JP2019053027A - Reaction promotion device and method for promoting reaction - Google Patents

Abstract

To provide such a method for promoting a reaction using magnetic force as a magnetic immunostaining technique, the method reducing variations in the promotion of a reaction generated by the position to which a sample is fixed and promoting reaction uniformly, and a reaction promotion device for realizing the method.SOLUTION: The present invention includes; a magnet 302 for drawing a magnetic complex to a sample located at the surface of a plate; and a control mechanism for making a predetermined range in which the sample is arranged on the surface of the plate and a magnetic pole surface 305 of the magnet 302 closer to each other. The magnetic pole surface 305 of the magnet 302 is broader than the predetermined range of the surface of the plate, and the magnetic pole surface 305 of the magnet 302, which is close to the plate, is inclined so that the center of magnetic pole surface is closer to the plate and the edges are more distant from the plate. This allows the difference of the magnetic force applied to the magnet 302 between the center part and the edges in a predetermined range of the plate where the sample is arranged.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a reaction promoting device and a reaction promoting method for promoting a specific binding reaction to a detection target by magnetism.

  By using a specific binding reaction with respect to a detection target, it is widely performed to check whether or not the detection target exists in the collected specimen, and when it exists, its position, amount, and the like. As one method for promoting this specific binding reaction, a magnetic immunostaining method is known. The magnetic immunostaining method is a method for detecting a target by labeling a specimen of a living tissue, and preparing fine particles in which a fluorescent dye and a magnetic particle are combined with an antibody specific to an antigen to be detected. By immersing the specimen in the suspended liquid and applying a magnetic field to the specimen to collect the microparticles on the specimen surface, the antigen-antibody reaction between the antigen to be detected and the microparticulate antibody is promoted, enabling rapid immunostaining. (For example, see Patent Document 1 and Non-Patent Document 1).

  An example of the procedure of the magnetic immunostaining method is as follows. First, a thin specimen such as a biological section is fixed to a slide glass. Next, an antigen-antibody reaction is performed by dropping a suspension of fluorescent magnetic particles with an antibody, which contains fine particles obtained by combining an antibody specific for the antigen to be detected and a fluorescent dye for labeling together with magnetic beads onto the surface of the specimen. I do. At this time, the magnetic pole surface of the magnet is brought close to the back surface of the slide glass (the surface opposite to the surface on which the specimen is fixed), thereby promoting the reaction by applying a magnetic field. Thereafter, the suspension is removed and washed, and fluorescence on the slide glass is detected, so that information on the antigen to be detected in the specimen can be obtained depending on the presence, intensity, and position of the fluorescence.

Japanese Patent No. 5401724

Sakamoto S, Omagari K et al. “Magnetically Promoted Rapid Immunoreactions Using Functionalized Fluorescent Magnetic Beads: A Proof of Principle”, Clinical Chemistry, 60: 4 2014 p.610-620

  In the above-described magnetic immunostaining method, the size of the specimen flake is often about 5 mm in diameter. Therefore, when the thin piece is fixed on the slide glass, it is difficult to place the thin piece at the same position with high accuracy every time. In the conventional magnetic immunostaining method, the progress of the antigen-antibody reaction varies depending on the position where the specimen is fixed, so the specimen may not be sufficiently stained or the reaction must be confirmed each time. There is a problem that there is. Such a problem can be avoided if the specimen can be fixed at the same position on the slide each time, but such work is burdensome for the operator and the work time is also long.

  The cause of the unevenness in the reaction is that the magnetic force applied by the magnet is not uniform. In magnetic immunostaining, generally, a magnet having a flat magnetic pole surface for attracting a magnetic material, such as a cylindrical magnet, is used. However, in such a magnet, the magnetic force at the end is stronger than the center of the magnetic pole surface, and the magnetic material is unevenly distributed at the end. Therefore, even when the antigen-antibody reaction is performed for the same time, the reaction is accelerated toward the end of the magnetic pole surface, but the reaction tends to be slow at the center, and the same specimen is immunostained. However, the result may differ depending on the fixed position. Therefore, a method for promoting the reaction by reducing the unevenness of the reaction caused by the position where the specimen is fixed is desired, and there is a problem that the present invention does not solve.

The present invention has been devised in order to solve the above-described problems, and the invention of claim 1 is a binding portion that specifically binds to a detection target to a specimen disposed within a predetermined range of the plate surface. And a magnetic complex comprising a magnetic substance is added, and the magnetic complex is attracted to a specimen on the surface of the plate by a magnetic force, thereby promoting a specific binding reaction between the binding part of the magnetic complex and a detection target. In this, a reaction promoting device comprising: a magnet for attracting the magnetic complex to the specimen on the plate surface; and a control mechanism for bringing the predetermined range where the specimen is arranged on the plate surface close to the magnetic pole surface of the magnet. The magnetic pole surface of the magnet is wider than a predetermined range of the plate surface, and the magnet is inclined so that the magnetic pole surface close to the plate is closer to the center and closer to the end portion from the plate. Therefore, in a predetermined range in which the specimen on the plate is arranged, the reaction promoting device is configured to reduce a difference in magnetic force applied by the magnet between the central portion and the end portion. It is.
According to a second aspect of the present invention, the magnet is composed of a plurality of small magnets, and the plurality of small magnets are arranged closer to the plate while being arranged at the center of the magnetic pole surface, and arranged at the end. The reaction promoting device according to claim 1, wherein the device is provided so as to be separated from the plate.
According to a third aspect of the present invention, there is provided a magnetic complex comprising: a specimen immobilized on a plate surface; a binding section that specifically binds to the specimen to be detected; and a magnetic substance that can be detected by a detection device. And a method for promoting the binding reaction by bringing a magnet from the back side of the plate closer to the position where the specimen of the plate is fixed, wherein the magnet has a magnetic pole surface close to the plate at the center. By tilting the end closer to the plate and away from the plate, the difference in the magnetic force applied by the magnet between the center and the end is reduced within a predetermined range where the specimen is placed on the plate. It is the reaction promotion method characterized by being comprised.

According to the first aspect of the present invention, when the binding reaction is promoted by the magnet, the magnetic force applied to a predetermined range where the specimen is arranged becomes uniform, and the reaction promoting device can uniformly promote the binding reaction. Can be provided.
According to the invention of claim 2, since the inclined magnetic pole surface can be composed of a plurality of small magnets, the manufacture of the inclined magnets is facilitated, and the degree of inclination can be adjusted by the arrangement and selection of the small magnets. For this reason, adjustment for obtaining an optimum magnetic pole face shape is facilitated.
According to the invention of claim 3, when the binding reaction is promoted by the magnet, the magnetic force applied to a predetermined range where the specimen is arranged becomes uniform so that the binding reaction can be promoted evenly. it can.

It is a schematic diagram of a reaction promoting device, (A) a plan view, (B) a front view. (A) Top view of magnet of 1st Embodiment, (B) Front view, (C) The top view which color-coded the 1st bar magnet and the 2nd bar magnet, (D) The 1st bar magnet and the 2nd bar magnet It is DD end view of (A) color-coded. It is the (A) top view of the magnet of a 2nd embodiment, (B) front view, CC sectional view of (C) and (B), and DD end view of (D) and (A). (A) Front view of magnet of third embodiment, (B) Front view of magnet of first modification of third embodiment, (C) Plan view of magnet of second modification of third embodiment, ( D) It is a front view of the magnet of the 2nd modification of 3rd Embodiment. It is a schematic diagram which shows the structure of a magnetic bead. It is a schematic diagram which shows the state which attracts | sucks a magnetic bead with a magnet, Comprising: (A) The state which attracts | sucks a magnetic bead to a test substance, (B) The state which peels off the remaining magnetic bead. The photograph figure which image | photographed the result of the experiment example using the magnet of each embodiment was displayed on a gray scale, (A) 1st Embodiment (no rocking), (B) 1st Embodiment (rocking) (C) Second embodiment (without swing), (D) Second embodiment (with swing), (E) Second modification of the third embodiment (without swing), (F ) The result of an experimental example of a flat cylindrical magnet (no swing). The photograph figure which image | photographed the result of the experiment example using the magnet of each embodiment is what dithered by the error diffusion, (A) 1st Embodiment (no rocking), (B) 1st implementation Form (with rocking), (C) Second embodiment (without rocking), (D) Second embodiment (with rocking), (E) Second modification of the third embodiment (without rocking) (F) is a result of an experimental example of a flat cylindrical magnet (without swinging). The photograph figure which image | photographed the result of the experiment example using the magnet of each embodiment displayed in simple monochrome two colors, (A) 1st Embodiment (no rocking), (B) 1st Embodiment (With rocking), (C) Second embodiment (without rocking), (D) Second embodiment (with rocking), (E) Second modification of the third embodiment (without rocking), (F) It is a result of the experiment example of a flat cylindrical magnet (no rocking | fluctuation).

  1st Embodiment of the reaction promotion method and reaction promotion apparatus of this invention is described. In the first embodiment, a thin piece of biological tissue is used as the specimen 1, and the specimen 1 is disposed within a predetermined range 2 a on the surface of the plate 2. Then, a suspension 6 a of magnetic beads 6, which is a magnetic complex having an antibody 4 that specifically reacts with the antigen 3 to be detected, and a magnetic body 5 is added to the specimen 1 on the plate 2. Thereafter, the magnetic beads 6 are attracted to the specimen 1 side by the magnet 102 from the back side of the plate 2, thereby promoting the antigen-antibody reaction between the antigen 3 and the antibody 4. Thereafter, the suspension 6a of the magnetic beads 6 is washed to remove the magnetic beads 6 that have not been bound to the antigen 3, and the magnetic beads 6 remaining on the plate 2 are detected as a label, whereby the specimen 1 is detected. The presence or absence of the antigen 3 can be confirmed. For detection of such an antigen, for example, by using an antibody that specifically binds to an antigen specific to cancer cells, it can be confirmed whether or not cancer cells are present in the specimen 1.

  As the plate 2, for example, a slide glass can be used. However, the plate 2 is not limited to a slide glass, and may be an appropriate one as long as it does not block magnetism and inhibit the magnetic beads 6 from being attracted by the magnet 102. Then, the specimen 1 is disposed within the predetermined range 2a of the plate 2. The predetermined range 2a can be an appropriate range according to the size of the sample 1 or the plate 2, but is a range in which the sample 1 can be disposed without difficulty when the sample 1 is disposed on the plate 2. For example, when the specimen 1 is a thin piece having a diameter of about 5 mm, it can be disposed without difficulty if it is within a circular range of about 20 mm in diameter. Therefore, this range may be set to the predetermined range 2a.

  Further, the plate 2 may be marked with a predetermined range 2a. This mark may be appropriate as long as the predetermined range can be recognized, for example, the boundary is provided with a groove, the boundary is printed, or the height of the predetermined range is recognized. it can.

  As shown in FIG. 5, the magnetic bead 6 is a magnetic complex including an antibody 4 that is a binding part that specifically binds to the antigen 3 to be detected, a magnetic body 5, and a fluorescent material 7. . The magnetic beads 6 hold the magnetic material 5 and the fluorescent material 7 inside, and the antibody 4 is fixed on the outer surface so that an antigen-antibody reaction with the antigen 3 is possible. Such a magnetic bead 6 and its manufacturing method are known, and are also described in, for example, Patent Document 1 described above, and appropriate ones can be adopted.

  The fluorescent substance 7 is a label for confirming that an antigen-antibody reaction has occurred. That is, the suspension 6a is finally washed away due to the presence of the fluorescent substance 7, and then the plate 2 is observed with a fluorescence microscope to determine whether or not the antigen 3 to be detected is present in the specimen 1 and its position. , Amount, etc. can be visually recognized. Therefore, in terms of “promoting the binding reaction”, it is not essential to add a label such as the fluorescent substance 7 to the magnetic beads 6 (magnetic complex). Further, the label is not limited to the fluorescent label, and for example, the presence or absence of the antigen 3 can be confirmed by detecting the magnetism of the magnetic beads 6 bound to the antigen 3. The fluorescent substance 7 may be directly labeled on the antibody 4 or may be labeled using a secondary antibody.

  101 is a reaction promoting device used for promoting the above-described reaction, and includes a magnet 102, a plate holding unit (not shown) that holds the plate 2, and a magnetic pole surface 105 of the magnet 102 on the plate 2 held by the plate holding unit. And a control mechanism 104 that controls to approach and separate under a predetermined condition.

  The magnet 102 includes a magnetic pole surface 105 close to the plate 2. As shown in FIG. 2, the magnet 102 is configured by continuously arranging the first bar magnets 102 a and the second bar magnets 102 b so as to be alternately adjacent to each other. In the first bar magnet 102a, the magnetic pole surface 105a on the magnetic pole surface 105 side has an N pole, while in the second bar magnet 102b, the magnetic pole surface 105b on the magnetic pole surface 105 side has an S pole. In FIGS. 2C and 2D, the first bar magnet 102a is shown in black. Since the magnetic field lines are directed from the N pole to the S pole, the first and second bar magnets 102a and 102b are alternately arranged in this manner, so that the magnetic pole surface 105 of the magnet 102 has the N pole of the first bar magnet 102a. A magnetic field line loop is completed from the magnetic pole surface 105a to the S-pole magnetic pole surface 105b of the adjacent second bar magnet 102b (see FIG. 2D). As a result, it is possible to prevent the magnetic force from increasing at the end of the magnetic pole surface 105 and to prevent the magnetic beads from being localized at the end.

  The first and second bar magnets 102a and 102b are bar magnets made of the same material with the top and bottom reversed. For example, the magnetic pole surfaces 105a and 105b have a rectangular column shape of 2 mm × 2 mm and have a high magnetic force such as a neodymium magnet. A magnet can be used. The smaller the area of the magnetic pole surfaces 105a and 105b of the first and second bar magnets 102a and 102b (that is, the greater the number of first and second bar magnets 102a and 102b), the more within the predetermined range 2a of the plate 2. The applied magnetic field becomes uniform. For example, in the case of immunostaining a specimen of about 5 mm, a sufficiently uniform magnetic field is applied by using the magnet having the 2 mm square magnetic pole face. However, even when the areas of the magnetic pole surfaces 105a and 105b are large (that is, the number of the first and second bar magnets 102a and 102b is small), the arrangement of the specimen is different from the case where the conventional magnets are used. The impact of will decrease.

  The first and second bar magnets 102a and 102b are not limited to a prismatic shape, and may be cylindrical. Moreover, it is not always necessary to be adjacent to each other, and a small gap may exist between them. As such a magnet 102, it is assumed that a plurality of bar magnets are integrally supported on the opposite side of the magnetic pole surface 105.

  The width of the magnetic pole surface 105 of the magnet 102 is set as a predetermined range 2a in such a range that an operator can arrange the specimen 1 without difficulty when arranging the specimen 1 in the plate 2, and the magnetic pole surface 105 has a predetermined range 2a. Be larger. For example, when a thin piece of living tissue having a diameter of about 5 mm is fixed to the center of the slide glass, the operator can fix the magnetic pole surface 105 of the magnet 102 within the range of about 20 mm in diameter without difficulty. The diameter may be about 22 mm.

  The plate holding part may be anything as long as it can hold the plate 2 in a predetermined position. For example, the plate holding part may be one that places and fixes the plate 2 on a flat plate, or one that grips and fixes the end of the plate 2. Good.

  The control mechanism 104 moves the magnetic beads 6 of the added suspension 6a to the specimen 1 by bringing the magnetic pole surface 105 of the magnet 102 from the back side of the plate 2 to the plate 2 held by the plate holding unit. The magnet 102 is controlled to be attracted. Thus, the state in which the magnetic pole surface 105 of the magnet 102 is brought close to the back surface side of the plate 2 is referred to as a proximity state, and the time and distance to bring the magnetic pole surface 105 close to each other depends on the magnetic force of the magnet 102, the reaction speed of the binding reaction, etc. An appropriate setting can be made in consideration. For example, it is possible to set the proximity state in which the plate 2 is close to the back surface at a position of about 1 mm for about 1 minute. Further, the control mechanism 104 does not necessarily move the magnet 102, and the plate 2 may be brought close to the magnet 102.

  The control mechanism 104 may include a swing mechanism that swings the magnet 102 in the horizontal direction in the proximity state. The oscillating mechanism may oscillate the magnet 102 in the horizontal direction along with the movement of the magnet to the proximity state when the control mechanism 104 brings the magnetic pole surface 105 close to the plate 2. It may start swinging after moving. The swing mechanism can move the magnet 102 in the horizontal direction, for example, at about 10 reciprocations per second. The horizontal movement of the magnet 102 may be a simple reciprocating motion or a circular motion. The swing mechanism may swing the plate 2 (or the plate holding mechanism) instead of the magnet 102, but it is usually preferable to swing the magnet 102. That is, the swing mechanism may be any mechanism that swings the magnet 105 relative to the plate 2.

  The swing range (stroke) of the magnet 102 by the swing mechanism may be about the area of the magnetic pole surfaces 105a and 105b of the first bar magnet 102a or the second bar magnet 102b. That is, when the magnet 102 is integrally formed by a plurality of first and second bar magnets 102a and 102b, the magnetic pole surfaces 105a and 105b of each bar magnet are as wide as a unit as shown in FIG. A shading pattern occurs in the accumulation of the magnetic beads 6. If this light and shade pattern is made sufficiently small with respect to the assumed size of the specimen 1, it will not affect the homogeneous reaction promotion, but a rocking mechanism to eliminate the light and shade and make it more uniform. The magnet 102 may be swung by. At this time, when a conventional cylindrical magnet is used, for example, if it is intended to make the magnetic force uniform within a predetermined range 2a having a diameter of about 20 mm, the stroke needs to be about 20 mm, the same as the diameter of the predetermined range 2a. . However, when the magnet 102 of the present embodiment is used, even if it is swung in order to reduce the unevenness of the magnetic force within a range of about 20 mm in diameter, the stroke is almost the same as the diameter of the magnetic pole surfaces 105a and 105b of each bar magnet. If so, it is possible to eliminate the shading and to homogenize. That is, since the stroke can be reduced when rocking, the load is small, and the durability of the reaction promoting device 101 is improved.

  The above is the basic configuration of the reaction promoting apparatus 101. Next, a reaction promoting method using the reaction promoting apparatus 101 will be described with a specific example. First, the specimen 1 collected from the living body is placed in a predetermined range 2a near the center of the upper surface of the plate 2 (slide glass), and the plate 2 is held by the plate holding portion of the reaction promoting apparatus 101. Thereafter, the specimen 1 on the plate 2 is immersed in a 10% formalin aqueous solution and fixed in formalin. The specimen 1 is washed with pure water for 15 minutes to remove formalin, and then immersed in skim milk / PBS (phosphate buffered saline) for blocking. Subsequently, the suspension 6a of the magnetic beads 6 on which the antibody 4 is immobilized is dropped onto the specimen 1 on the plate 2, and the magnet 102 is moved about 1 mm from the back side of the predetermined range 2a of the plate 2 by the control of the control mechanism 104. The magnetic beads 6 are magnetically attracted and attracted to the specimen 1 by bringing them close to a certain distance, and the antigen-antibody reaction between the antigen 3 and the antibody 4 is promoted. The magnet 102 is separated from the plate 2 by the control of the control mechanism 104 through a proximity state of about 1 minute. Thereafter, the suspension 6a is removed, and the specimen 1 is rinsed with PBS. Then, by fluorescence observation of the specimen 1 on the plate 2 using a fluorescence microscope, if fluorescence is detected, it can be confirmed that the antigen 3 to be detected is present in the specimen 1, and depending on the fluorescence intensity and distribution, the antigen 1 The amount and position of 3 can be confirmed.

  Ideally, the presence or absence of the antigen 3 can be confirmed by the above method. However, in actual operation, the magnetic beads 6 that did not bind to the antigen 3 may be adsorbed and remain on the surface of the plate 2, which may be erroneously detected when fluorescence is detected. Typically, when the magnetic body 5 is a ferromagnetic body, the magnetic beads 6 are bonded and remain. In order to avoid such a situation, it is desirable to perform the following steps. After the reaction, the specimen 1 is rinsed with PBS and then immersed in PBS, and the magnet 102 is brought close to the surface side of the plate 2 to which the specimen 1 is fixed to draw the non-specifically adsorbed magnetic beads 6. It is a process of peeling and removing.

  Even when the magnetic beads 6 are peeled off, the magnetic beads 6 can be peeled off and removed uniformly by using the magnet 102. The magnet 102 used for peeling may be controlled to be moved by the control mechanism 104. However, it is not always necessary to use the same magnet 102 for attraction and peeling, and different magnets 102 may be used, or they may be used in combination with magnets of other embodiments described later.

  The above is a magnetic immunostaining method including a reaction promoting method using the reaction promoting device 101 having a basic configuration. The reaction promoting device 101 is an automated method including steps before and after promoting the reaction by the magnet 102. Further explanation will be given.

  First, as shown in FIG. 1, a case 10 that accommodates the plate 2 is used. The case 10 has a box shape in which a liquid inlet 10a and a liquid outlet 10b are provided at both left and right ends, and an inlet 10c for a suspension 6a is formed in the center of the upper surface. The inlet 10c is positioned above the predetermined range 2a where the specimen 1 is placed when the plate 2 is placed inside. Further, the upper portion of the predetermined range 2 a is so close to the surface of the plate 2 that the upper surface of the case 10 is raised. As a result, the suspension 6a injected from the injection port 10c is widely added to the specimen 1 in a small amount.

  The reaction promoting device 101 is connected to a pump 110 for filling and discharging PBS into the case 10, a liquid inlet path 111 connecting the pump 110 and the liquid inlet 10a of the case, and a liquid outlet 10b of the case. And a drainage path for discharging PBS or the like in the case. In addition, an appropriate means for automating the reaction, such as an injection means for automatically injecting the suspension, may be provided.

  By using such a reaction accelerating device 101, it is possible to automate the process including the steps of blocking the above-described magnetic immunostaining and rinsing with PBS, so that the work efficiency is improved.

  Next, experimental examples and results for confirming that the reaction can be promoted without unevenness by using the reaction promoting apparatus 101 will be described. This experimental example was performed according to the following procedure. First, a glass slide is held as a plate 2 on a plate holder, and about 300 μl of a suspension 6a containing magnetic beads 6 diluted 50 times with skim milk is dropped on the top surface of the glass slide. Thereafter, the magnetic pole surface 105 of the magnet 102 is brought close to a position about 1 mm away from the back surface of the slide glass and is allowed to stand for 30 seconds. Then, the state of the slide glass in that state is observed with a fluorescence microscope to confirm the state of accumulation of the magnetic beads 6. In addition, as a control experiment, the same operation is performed using a cylindrical magnet with a flat magnetic pole face instead of the magnet 102.

  In this experimental example, the first and second bar magnets 102a and 102b are both neodymium whose magnetic pole faces 105a and 105b have a prismatic shape of 2 mm × 2 mm, a holding force of about 955 kA / m, and a residual magnetic flux density of about 1.3 T. A magnet (manufactured by Sagami Chemical Metal Co., Ltd., NF40) was used. As shown in FIG. 2, the magnet 102 has the first and second bar magnets 102a and 102b arranged adjacent to each other so as to be integrated. As the magnetic beads 6, FF beads (particle diameter 200 nm) manufactured by Tamagawa Seiki Co., Ltd. were used. The cylindrical magnet of the control experiment was a neodymium magnet having a magnetic pole face of 22 mm in diameter.

  7A, FIG. 8A, and FIG. 9A (hereinafter referred to as “FIGS. 7 to 9A” and the same applies to FIGS. 7B and 7F) are described above. 5 is a photograph data obtained by photographing a state in which the magnetic beads 6 are accumulated on the slide glass according to the experimental example. 7A to 9A are images obtained by performing image processing on the same photographic data. FIG. 7A is a gray scale, FIG. 8A is a dithered black and white two-color display, and FIG. (A) is a simple monochrome two-color display. FIGS. 7A and 8A show that the white portion is more intensely fluorescent, and thus more magnetic beads 6 are accumulated. Further, in FIG. 9A, a portion having a certain fluorescence intensity or more is displayed in white. The photographic data resulting from the control experiment are also represented in FIGS. 7 to 9 (F).

  As a result of the control experiment, as can be confirmed also from FIGS. 7 to 9F, the fluorescence intensifies in a ring shape along the end of the magnet. That is, when a cylindrical magnet having a flat magnetic pole surface was used, it was confirmed that magnetic beads were unevenly distributed at the end of the magnetic pole surface. On the other hand, in the result of the experimental example based on the first embodiment, as can be confirmed from FIGS. 7 to 9A, the fluorescence is distributed in a lattice pattern in units of the first and second bar magnets. Therefore, in this experimental example, unlike the control experiment, it can be said that the magnetic beads 6 are not unevenly distributed in a ring shape, and are attracted and accumulated uniformly over the entire magnetic pole surface 105. This can be said to show that the reaction promotion device 1 can promote the reaction without unevenness.

  7A to 9A, the black circular portions appear to be continuous. The black circular portions are adjacent to the first and second bar magnets 102a. , 102b are adjacent to each other. That is, the magnetic pole surfaces 105a and 105b of the first and second bar magnets 102a and 102b are positioned around a central portion surrounded by a black circular portion in the horizontal direction.

  In order to confirm the effect of swinging the magnet 102 by the swing mechanism, in the above experimental example, “the magnetic pole surface 105 of the magnet 102 is brought close to a position about 1 mm away from the back surface of the slide glass. , The magnetic pole face 105 of the magnet 102 is moved to the position spaced about 1 mm away from the back surface of the slide glass by moving it back and forth 10 mm reciprocally every second with a stroke of 2 mm and rocked for 30 seconds. An experimental example was performed as a process. The photograph data which image | photographed the result of the experiment example about this rocking | fluctuation are shown by FIGS. 7-9 (B). As can be confirmed from FIGS. 7 to 9 (B), the results of the oscillating experimental example showed that the intensity of fluorescence disappeared over the entire area where the magnetic pole face 105 was close even when the oscillating movement was caused by a stroke of 2 mm. .

  Next, a second embodiment of the present invention will be described. The second embodiment can be the same as the first embodiment except that the structure of the magnet is different from the first embodiment, and the other description will be omitted. As shown in FIG. 3, the magnet 202 of the second embodiment is provided with uniform unevenness on the magnetic pole surface 205. The magnet 202 is composed of a plurality of bar magnets, and the first bar magnets 202a and the second bar magnets 202b shorter than the first bar magnets 202a are continuously adjacent to each other. The second bar magnet 202b enters the center in the longitudinal direction by a predetermined distance from the first bar magnet 202a and is adjacent to the first bar magnet 202a so that the magnetic pole surface 205 of the magnet 202 has an uneven shape. .

  Thus, by making the magnetic pole surface 205 of the magnet 202 uneven, the magnetic force acts uniformly at the center and the end of the magnetic pole surface 205, and the staining of the specimen 1 is arranged on the plate 2. The unevenness due to the difference in position is less likely to occur. Further, when the swing mechanism is provided, the stroke may be the same as that of the magnetic pole surfaces 205a and 205b of the first or second bar magnets 202a and 202b, as in the first embodiment.

  In the second embodiment, when the magnetic pole surface 205 is provided with irregularities, the magnet 202 is composed of a plurality of long and short bar magnets, but is not necessarily limited thereto. A plurality of bar magnets having the same length may be used to provide unevenness on the magnetic pole surface 205, or the unevenness may be obtained by processing the magnetic pole surface 205 of one magnet into an uneven shape. Further, the uneven shape may be a sawtooth shape in which the inclination at a predetermined angle is repeated. From the viewpoint of promoting the reaction with less unevenness, the shape of the irregularities is preferably a continuous and regular shape.

  The smaller the size of one concave portion or convex portion in plan view is, the smaller the applied magnetic field becomes. For example, when immunostaining a specimen of about 5 mm, a sufficiently uniform magnetic field is applied by using a magnet in which a 2 mm square recess and projection are continuously repeated. However, even when the size of each concave portion and convex portion is large, the influence of the arrangement of the specimen 1 is reduced as compared with the case where a conventional magnet without irregularities is used. Further, the depth of the concave portion can be set appropriately, but in the case of the above example, the concave portion can be recessed by about 2 mm with respect to the convex portion.

  Also in the reaction promoting device of the second embodiment, an experimental example using the same method as that of the first embodiment is performed, and photograph data obtained by photographing the result are shown in FIGS. 7 to 9C (in the case of no swing), FIG. 7-9 (D) (when there is rocking). The first bar magnet 202a and the second bar magnet 202b have the same polarity on the magnetic pole face 205 side, and are arranged adjacent to each other continuously as shown in FIG. In a certain point, this experimental example is different from the conditions of the experimental example in the first embodiment.

  As a result of the present experimental example based on the second embodiment, as shown in FIGS. 7 to 9 (C), a place where the fluorescence is strong so as to correspond to the positions of the first and second bar magnets 202a and 202b that are close to each other. And weak points were staggered. Therefore, unlike the control experiment (FIGS. 7 to 9 (F)), the magnetic beads 6 are not unevenly distributed in a ring shape and are attracted and accumulated uniformly over the entire magnetic pole face 205. It can be said. This can be said to show that the reaction promotion device 1 can promote the reaction without unevenness. Further, as can be seen from FIGS. 7 to 9D, when the magnet 202 is swung by the swing mechanism, the grayscale pattern of the fluorescence disappears over the entire area where the magnetic pole face 205 is close even if the stroke is reduced. This is the same as in the case of the first embodiment.

  7 to 9C, a white circular shape and a black circular shape are alternately arranged. The white circular portion is a first bar magnet 202a that is a convex portion, and the black circular portion is a concave portion. Each of the second bar magnets 202b corresponds to a position close to the second bar magnet 202b.

  A third embodiment of the present invention will be described. Since the third embodiment can be the same as the first embodiment except that the structure of the magnet 302 is different, other description is omitted. As shown in FIG. 4A, the magnet 302 of the present embodiment is formed in a paraboloidal shape that is inclined so that the magnetic pole surface 305 is closer to the plate 2 toward the center and further away from the plate 2 toward the end. Yes.

  In this manner, the magnetic pole surface 305 is inclined closer to the plate 2 toward the center and away from the plate 2 toward the end, so that the magnetic pole surface 305 is applied at the center and the end within the predetermined range 2a of the plate 2. The difference in magnetic force will be reduced. In the case of a normal cylindrical magnet, the magnetic force is weaker at the center and stronger at the end. On the other hand, in the magnet 302 of the present embodiment, the magnetic force is increased at the end of the magnetic pole surface 305 by making the magnetic pole surface 305 closer to the plate 2 while being separated from the plate 2 toward the end. The antigen-antibody reaction can be promoted uniformly.

The paraboloid shape of the magnetic pole surface 305 can be appropriately determined based on the distance between the plate 2 and the magnet 302, the magnetic force of the magnet 302, and the like. For example, regarding the shape of the paraboloid, the paraboloid satisfying z = −kx ² with the center as the origin when the vertical direction of the magnetic pole surface 305 is z (mm) and the arbitrary horizontal direction is x (mm). When making the surface shape, when the predetermined range 2a in which the specimen 1 is arranged is a circular shape having a diameter of 20 mm, k is preferably set to about 0.02 to 0.03.

  Further, in this embodiment, since the density in the bar magnet unit as in the first and second embodiments does not occur, it is not necessary to swing the magnet 302 by the swing mechanism, and the life of the reaction promoting device is extended. be able to. However, a rocking mechanism may be provided to ensure completeness, and the magnet 302 may be rocked relative to the plate 2.

  Further, as a first modification of the third embodiment, as shown in FIG. 4B, a magnet 402 is configured by a plurality of bar magnets 402a adjacent to each other, and a magnetic pole surface 405 is formed. It is good also as what was comprised so that the center magnet might adjoin to the plate 2 so that it might become the paraboloid shape of 3rd Embodiment. The bar magnet 402a has a prismatic shape, and all the magnetic poles on the magnetic pole surface 405 side are aligned. The shape in plan view is the same as in FIG. 2A, and the magnetic pole surface 405 has a circular shape as a whole in plan view. However, the magnetic pole surface 405 may have a rectangular shape as a whole in plan view.

  Furthermore, as a second modification of the third embodiment, as shown in FIGS. 4C and 4D, the magnet 502 may be configured by overlapping a plurality of flat first columnar magnets 502a. In this modification, a plurality of first columnar magnets 502a are stacked to be overlapped, and the plurality of first columnar magnets 502a are arranged concentrically in a plan view and arranged on the upper side (plate 2 side). The diameter is shorter. In addition, the outermost second cylindrical magnet 502b in a plan view is not flat but has a long shape in the axial direction. In other words, the first columnar magnet 502a that is flatter and has a smaller diameter is stacked on the magnetic pole surface of the second columnar magnet 502b to constitute the magnet 502. As a result, the magnetic pole surface 505 of the magnet 502 is inclined so as to be closer to the plate 2 toward the center and away from the plate 2 toward the end. A long cylinder such as the second columnar magnet 502b is not essential, and the magnet 502 may be configured by laminating only the flat first columnar magnet 502a. Further, the thicknesses of the first columnar magnets 502a may all be equal, or may be different, for example, the magnetic pole surface 505 may be a paraboloid as a whole.

  In the first and second modified examples of the third embodiment, the magnets 402 and 502 are composed of a plurality of small magnets (bar magnets 402a and first cylindrical magnets 502a), and small magnets arranged in the center of the magnets. The closer the magnet is to the plate 2, the smaller the magnet disposed at the end of the magnet, the farther away from the plate 2. By doing in this way, the inclination of the magnetic pole surface (405, 505) of a magnet can be provided easily.

  Also in the reaction promoting apparatus according to the second modification of the third embodiment, an experimental example using the same method as in the first and second embodiments is performed, and photograph data obtained by photographing the result are shown in FIGS. It is represented. In this experimental example, the rocking mechanism is not used for rocking. A plurality of first cylindrical magnets 502a are arranged as shown in FIG. 4C in a plan view and FIG. 4D in a front view, and are closer to the plate 2 as a whole and farther away from the plate 2 as ends. This experimental example is different from the conditions of the experimental example in the first and second embodiments in that the inclined magnetic pole surface 505 is formed. The first cylindrical magnets 502a each have a thickness (stacking direction) of 1 mm, the diameter of the magnetic pole surface of the uppermost part closest to the plate 2 is 3 mm, and the seven first cylindrical magnets 502a are increased by 4 mm thereafter. Are stacked. That is, eight first columnar magnets 502a are arranged in total. Under the eight stacked first cylindrical magnets 502a, a long second cylindrical magnet 502b having a magnetic pole surface of 35 mm in diameter is disposed. Therefore, the magnetic pole surface 505 is inclined so that the center is close to the plate 2 by about 8 mm with respect to the end portion. Both the first and second cylindrical magnets were neodymium magnets.

  As a result of the experimental example, the fluorescence did not increase in a ring shape as a whole, and the fluorescence was observed almost uniformly as a whole. This can be confirmed also from FIGS. Therefore, it can be said that the magnetic beads 6 are not unevenly distributed. This indicates that according to the reaction promoting device of the present embodiment, the magnetic beads 6 can be attracted uniformly over the entire magnetic pole surface 505 of the magnet 502, and as a result, the reaction without unevenness. It can be said that promotion is possible.

  Although the embodiments of the present invention have been described above, the present invention is not limited to these embodiments, specific numerical values, reactions, and the like. Specific binding promoted by the reaction promoting method of the present invention is not limited to antigen-antibody reaction. For example, binding between an enzyme and a substrate, complementary binding between DNA, specific binding between proteins, nucleic acid and protein There are various types such as specific binding between them.

  Further, the fact that the magnetic force is “uniform” does not mean that the magnetic force is completely equal within a predetermined range, and part of the magnetic force does not become extremely strong. It is about attracting. Furthermore, the reaction promotion being “homogeneous” means that the same degree of reaction promotion is possible regardless of the arrangement of the specimen 1 (or magnetic beads 6) within the predetermined range 2a.

  In the first embodiment of the present invention configured as described above, the antibody 4 specifically binds to the antigen 3 to be detected on the specimen 1 which is a section of the biological tissue placed within the predetermined range 2a on the surface of the plate 2. And a magnetic bead 6 having a magnetic substance 5 is added, and the magnetic bead 6 is attracted to the specimen 1 on the plate 2 by a magnetic force, whereby a specific antigen antibody between the antibody 4 of the magnetic bead 6 and the antigen 3 is obtained. The reaction can be promoted. Then, the reaction promoting device 101 controls the magnet 102 for attracting the magnetic beads 6 to the specimen 1 on the surface of the plate 2 and the control mechanism 104 for bringing the predetermined range 2a of the plate 2 and the magnetic pole surface 105 of the magnet 102 into close proximity. It has. The magnetic pole surface 105 of the magnet 102 is wider than the predetermined range 2 a of the plate 2, and the magnet 102 has a first magnetic pole surface 102 a having the N-pole magnetic pole surface 105 a on the magnetic pole surface 105 side and a magnetic pole surface 105 side. A plurality of second bar magnets 102b having magnetic pole surfaces 105b and S poles are alternately adjacent to each other. With such a configuration of the magnet 102, a loop of magnetic lines of force is completed from the magnetic pole surface 105a of the adjacent first bar magnet 102a to the magnetic pole surface 105b of the second bar magnet 102b, and within the predetermined range 2a. In this case, the magnetic force applied at the center is uniform between the central portion and the end portion, and the binding reaction by the antigen-antibody reaction between the antigen 3 and the antibody 4 can be uniformly promoted.

  Further, in the second embodiment, a plurality of first rod magnets 202a having long magnets 202 and a plurality of short second bar magnets 202b are alternately adjacent to each other. Is provided. As a result, end portions are formed on each of the irregularities, and the magnetic pole surface 205 of the magnet 202 as a whole has a uniform magnetic force at the center portion and the end portions, and is therefore applied within the predetermined range 2a. The magnetic force is uniform between the central portion and the end portion, and the antigen-antibody reaction can be promoted uniformly.

  In the third embodiment, the magnetic pole surface 305 of the magnet 302 is inclined in a parabolic shape so that the magnetic pole surface 305 of the magnet 302 is closer to the plate 2 and is separated from the plate 2 toward the end portion. Thus, the difference in magnetic force applied by the magnet 302 between the two ends is reduced. As a result, the end portion where the magnetic force of the magnetic pole surface 305 is separated from the plate 2 in the proximity state, while the central portion where the magnetic force is weak is close to the plate 2, and the end antigen-antibody reaction can be promoted evenly. Become.

  Furthermore, in each modification of 3rd Embodiment, the magnets 402 and 502 are comprised by the bar magnet 402a and the 1st cylindrical magnet 502a which are several small magnets, and the bar magnet 402a and the 1st cylindrical magnet 502a are magnetic pole surfaces. The one arranged at the center of 405 and 505 is closer to the plate 2, while the one arranged at the end is provided away from the plate 2. Thereby, since the inclination of the magnetic pole surfaces 405 and 505 can be formed by a combination of a plurality of small magnets not provided with the inclined surfaces, the magnet can be easily manufactured and the magnetic pole surface of a single magnet is inclined. Compared to the case of machining, the optimum inclination shape adjustment to promote the reaction without unevenness can be adjusted by the arrangement and selection of the small bar magnet 402a and the first cylindrical magnet 502a. Adjustment becomes easy.

  The present invention can be used in a technical field using reaction promotion that promotes a specific binding reaction to a detection target by magnetism, such as magnetic immunostaining.

1 specimen 2 plate 2a predetermined range 3 antigen (detection target)
4 Antibody (binding part)
5 Magnetic body 6 Magnetic beads (magnetic composite)
7 Fluorescent substance 101 Reaction accelerating device 102, 202, 302, 402, 502 Magnet 102a, 202a First bar magnet 102b, 202b Second bar magnet 402a Bar magnet (small magnet)
502a First cylindrical magnet (small magnet)
104 Control mechanism 105, 205, 305, 405, 505 Magnetic pole surface

Claims (3)

A magnetic complex having a binding part that specifically binds to a detection target and a magnetic substance is added to a specimen arranged within a predetermined range on the plate surface, and the magnetic complex is applied to the specimen on the plate surface by magnetic force. By attracting, in promoting the specific binding reaction between the binding part of the magnetic complex and the detection target,
A magnet for attracting the magnetic complex to the specimen on the plate surface;
A control mechanism for bringing the predetermined range in which the specimen is arranged on the plate surface and the magnetic pole surface of the magnet close to each other,
The magnetic pole surface of the magnet is wider than a predetermined range of the plate surface,
The magnet is inclined such that the magnetic pole surface close to the plate is closer to the center and closer to the plate and is separated from the plate toward the end. A reaction promoting device configured to reduce a difference in magnetic force applied by a magnet between parts.   The magnet is composed of a plurality of small magnets. The plurality of small magnets are arranged closer to the plate as they are arranged in the center of the magnetic pole surface, while those arranged at the ends are closer to the plate. The reaction promoting device according to claim 1, wherein the reaction promoting device is spaced apart. Fix the sample on the plate surface,
A magnetic complex comprising a binding part that specifically binds to a detection target and a magnetic substance that can be detected by a detection device is added to the fixed specimen,
A reaction promoting method for promoting a binding reaction by bringing a magnet from the back side of the plate to a position where the specimen of the plate is fixed,
The magnet is inclined such that the magnetic pole surface close to the plate is closer to the center and closer to the plate and is separated from the plate toward the end. A reaction promoting method, characterized in that a difference in magnetic force applied by a magnet between parts is reduced.

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