture of many proteins, rather than separating individual proteins into their components based on charge alone or molecular weight alone. As a result, more proteins can be separated with high resolution.

  Although proteins are separated by electrophoresis and / or molecular weight by electrophoresis, it is difficult to specify biological properties from the separation position of proteins separated only by such physical properties. In addition, it is known that the function of a protein is controlled by being subjected to chemical modification (post-translational modification) such as phosphorylation after being synthesized. It is difficult to obtain information on such post-translational modifications by electrophoresis alone.

  Western blotting is a method of transferring proteins in a slab gel separated by electrophoresis to a membrane. If a specific antibody is overlaid on the membrane onto which the protein has been transferred by Western blotting, the protein can be specified to some extent based on the antigen-antibody reaction. In addition, regarding phosphorylation, which is one of post-translational modifications, it is possible to detect the presence or absence of phosphorylation and the difference in phosphorylation sites by overlaying an anti-phosphorylated protein antibody on the membrane onto which the protein has been transcribed. .

The method of overlaying a specific antibody on a membrane onto which a protein has been transferred is usually performed by reacting the transfer membrane with a blocking buffer, a primary antibody, a washing solution, and a secondary antibody in tupperware or a vinyl bag, respectively. These are usually manual operations. At this time, in order to promote the reaction, the tupperware or the plastic bag is also reciprocated or rotated. A detection method as described in Patent Document 1 has also been developed.
JP 2004-163146 A (published on June 10, 2004)

  However, in the prior art, it has been difficult to uniformly react the sample transferred to the transfer film and the reaction solution.

  The present invention has been made in view of the above problems, and a main object of the present invention is to provide a sample reaction apparatus that uniformly reacts a sample transferred to a transfer film and a reaction solution.

  In order to solve the above problems, a sample reaction apparatus according to the present invention stores an adsorption structure having a sample adsorption region for adsorbing a sample, a reaction solution, and the sample adsorption region is in contact with the reaction solution. A reaction tank that houses the adsorption structure; and a drive unit that rotates the adsorption structure in the reaction tank. The sample adsorption region is provided on a surface that covers the rotation axis of the rotation. It is characterized by being.

  According to said structure, the said sample adsorption | suction area | region rotates in the said reaction liquid seamlessly like a water wheel. Therefore, compared to the case where the sample is reciprocated as in the prior art, the sample and the reaction solution can be reacted uniformly, and compared with the case where the entire reaction vessel is rotated as in the conventional technique, Since unevenness of the reaction solution accompanying the movement of the rotating tank can be avoided, there is an effect that the sample and the reaction solution can be reacted uniformly.

  In the sample reaction apparatus, it is preferable that the surface covering the rotation shaft is a surface parallel to the rotation shaft.

  According to said structure, the said sample adsorption | suction area | region is provided on the surface parallel to a rotating shaft. Therefore, the sample adsorption region is composed of a line orthogonal to the rotation about the rotation axis. Therefore, the sample 111 on each line has the same moving speed associated with the rotation, and the sample 111 and the reaction liquid 121 can be uniformly contacted.

  In the sample reaction apparatus, the surface covering the rotation axis is preferably a cylindrical surface or an elliptical cylindrical surface.

  According to said structure, since the motion of the said sample adsorption | suction area | region in the said reaction liquid can be made smoother, the nonuniformity of reaction with the said sample and this reaction liquid can be suppressed.

  In the sample reaction apparatus, the adsorption structure may include a transfer film provided with the sample adsorption region and a transfer film support that supports the transfer film.

  According to said structure, since the said sample adsorption | suction area | region is provided on the said transfer film, there exists an effect that the said sample can be made to adsorb | suck to the said adsorption | suction structure easily.

  In the sample reaction apparatus, a gap is preferably provided between the transfer film support and the transfer film. Further, it is preferable that the transfer film support is provided with a groove along a direction obliquely intersecting with a straight line parallel to a rotation axis of rotation by the driving unit.

  According to said structure, it becomes possible for the said reaction liquid to pass the back side of the said transfer film. Therefore, the flow of the reaction liquid becomes easy, and there is an effect that the sample and the reaction liquid can be reacted more uniformly.

  In the sample reaction apparatus, the adsorption structure may further include a cover that covers the sample adsorption region.

  According to said structure, since the cover is provided in the outer side of the said sample adsorption | suction area | region, there exists an effect that the said sample can be preserve | saved with adsorb | sucking to the said adsorption | suction structure.

  In the sample reaction apparatus, it is preferable that the reaction liquid is transmitted through the cover.

  According to said structure, since the said cover permeate | transmits the said reaction liquid, the said adsorption | suction structure is accommodated in the said reaction tank with this cover attached, and by rotating, the said sample, the said reaction liquid, There is an effect that can be reacted.

  In the sample reaction apparatus, the inner surface of the reaction vessel may be a semi-cylindrical surface shape or a wall surface shape when a polygonal column equal to or higher than a hexagonal column is cut by a plane passing through a central axis parallel to the length direction. preferable.

  According to said structure, since the said reaction tank has a shape close | similar to a semi-cylindrical surface or a semi-cylindrical surface, the said adsorption | suction structure which rotates can be accommodated without waste of space, thereby There is an effect that the required amount of the reaction solution can be suppressed.

  In the sample reaction apparatus, a groove or a protrusion along a direction obliquely intersecting with a straight line parallel to the rotation axis may be provided on the inner surface of the reaction tank.

  According to the above configuration, the flow of the reaction liquid generated by the rotation of the adsorption structure along the direction of the rotation is slanted by the grooves or protrusions along the direction that obliquely intersects the straight line parallel to the rotation axis. Since the reaction solution is flowed not only in the direction of rotation but also in an oblique direction and the reaction solution is further stirred, the sample and the reaction solution are reacted more uniformly. There is an effect that can be.

  In the sample reaction apparatus, the reaction tank may further include an adding unit for adding the reaction liquid and a waste liquid tank for receiving the reaction liquid flowing out from the reaction tank.

  According to the above configuration, since the unreacted reaction solution can be newly added and the reacted reaction solution can be recovered, the sample and the unreacted reaction solution can be reacted. There is an effect.

  In the sample reaction apparatus, the sample may be a protein, and the reaction solution may contain an antibody of the protein.

  According to said structure, there exists an effect that the sample in which it is important to make it react with an antibody uniformly like the protein isolate | separated by two-dimensional electrophoresis can be made to react with this antibody uniformly.

  The sample reaction apparatus may include a plurality of the reaction vessels, and the driving unit may move the adsorption structure to any reaction vessel.

  According to the above configuration, since there are a plurality of reaction vessels, a series of reactions (for example, a blocking reaction, a primary antibody reaction, a secondary antibody reaction, etc. in an antibody immune reaction) can be performed continuously. There is an effect.

  In the sample reaction apparatus, the sample is protein, and the plurality of reaction tanks are a first reaction tank that stores a blocking buffer, a second reaction tank that stores a reaction liquid containing a primary antibody that binds to the protein, and the primary reaction tank. You may include the 3rd reaction tank which stores the reaction liquid containing the secondary antibody couple | bonded with an antibody, and the 4th reaction tank which stores a washing | cleaning liquid.

  According to said structure, the detection of the said sample after the western blotting of the protein isolate | separated by two-dimensional electrophoresis can be performed suitably.

  The sample reaction apparatus according to the present invention also includes a transfer film support that supports a transfer film that adsorbs a sample and a reaction liquid, and the transfer film held by the transfer film support is in contact with the reaction liquid. A reaction tank containing the film support; and a rotating means for rotating the transfer film support in the reaction tank. The transfer film support covers the rotation axis of the rotation. You may support on a surface.

  According to said structure, the sample adsorption | suction area | region provided on the said transfer film rotates in the said reaction liquid, when this transfer film is supported by the said transfer film support body. Therefore, there is an effect that the sample and the reaction solution can be reacted uniformly.

  The sample reaction method according to the present invention includes a step of rotating an adsorption structure including a sample adsorption region for adsorbing a sample in a reaction tank in which a reaction solution is stored, and the sample adsorption region includes: It is characterized by being provided on a surface covering the rotation axis of the rotation.

  According to said structure, there can exist an effect equivalent to the sample reaction apparatus which concerns on this invention.

  A sample storage chip according to the present invention includes a sample adsorption region for adsorbing a sample and a cover for covering the sample adsorption region, which are provided so as to cover one axis.

  According to said structure, it is an adsorption | suction structure for incorporating in the sample reaction apparatus which concerns on this invention, Comprising: The chip | tip for sample preservation | save which can preserve | save while a sample is adsorbed can be provided.

  A second sample reaction apparatus according to the present invention includes an adsorption structure having a sample adsorption region for adsorbing a sample, a reaction solution, and the adsorption structure so that the sample adsorption region is in contact with the reaction solution. It may be provided with a plurality of reaction tanks to be stored and a driving means for moving the adsorption structure to an arbitrary reaction tank.

  According to said structure, since it is equipped with the said several reaction tank and the drive means which moves the said adsorption | suction structure between this reaction tank, a series of reaction (For example, blocking reaction in an antibody immune reaction, primary reaction) Antibody reaction, secondary antibody reaction, etc.) can be performed continuously.

  A second sample reaction method according to the present invention includes a first reaction step of moving an adsorption structure having a sample adsorption region for adsorbing a sample into a first reaction tank in which a first reaction liquid is stored; It is characterized by including the 2nd reaction process of moving the said adsorption structure to the 2nd reaction tank in which the 2nd reaction liquid was stored after the 1st reaction process.

  According to said structure, there can exist an effect equivalent to the 2nd sample reaction apparatus which concerns on this invention.

  In the sample reaction apparatus according to the present invention, the adsorption structure having the sample adsorption region for adsorbing the sample is rotated in the reaction tank for storing the reaction solution, so that the sample can be reacted uniformly with the reaction solution. it can.

  The present invention provides a sample reactor. In this specification, the sample reaction apparatus is intended to be an apparatus for bringing a sample into contact with a reaction solution.

  The term “sample” is used interchangeably with specimens, preparations in the art, and as used herein, a “biological sample” or equivalent thereof is intended. A “biological sample” is intended to be any preparation obtained from biological material as a source (eg, an individual, body fluid, cell line, tissue culture or tissue section). Biological samples include body fluids (eg, blood, saliva, plaque, serum, plasma, urine, synovial fluid, and fluids) and tissue sources. A preferred biological sample is a subject sample. Preferred subject samples are skin lesions, sputum, pharyngeal mucus, nasal mucus, pus, or secretions obtained from the subject. As used herein, the term “tissue sample” intends a biological sample obtained from a tissue source. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. As used herein, the term “sample” includes, in addition to the biological sample and the tissue sample, a protein sample, genomic DNA sample and / or extracted from the biological sample and the tissue sample. A total RNA sample is also included.

  In the present specification, the term “reaction solution” intends a liquid reagent for reacting with a sample. As a reaction solution for use in the sample reaction apparatus according to the present invention, for example, an antibody solution, an enzyme solution, a staining solution, a washing solution, a buffer solution, and the like can be used, but it is not particularly limited, depending on the sample and purpose to be used. Conventional reagents well known in the art can be used. For example, when the sample reaction apparatus according to the present invention is used for detecting a protein transferred to a transfer membrane by a Western blotting method using an antibody, the reaction solution is specifically a blocking buffer, and the above protein. Any of a primary antibody that binds, a secondary antibody that specifically binds to the primary antibody, a washing solution, and the like can be suitably used. When the sample reaction apparatus according to the present invention is used to detect DNA transferred to a transfer film by the Southern blotting method using probe DNA, a solution containing probe DNA can be used as a reaction solution. Furthermore, by using a staining solution as the reaction solution, the sample reaction apparatus according to the present invention can be used as a fully automatic protein staining apparatus. As the staining solution, one that stains a protein itself, one that stains a sugar residue, a phosphate group, a nitro group, or the like of a protein can be used. Such staining solutions include, but are not limited to, commercially available SYPRO Ruby (Invitrogen Corporation), MemCode Reversible Protein Stain Kit (Takara Bio Inc.), GelCode Glycoprotein Staining Kit (Technochemical Co., Ltd.) )), Deep Purple Total Protein Stain (GE Healthcare), etc. can be used.

  FIG. 1 is a cross-sectional view showing a schematic configuration of a sample reaction apparatus 100 according to an embodiment of the present invention. FIG. 2 is a perspective view showing a schematic configuration of the sample reaction apparatus 100. As shown in FIGS. 1 and 2, the sample reaction apparatus 100 according to this embodiment includes an adsorption structure 110, a reaction tank 120, and a rotation drive unit (drive means) 130. The adsorption structure 110 includes a transfer film 115 and a transfer film support 114, and a sample adsorption region 112 that adsorbs the sample 111 is provided on the transfer film 115. The reaction tank 120 stores a reaction liquid 121. The rotation drive unit 130 includes a motor 132 and a gear set 133 provided on the reaction tank 120 side, and a gear 134 coupled to the adsorption structure 110.

  As described above, the sample reaction apparatus 100 includes the sample adsorption region 112 that adsorbs the sample 111, and the sample 111 adsorbed on the sample adsorption region 112 and the reaction liquid 121 are brought into contact with each other. As will be described later, according to the present invention, the sample and the reaction liquid can be contacted uniformly over the entire area of the sample adsorption region. Therefore, it is possible to uniformly react the sample with the reaction solution, which could not be achieved conventionally.

  The sample adsorption region 112 only needs to adsorb the sample 111, and chemical, electrical, or physical characteristics can be used for adsorption of the sample 111.

  In the present embodiment, the adsorption structure 110 includes a transfer film 115 and a transfer film support 114 that supports the transfer film 115, and a sample adsorption region 112 is provided on the transfer film 115. The adsorption structure 110 does not need to be composed of the transfer film support 114 and the transfer film 115. For example, the adsorption structure 110 has a rod shape or a pipe shape, and a sample adsorption region on the surface. 112 may be provided.

  The method for adsorbing the sample on the transfer film is not particularly limited, and a well-known and commonly used method (for example, Western blotting, dropping a cell lysate or the like) can be used. Further, when the adsorption structure 110 has a rod shape or a pipe shape and the sample adsorption region 112 is provided on the surface, the adsorption structure 110 is made of a gel or the like on which the sample 111 is adsorbed. You may make it adsorb | suck by rolling up.

  The reaction tank 120 stores the reaction solution 121 and stores the adsorption structure 110 so that the sample adsorption region 112 is in contact with the reaction solution 121. Depending on the properties of the sample 111 or the reaction solution 121, a temperature control may be performed by arranging a Peltier element or the like on the bottom surface of the reaction tank 120.

  The rotation drive unit (drive means) 130 may be anything that rotates the adsorption structure 110 around the rotation shaft 131 in the reaction tank 120. In the present embodiment, the rotation of the motor 132 is transmitted to the gear set 133, and the gear set 133 and the gear 134 are engaged with each other, whereby the adsorption structure 110 can be rotated. At this time, the rotation shaft 131 is set in the reaction tank 120, and the gear set 133 is provided so as to mesh with the gear 134 at a position where the center of the gear 134 coincides with the rotation shaft 131. The motor 132 may be provided inside the adsorption structure 110. In that case, the gear 134 is engaged with a gear set 133 fixed to the processing tank 120, thereby rotating the adsorption structure 110. .

  Here, as shown in FIGS. 1 and 2, the sample adsorption region 112 is provided on a surface covering the rotation shaft 131. Therefore, when the adsorption structure 110 rotates, the sample reaction region 112 and the reaction liquid 121 can be brought into good contact with each other. Note that the surface covering the rotation shaft 131 is an outer surface of a surface provided so as to surround the rotation shaft 131.

  FIG. 3 is a perspective view showing a variation of the structure of the transfer film support 114. The transfer film support 114 may be rod-shaped as shown in FIG. 3 (a), or may be a pipe-shaped structure as shown in FIG. 3 (b). By using a screw-like structure as shown in FIG. 3 and a mesh-like structure as shown in FIG. 3D, the transfer film 115 can be supported so as to have a gap between the transfer film 115 and the transfer film 115. When the transfer film support 114 supports the transfer film 115 so as to have a gap between the transfer film 115 and the transfer film 115, the reaction liquid 121 also flows on the back side of the transfer film 115 during the rotation. Therefore, the reaction liquid 121 does not stay, and the sample 111 and the reaction liquid 121 can be brought into contact more suitably.

  FIG. 4 is a cross-sectional view showing variations in the coupling mode between the transfer film support 114 and the transfer film 115. The transfer film 115 is coupled to a coupling portion 116 provided on the transfer film support 114. The structure of the coupling portion 116 may be, for example, a slit that sandwiches the transfer film 115 as shown in FIG. 4A, or an adhesive that adheres the transfer film 115 as shown in FIG. 4B. (A magnet, a double-sided tape, or the like), or as shown in FIG. 4C, the transfer film support 114 may be a divided portion that sandwiches the transfer film 115, and supports the transfer film 115. The structure is not particularly limited as long as it can be structured.

  5 to 7 are schematic diagrams illustrating the surface 113 on which the sample adsorption region 112 is provided. The surface 113 may be provided so as to cover the rotation shaft 131, and is preferably a surface parallel to the rotation shaft 131. If the surface 113 is a surface parallel to the rotation axis 131, the sample adsorption region 112 is provided on the surface 113 configured by a line orthogonal to the moving direction. Therefore, the sample 111 on each line has the same moving speed with the rotation, and the sample 111 and the reaction liquid 121 can be uniformly contacted. Note that a plane parallel to a specific straight line refers to a plane that does not intersect the straight line.

  In one aspect, the surface 113 is a cylindrical surface having a rotation axis 131 as a central axis 119, as shown in FIG. In this case, as shown in FIG. 5B, each point on the surface 113 moves smoothly in the reaction solution 121 under the same conditions. Therefore, the sample 111 and the reaction liquid 121 can be contacted more uniformly.

  In another aspect, the surface 113 is a cylindrical surface having a central axis 119 as an axis parallel to the rotation axis 131 as shown in FIG. In this case, as shown in FIG. 5B, each point on the surface 113 moves smoothly in the reaction solution 121. Furthermore, the reaction liquid 121 is agitated by the rotation of the surface 113, and the sample 111 and the reaction liquid 121 can be brought into contact more suitably. The distance between the rotary shaft 131 and the central axis 119 of the surface 113 is preferably 50% or less of the radius of the surface 113. When the distance exceeds 0% of the radius, the reaction liquid 121 can be stirred. When the distance is 50% or less of the radius, the reaction solution 121 and the sample 111 can be more reliably brought into contact with each other.

  In yet another aspect, the surface 113 is an elliptical cylinder surface having a rotation axis 131 as a central axis 119, as shown in FIG. In this case, as shown in FIG. 5B, each point on the surface 113 moves smoothly in the reaction solution 121. Furthermore, the reaction liquid 121 is agitated by the rotation of the surface 113, and the sample 111 and the reaction liquid 121 can be brought into contact more suitably. The flatness of the surface 113 (long axis length (a) −short axis length (b) / long axis length (a)) is preferably 0.5 or less. When the flatness exceeds 0, the reaction liquid 121 can be stirred. When the flatness is 0.5 or less, the reaction liquid 121 and the sample 111 can be brought into contact more uniformly.

  Further, the adsorption structure 110 may include a structure for protecting the sample adsorption region 112. FIG. 8 is a perspective view illustrating a schematic configuration of the adsorption structure 110 including the cover 117. FIG. 9 is a cross-sectional view illustrating a schematic configuration of the adsorption structure 110 including the cover 117. As shown in FIGS. 8 and 9, the cover 117 is provided outside the transfer film 115 and covers the sample adsorption region 112. Therefore, the sample 111 adsorbed on the sample adsorption region 112 can be prevented from coming into contact with other articles. Therefore, the adsorption structure 110 can be stored while the sample 111 is adsorbed. Such an adsorption structure 110 provided with the cover 117 can be reused for diagnosing a disease by storing it in a refrigerated state as a 2DE chip in advance and setting it in a device when it becomes ill. . That is, the present invention also provides a sample storage chip having the above-described configuration.

  The cover 117 also preferably allows the reaction liquid 121 to pass therethrough. Such a cover 117 is not limited to this, but may have an opening 118 so that the reaction liquid 121 can enter and exit from the opening 118. The opening 118 may be a notch or a mesh. At this time, as shown in FIG. 9, even when the adsorption structure 110 is housed in the reaction tank 120 and rotated with the cover 117 set, the reaction solution 121 is passed through the opening 118. However, since it reaches the sample adsorption region 112, the sample 111 and the reaction liquid 121 can be reacted. If the cover 117 does not pass through the reaction solution 121, the cover 117 may be removed before the adsorption structure 110 is stored in the reaction tank 120.

  FIG. 10 is a perspective view showing a variation of the reaction tank 120. As shown in FIGS. 10 (a) and (b), the inner surface of the reaction vessel 120 has a semi-cylindrical surface, a semi-cylindrical surface shape, or a central axis parallel to the length direction of a polygonal column greater than a hexagonal column. It is preferable that it is a wall surface shape cut | disconnected by the surface to pass. If the inner surface of the reaction tank 120 is a semi-cylindrical surface, the adsorption structure 110 can be suitably accommodated by setting the central axis of the semi-cylindrical surface and the rotation axis of the adsorption structure 110 to the same position. In particular, when the diameter of the adsorption structure 110 and the diameter of the inner surface of the reaction tank 120 are substantially equal, the adsorption structure 110 can be accommodated in the reaction tank 120 with a slight gap. By filling the reaction liquid 121 in this slight gap, the reaction liquid 121 can be brought into contact with a wide range of the adsorption structure 110 with a small liquid volume, and the required amount of the reaction liquid 121 can be reduced. it can. In addition, even if the inner surface of the reaction tank 120 is not a semi-cylindrical surface but the above wall surface shape, an effect close to the semi-cylindrical surface can be obtained.

  The inner surface of the reaction tank 120 is preferably provided with a groove or a protrusion along a direction obliquely intersecting with a straight line parallel to the rotation shaft 131 as shown in FIG. FIG. 11 is a top view of the reaction layer 120 provided with such grooves or protrusions. FIG. 11A shows a configuration in which grooves or protrusions are provided on one side of the reaction tank 120, and FIG. 11B shows a configuration in which grooves or protrusions are provided on both sides of the reaction tank 120. At this time, the reaction liquid 121 flows along the direction of the rotation due to the rotation of the adsorption structure 110, but the direction of the flow is changed obliquely along the grooves or protrusions (arrows in the figure). Direction). Therefore, the flow direction of the reaction liquid 121 is not unidirectional and is stirred, so that the sample 111 and the reaction liquid 121 can be brought into contact more uniformly.

  The sample reaction apparatus 100 according to the present embodiment can be used for, for example, the transfer film 115 to which the protein separated by two-dimensional electrophoresis is transferred. Since such a transfer film has a large area, it has been difficult for the transfer film to uniformly react with the reaction liquid 121 by the conventional technique. For the analysis of two-dimensional electrophoresis, it is particularly important that the concentration of each part is uniform, so that the sample reaction apparatus 100 according to the present embodiment can be used particularly suitably.

  The sample reaction apparatus according to the present invention may also be configured to include a plurality of reaction vessels. FIG. 12 is a schematic diagram showing a schematic configuration of a sample reaction apparatus 200 according to another embodiment of the present invention, in which FIG. 12 (a) shows a sectional view and FIG. 12 (b) shows a top view. As shown in FIG. 12, the sample reaction apparatus 200 according to this embodiment includes an adsorption structure 210, reaction tanks 220, 222, 224, and 226, a waste liquid tank 228, and a drive unit (drive means) 230. I have. Reaction liquids 221, 223, 225, and 227 are stored in the reaction tanks 220, 222, 224, and 226, respectively. The drive unit 230 includes a motor 232, a gear set 233, and an arm 234.

  As the adsorption structure 210, the same adsorption structure 110 as that in the sample reaction apparatus 100 described above can be used. The arm 234 moves the adsorption structure 210 to an arbitrary reaction tank.

  Therefore, if the sample reaction apparatus 200 according to the present embodiment is used, a series of reactions can be sequentially performed by moving the adsorption structure 210 to a predetermined reaction tank. For example, the sample reaction device 200 can be used for a series of reactions of blocking treatment, reaction with a primary antibody, washing, and reaction with a secondary antibody. In this case, the reaction solution 221 is a blocking buffer solution, the reaction solution 223 is a blocking buffer solution containing a primary antibody, the reaction solution 225 is a blocking reaction solution containing a secondary antibody, and the reaction solution 227 is a washing solution. It can be.

  In addition, a cleaning liquid as the reaction liquid 227 may be sequentially added to the reaction tank 226 at, for example, the addition portion 229. In that case, a waste liquid tank 228 for collecting the overflowing reaction liquid 227 may be provided adjacent to the reaction tank 226 as shown in FIG.

  As described above, if the sample reaction apparatus 200 according to the present embodiment is used, a series of reactions can be performed sequentially, so that complicated processes such as detection of the Western blotting result can be automated. A photograph of an example of the sample reactor 200 is shown in FIG.

  That is, from another point of view, the sample reaction apparatus 200 includes a plurality of reaction vessels, and an object of the present invention is to automate a series of reactions by sequentially moving an adsorption structure that adsorbs a sample in each reaction vessel. One.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

(Preparation of transfer film with sample adsorbed)
As the transfer film, a PVDF film (Immobilon-pSQ, Millipore) was used. As a sample, dilution series (# 1: 5.63 nmole, # 2: 563 pmole, # 3: 56.3 pmole, # 4: 5.63 pmole, # 5 of Carbon anhydrase from Bovine Erythrocytes (C-3934, Sigma Co.) : 563 fmol, # 6: 56.3 fmol). Distilled water was used for dilution.

  The transfer film was placed on a screener blotter (Samplatech), and 85 μl of each sample was applied and allowed to stand for 40 minutes to adsorb the sample onto the transfer film.

(Blocking process)
Next, the transfer membrane is treated with a blocking buffer (TBS-T containing 1.5% skim milk (0.05% Tween 20, 50 mM Tris (24.2 g / 4 L), 150 mM NaCl (35 g / 4 L))) for 30 minutes. Reacted.

(Reaction process with primary antibody)
After the blocking treatment, the transfer membrane was reacted with a primary antibody diluted with a blocking buffer (anti- carbonic anhydrase from bovine erythrocytes rabbit antibody (NE023 / 7S, Nordic Immunological Lab.)).

(Washing process-1)
After the reaction with the primary antibody, the transfer film was washed with a washing solution (TBS-T). Washing was performed three times for 5 minutes each.

(Reaction process with secondary antibody)
After washing, the transfer membrane was reacted with a secondary antibody (Alexa Fluor 647 goat anti-rabbit IgG (H + L) (A21244, Invitrogen Co.)) diluted with a blocking buffer.

(Washing process-2)
After the reaction with the primary antibody, the transfer film was washed with a washing solution (TBS-T) three times for 5 minutes each. Thereafter, it was further washed with a washing solution (TBS (50 mM Tris (24.2 g / 4 L), 150 mM NaCl (35 g / 4 L)) for 5 minutes.

  A band on the obtained transfer film was detected by a fluorescence scanner (Typhoon TRIO +, GE Healthcare).

[Example 1]
The blocking treatment, the reaction with the primary antibody, the washing, and the reaction with the secondary antibody described above are shown in FIG. 12 in the blocking treatment, the reaction with the primary antibody, the washing, and the reaction with the secondary antibody. The reaction was carried out using a sample reaction apparatus according to the present invention equipped with a reaction tank for each reaction. The transfer film support used was a screw having a diameter of 19 mm as shown in FIG. In addition, as a reaction vessel for blocking treatment, reaction with the primary antibody, and reaction with the secondary antibody, a semi-cylindrical shape having a diameter of 20 mm is used, and as a reaction vessel for washing, a semi-cylindrical shape having a diameter of 22 mm is used. The thing of was used. All reactions were carried out at 20 ° C. and 70%.

  In the blocking step, the reaction step with the primary antibody, the washing step, and the reaction step with the secondary antibody, the support for supporting the transfer film is rotated at a speed of 18 revolutions / minute in the reaction tank for each. It went by.

  The amount of blocking buffer in the blocking step was 5 ml. The reaction solution in the reaction step with the primary antibody or the secondary antibody was prepared by diluting 1 μl of the above antibody so that the total amount became 5 ml, and the reaction time was 60 minutes.

  The results are shown in FIG.

[Example 2]
The reaction was performed in the same manner as in Example 1 except that the reaction time in the reaction step with the primary antibody or the secondary antibody was 15 minutes. The results are shown in FIG.

Example 3
As in Example 1, except that the reaction solution in the reaction step with the primary antibody or the secondary antibody was prepared by diluting 1 μl of the above antibody to a total volume of 1.23 ml, and setting the reaction time to 15 minutes. Reaction was performed. The results are shown in FIG.

[Comparative Example 1]
The above-described blocking treatment, reaction with the primary antibody, washing, and reaction with the secondary antibody were performed by conventional methods. Specifically, after the transfer film and each of the reaction liquids were put into a bag, the bag was sealed and rotated at a speed of 18 rotations / minute together with the bag. All reactions were performed at room temperature.

  The amount of blocking buffer in the blocking step was 25 ml. The reaction solution in the reaction step with the primary antibody or the secondary antibody was prepared by diluting 5 μl of the above antibody so that the total amount became 5 ml, and the reaction time was 60 minutes. The amount of the cleaning liquid in the cleaning process was 25 ml. The result is shown in FIG.

  As can be seen from a comparison between FIGS. 14A to 14C and FIG. 14D, the band obtained by the conventional method is thin at the center (see FIG. 14D). On the other hand, when the sample reaction apparatus according to the present invention is used, the whole color is uniformly developed (see FIGS. 14A to 14C).

  In addition, when the sample reaction apparatus according to the present invention is used, color development substantially equivalent to the band obtained by the conventional method (see FIG. 14 (d)) is obtained when the concentrations of the primary antibody and the secondary antibody are reduced (FIG. 14). 14 (a)), when the concentration is decreased and the reaction time with the primary antibody and the secondary antibody is further shortened (see FIG. 14 (b)), and in the reaction step with the primary antibody and the secondary antibody. It was also obtained when the amount of the reaction solution was reduced and the reaction time was further shortened (see FIG. 14 (c)).

  The present invention can be used in fields such as various experimental apparatuses, inspection apparatuses, and measurement apparatuses.

It is sectional drawing which shows schematic structure of the sample reaction apparatus which concerns on one Embodiment of this invention. It is a perspective view which shows schematic structure of the sample reaction apparatus which concerns on one Embodiment of this invention. (A) is a perspective view which shows an adsorption | suction structure provided with the rod-shaped transfer film support body based on one Embodiment of this invention, (b) is a pipe-shaped transfer based on one Embodiment of this invention. It is a perspective view showing an adsorption structure provided with a membrane support, and (c) is a perspective view showing an adsorption structure provided with a screw-like transfer membrane support concerning one embodiment of the present invention, and (d) FIG. 4 is a perspective view showing an adsorption structure including a mesh-like transfer film support according to an embodiment of the present invention. (A) is a perspective view which shows the adsorption | suction structure whose coupling | bond part which concerns on one Embodiment of this invention is a slit, (b) is a coupling | bond part which concerns on one Embodiment of this invention, and is an adhesive material. It is a perspective view which shows a certain adsorption | suction structure, (c) is a perspective view which shows the adsorption | suction structure which a connection part is an insertion division part based on one Embodiment of this invention. (A) is a perspective view which shows the cylindrical adsorption structure which concerns on one Embodiment of this invention, (b) is a sample provided with the cylindrical adsorption structure which concerns on one Embodiment of this invention. It is sectional drawing explaining operation | movement of a reaction apparatus. (A) is a perspective view which shows the adsorption | suction structure which the center axis and rotation axis which concern on one Embodiment of this invention have shifted | deviated, (b) is a center axis | shaft which concerns on one Embodiment of this invention, It is sectional drawing explaining operation | movement of the sample reaction apparatus provided with the adsorption structure which has shifted | deviated from the rotating shaft. (A) is a perspective view which shows the elliptical columnar adsorption structure concerning one embodiment of the present invention, and (b) is a sample provided with the elliptical columnar adsorption structure concerning one embodiment of the present invention. It is sectional drawing explaining operation | movement of a reaction apparatus. It is a perspective view showing a schematic structure of an adsorption structure provided with a cover concerning one embodiment of the present invention. It is sectional drawing which shows schematic structure of the sample reaction apparatus provided with the adsorption | suction structure provided with the cover based on one Embodiment of this invention. (A) is a perspective view which shows schematic structure of the semi-cylindrical reaction tank which concerns on one Embodiment of this invention, (b) is the wall surface shape of the cut | disconnected polygonal column which concerns on one Embodiment of this invention It is a perspective view which shows schematic structure of this reaction tank, (c) is a perspective view which shows schematic structure of the reaction tank provided with the groove | channel diagonally which concerns on one Embodiment of this invention. (A) is a top view which shows schematic structure of the reaction tank by which the groove | channel was diagonally provided in one side which concerns on one Embodiment of this invention, (b) is slanting in the both sides which concern on one Embodiment of this invention It is a top view which shows schematic structure of the reaction tank provided with the groove | channel. (A) is sectional drawing which shows schematic structure of the sample reaction apparatus provided with the several reaction tank based on one Embodiment of this invention, (b) is the some according to one Embodiment of this invention. It is a top view which shows schematic structure of the sample reaction apparatus provided with the reaction tank. It is a photograph which shows the sample reaction apparatus which concerns on one Embodiment of this invention. (A)-(c) is a photograph which shows the result of having contacted the sample with the reaction liquid using the sample reaction apparatus which concerns on one Embodiment of this invention, (d) is a sample using a prior art. It is a photograph which shows the result made to react with the reaction liquid.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Sample reactor 110 Adsorption structure 111 Sample 112 Sample adsorption area 113 Surface 114 Transfer film support body 115 Transfer film 116 Coupling part 117 Cover 118 Opening part 119 Central axis 120 Reaction tank 121 Reaction liquid 122 Groove 130 Rotation drive part (drive means) )
131 Rotating shaft 132 Motor 133 Gear set 134 Gear 200 Sample reaction device 210 Adsorption structure 220 First reaction tank 221 First reaction liquid 222 Second reaction tank 223 Second reaction liquid 224 Third reaction tank 225 Third reaction liquid 226 First 4 reaction tank 227 5th reaction liquid 228 waste liquid tank 229 addition part 232 motor 233 gear set 234 arm

Claims (15)

An adsorption structure having a sample adsorption area for adsorbing a sample;
A reaction tank for storing the reaction solution and storing the adsorption structure so that the sample adsorption region is in contact with the reaction solution;
Drive means for rotating the adsorption structure in the reaction vessel,
The sample adsorption region is provided on a surface covering the rotation axis of the rotation ,
The adsorption structure includes a transfer film provided with the sample adsorption region, and a transfer film support that supports the transfer film,
A sample reaction apparatus, wherein a gap is provided between the transfer film support and the transfer film .   The sample reaction apparatus according to claim 1, wherein a surface covering the rotation shaft is a surface parallel to the rotation shaft.   The sample reaction apparatus according to claim 1 or 2, wherein the surface covering the rotation axis is a cylindrical surface or an elliptical cylinder surface. To the transfer membrane support, sample reaction apparatus according to any one of claims 1 to 3, characterized in that a groove along a direction intersecting obliquely to a line parallel to the rotary shaft. The sample reaction apparatus according to any one of claims 1 to 4 , wherein the adsorption structure further includes a cover that covers the sample adsorption region. The cover, sample reaction apparatus according to claim 5, characterized in that to transmit the reaction solution. The inner surface of the reaction tank is a semi-cylindrical surface shape or a wall surface shape when a polygonal column equal to or higher than a hexagonal column is cut by a plane passing through a central axis parallel to the length direction. The sample reaction apparatus according to any one of to 6 . The sample reaction according to any one of claims 1 to 7 , wherein a groove or a protrusion along a direction obliquely intersecting a straight line parallel to the rotation axis is provided on an inner surface of the reaction tank. apparatus. And adding means for adding the reaction solution to the reactor, according to any one of claims 1-8, characterized in that it further comprises a waste reservoir for receiving the reaction solution flowing out from the reaction vessel Sample reactor. A plurality of the reactor, and the upper Symbol samples reactor of any one of claim 1 to 9, characterized in that it comprises a means for moving the suction structure of any of the reaction vessel. The sample reaction apparatus according to any one of claims 1 to 10 , wherein the sample is a protein, and the reaction solution contains an antibody of the protein. The sample is a protein, and the plurality of reaction tanks store a first reaction tank that stores a blocking buffer, a second reaction tank that stores a reaction liquid containing a primary antibody that binds to the protein, and a secondary that binds to the primary antibody. The sample reaction apparatus according to claim 10 , further comprising a third reaction tank for storing a reaction liquid containing an antibody and a fourth reaction tank for storing a washing liquid. A transfer film support that supports the transfer film that adsorbs the sample;
A reaction tank for storing the reaction liquid and storing the transfer film support so that the transfer film supported by the transfer film support is in contact with the reaction liquid;
Drive means for rotating the transfer film support in the reaction vessel,
The transfer film support supports the transfer film on a surface covering the rotation axis of the rotation ,
Sample reaction apparatus characterized that you have gap is provided between the transfer membrane support and the transfer membrane. Including a step of rotating an adsorption structure having a sample adsorption region for adsorbing a sample in a reaction tank in which a reaction solution is stored;
The sample adsorption region is provided on a surface covering the rotation axis of the rotation ,
The adsorption structure includes a transfer film in which the sample adsorption region is provided, and a transfer film support that supports the transfer film,
Sample reaction wherein the voids are provided between the transfer membrane support and the transfer membrane. An adsorption structure provided with a sample adsorption area for adsorbing a sample, and a cover for covering the sample adsorption area, provided so as to cover one axis ;
The adsorption structure includes a transfer film in which the sample adsorption region is provided, and a transfer film support that supports the transfer film,
A sample storage chip, wherein a gap is provided between the transfer film support and the transfer film .