JP2014069149A - Spacer and processing unit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a spacer which is included in a processing unit and thereby enables the processing unit to perform highly accurate processing of a liquid to be processed therein with no waste, and to provide the processing unit including the spacer.SOLUTION: A spacer 700 of this invention is disposed between a multi-well filter plate 100 and a multi-well plate 200 disposed below the multi-well filter plate 100 and has a cylindrical body 701 corresponding to a well 101. When the spacer 700 is disposed between the multi-well filter plate 100 and the multi-well plate 200, a lower end of the cylindrical body 701 corresponds to an upper end of a well 201 in a vertical direction or is set so as to be positioned at the lower side than the upper end of the well 201. A bottom surface 103 in the multi-well filter plate 100 that the cylindrical body 701 faces corresponds to an upper end of the cylindrical body 701 in the vertical direction or is positioned at the lower side than the upper end of the cylindrical body 701.

Description

  The present invention relates to a spacer and a processing apparatus, and more particularly to a spacer and a processing apparatus used when purifying a sugar chain possessed by a glycoprotein.

  A sugar chain is a general term for molecules in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid, and derivatives thereof are linked in a chain by glycosidic bonds. .

  Sugar chains are very diverse and are substances that are involved in various functions of naturally occurring organisms. Sugar chains often exist as complex carbohydrates bound to proteins, lipids, and the like in vivo, and are one of the important components in vivo. It is becoming clear that sugar chains in living organisms are deeply involved in cell-to-cell information transmission, protein functions and interactions.

  Examples of biopolymers having sugar chains include proteoglycans on the cell wall of plant cells that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and cell-cell interactions and cells. Glycoproteins involved in recognition can be mentioned, but the mechanism by which these high-molecular sugar chains control advanced and precise biological reactions while acting, assisting, amplifying, regulating, or inhibiting each other's functions gradually. It is being revealed. Furthermore, if the relationship between such sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, this sugar chain engineering and medicine, cell engineering, or organ engineering are closely related. It can be expected that a new development will be made in relation to (Non-patent Document 1).

  In particular, it has been revealed that sugar chains present on the cell surface play an important role as scaffolds for various biological reactions. For example, it is said to be involved in the occurrence of diseases due to abnormal interaction with the receptor, infection with AIDS virus, influenza virus, etc., and invasion of pathogenic E. coli O157 toxin and cholera toxin into cells. In addition, specific sugar chains appear on the cell surface in certain types of cancer cells, and the cell surface sugar chains are considered to be important molecules that give the cells individuality.

  For these analyses, sugar chain structure analysis techniques have been developed, which combine processes such as sugar chain excision from glycoconjugates, separation and purification of sugar chains, and labeling of sugar chains. However, these steps are extremely complicated.

  Specifically, as a method for analyzing sugar chains on the cell surface, for example, a step of preparing a solution containing sugar chains, and a solution containing sugar chains are brought into contact with a capture carrier that specifically binds to sugar chains. The step of capturing the sugar chain on the capture carrier, the step of removing substances other than the sugar chain bound to the capture carrier, the step of re-releasing the sugar chain bound to the capture carrier, and the re-released sugar chain Has been proposed (see, for example, Patent Literature 1).

  In such a sugar chain analysis method, for example, in the step of separating and purifying the re-released sugar chain from the capture carrier, a pressure difference between the upper and lower sides of the well is obtained using a multiwell filter plate having a plurality of wells with filters. By separating the re-released glycan contained in the solution and the capture carrier by the filter, a multiwell plate having a plurality of wells is placed below the multiwell filter plate. The re-released sugar chain is purified by selectively supplying a solution containing the re-released sugar chain to the well of the multi-well plate corresponding to the well of the multi-well filter plate.

  In order to improve the purification rate in the step of purifying the re-released sugar chain, the solution containing the re-released sugar chain is changed from the well of the multi-well filter plate to the well of the corresponding multi-well plate. It is required to be delivered with excellent accuracy. However, the transfer of the solution containing the re-released sugar chain between the wells is performed using the pressure difference generated above and below the wells of the multi-well filter plate. Depending on the size, the solution containing sugar chains that have passed through the filter is scattered. As a result, since contamination occurs between adjacent wells of the multi-well plate, there arises a problem that the purification rate of the re-released sugar chain is lowered.

  Such problems are not limited to the purification of sugar chains using multiwell filter plates and multiwell plates, but include size removal, bead / resin / base assay, sample concentration / desalting / purification, etc. The same is true for.

International Publication No. 2009-133696

Ioffe E., Stanley P., Proc. Natl. Acad. Sci., 91, pp. 728-732 (1994)

  An object of the present invention includes a spacer that can process a liquid to be processed by the processing apparatus with high accuracy without waste by providing the processing apparatus with a spacer, and the spacer. It is to provide a processing apparatus.

Such an object is achieved by the present invention described in the following (1) to (7).
(1) a multi-well filter plate comprising a plurality of first wells, a filter housed in the bottom of the first well, and a through-hole penetrating the bottom surface of the first well;
A multi-well plate disposed below the multi-well filter plate and having a plurality of second wells corresponding to the plurality of first wells;
A spacer having a plurality of cylinders corresponding to the plurality of first wells,
When arranged between the multi-well filter plate and the multi-well plate, the lower end of the cylinder is vertically aligned with the upper end of the second well or below the upper end of the second well. The bottom surface of the multi-well filter plate facing the cylinder body is set to be aligned with the upper end of the cylinder body or positioned below the upper end of the cylinder body. A spacer characterized by being set.

  (2) The cylindrical body flows out to the lower side of the first well through the filter and the through hole based on the force difference generated above and below the first well of the multi-well filter plate. The spacer according to (1), wherein the spacer functions as a liquid guiding unit that guides the liquid to the second well of the multi-well plate.

(3) The multi-well filter plate includes a projecting portion projecting downward on the bottom surface where the cylindrical body faces,
The spacer according to (1) or (2), wherein a lower end of the projecting portion coincides with an upper end of the cylindrical body in a vertical direction or is positioned below the upper end of the cylindrical body.

  (4) The spacer according to (3), wherein the protrusion is set to a size that allows the protrusion to be inserted.

  (5) The spacer according to any one of (1) to (4), wherein the cylindrical body is set to a size that can be inserted into the second well.

  (6) The spacer according to any one of (1) to (5), wherein the force difference is a pressure difference.

(7) A multi-well filter plate comprising a plurality of first wells, a filter housed in the bottom of the first well, and a through-hole penetrating the bottom surface of the first well;
A multi-well plate disposed under the multi-well filter plate and having a plurality of second wells corresponding to the plurality of first wells;
A processing apparatus comprising: the multi-well filter plate; and a spacer having a plurality of cylindrical bodies disposed between the multi-well plate and corresponding to the plurality of first wells,
When the spacer is disposed between the multiwell filter plate and the multiwell plate, the lower end of the cylindrical body coincides with the upper end of the second well in the vertical direction, or the second spacer The bottom surface of the multi-well filter plate, which is set to be positioned below the upper end of the well and faces the cylindrical body, coincides with the upper end of the cylindrical body in the vertical direction, or from the upper end of the cylindrical body A processing apparatus which is set to be positioned on the lower side.

  According to the present invention, since the processing apparatus includes the spacer, the liquid to be processed by the processing apparatus can be processed with high accuracy without waste, so that the waste of liquid is accurately prevented or suppressed. be able to. In addition, a plurality of types of liquids (multiple samples) can be processed at the same time.

  Therefore, when this treatment apparatus is used for separation of sugar chains, it is possible to separate sugar chains with high throughput with simple operations and without waste.

It is a disassembled perspective view which shows an example of the refiner | purifier used for the refinement | purification method of sugar_chain | carbohydrate. It is a perspective view which shows an example of the refiner | purifier used for the refinement | purification method of sugar_chain | carbohydrate. It is the sectional view on the AA line of the refiner | purifier shown in FIG. It is a longitudinal cross-sectional view for demonstrating the refinement | purification method of the sugar_chain | carbohydrate which refine | purifies sugar_chain | carbohydrate from glycoprotein. It is a longitudinal cross-sectional view for demonstrating the refinement | purification method of the sugar_chain | carbohydrate which refine | purifies sugar_chain | carbohydrate from glycoprotein. It is a longitudinal cross-sectional view for demonstrating the refinement | purification method of the sugar_chain | carbohydrate which refine | purifies sugar_chain | carbohydrate from glycoprotein. It is a longitudinal cross-sectional view for demonstrating the refinement | purification method of the sugar_chain | carbohydrate which refine | purifies sugar_chain | carbohydrate from glycoprotein.

  Hereinafter, the spacer and the processing apparatus of the present invention will be described in detail based on preferred embodiments.

  In the following, prior to describing the spacer of the present invention, first, an example in which the processing apparatus of the present invention is applied to a purification apparatus used in a method for purifying a sugar chain of a glycoprotein will be described. The spacer of the present invention is used by being attached to the purification apparatus (the processing apparatus of the present invention) described here.

<Purification equipment>
FIG. 1 is an exploded perspective view showing an example of a purification apparatus used in a sugar chain purification method, FIG. 2 is a perspective view showing an example of a purification apparatus used in a sugar chain purification method, and FIG. FIG. 3A shows a case where a multiwell plate is attached to the purification apparatus, and FIG. 3B shows a case where a tray is attached to the purification apparatus. .) For convenience of description, in the following description, the upper side in FIGS. 1 to 3 is referred to as “upper” and the lower side is referred to as “lower”.

  1 to 3 includes a multi-well filter plate 100, a spacer 700, a first support base 400 that supports the multi-well filter plate 100 and the spacer 700, a multi-well plate 200, and a tray. 300 and a second support base 500 that supports the multi-well plate 200 or the tray 300.

  The multi-well filter plate 100 is used by being inserted into a recess 405 provided in the first support base 400. The multi-well filter plate 100 has a flat shape as a whole and is provided in the thickness direction (vertical direction). (In this embodiment, 96) wells (holes) 101 are provided.

  As shown in FIG. 3, each of these wells (first wells) 101 has a structure having a bottom surface 103 on the lower side thereof, and further, a through hole corresponding to substantially the center of the bottom surface 103. 104 is provided. Thereby, the outflow of the liquid supplied (stored) to the well 101 through the through hole 104 to the outside is permitted from the bottom surface 103 side.

  The hole diameter of the through hole 104 is preferably set to about 0.1 mm to 1 mm, more preferably about 0.2 mm to 0.7 mm.

  In addition, the multiwell filter plate 100 includes a plurality of (in this embodiment, 96) protrusions 107 at positions corresponding to the through holes 104 of the bottom surface 103, that is, positions where the cylindrical body 701 included in the spacer 700 faces. . More specifically, in the present embodiment, the protruding portion 107 is provided to protrude so as to surround the opening portion of the through hole 104 opened at the bottom surface 103 as shown in FIG.

  The protruding portion 107 has an outer diameter smaller than an inner diameter of a cylindrical body 701 provided in a spacer 700 described later. That is, the protruding portion 107 is set to a size that can be inserted into the cylindrical body 701. As a result, when the spacer 700 is disposed between the multiwell filter plate 100 and the multiwell plate 200, the protruding portion 107 is formed in the cylindrical body 701 in the thickness direction (vertical direction) of the multiwell filter plate 100. The upper opening (upper end) 702 can be positioned below the upper opening 702.

  Furthermore, a filter 105 is disposed below the well 101 (on the bottom surface 103 side) so as to close the through hole 104. Due to the relationship between the filter 105 and the hole diameter of the through hole 104, the liquid supplied to the well 101 is prevented from flowing out to the outside through the through hole 104 when the multiwell filter plate 100 is left standing.

  On the other hand, as will be described later, when the multiwell filter plate 100 is attached to the purification apparatus 1 and the inside of the concave portion 505 of the second support base 500 is sucked using a suction pump, the liquid Permeation of the filter 105 is allowed. Therefore, the liquid flows out from the bottom surface 103 side to the outside through the through hole 104.

  Although it does not specifically limit as a raw material of the filter 105, For example, a porous film, a nonwoven fabric, etc. are mentioned. In addition, examples of the constituent material include polytetrafluoroethylene, cellulose ester, vinylidene fluoride, polycarbonate, polyethylene, polypropylene, nylon, and the like, and one or more of these can be used in combination.

  The filter 105 is preferably subjected to a hydrophilic treatment. Thereby, the permeability | transmittance of the said liquid can be aimed at.

  This hydrophilic treatment can be performed, for example, by applying (coating) a surfactant, water-soluble silicon, polypropylene glycol, or the like.

  The pore diameter of the pores provided in the filter 105 is preferably set to about 0.1 to 50 μm, more preferably about 0.2 to 20 μm, and further preferably 0.4 to 10 μm. Thereby, the permeation of the liquid component contained in the liquid is allowed and the permeation of the polymer particles 20 as will be described later is definitely not allowed.

  In addition, although it does not specifically limit as a constituent material of each part except the filter 105 of the multiwell filter plate 100, For example, a polypropylene, polyethylene, a polystyrene, a polyvinyl chloride, a polytetrafluoroethylene etc. are mentioned, Of these, 1 Species or a combination of two or more can be used.

  The spacer 700 is used by being inserted into the recess 405 provided in the first support base 400, and has a flat plate-like support body 705 and a plurality of (this embodiment) provided in the thickness direction (vertical direction). 96 cylinders 701 in the form.

  The support body 705 has a flat plate shape as a whole, and includes a plurality of (96 in this embodiment) through-holes 706 that penetrate in the thickness direction (vertical direction).

  The cylindrical body 701 has a shape corresponding to the shape of the well 201. In this embodiment, the cylindrical body 701 has a cylindrical shape, and the upper opening 702 is disposed so as to correspond to the through hole 706. Thereby, the cylindrical body 701 protrudes from the lower surface side of the support body 705 toward the thickness direction, and opens at the upper opening 702 and the lower opening 703.

  Each well 101 included in the multiwell filter plate 100 and each well 201 included in the multiwell plate 200 described later correspond to the vertical direction when the plates 100 and 200 are overlapped in the vertical direction. Furthermore, when the spacer 700 is inserted between the plates 100 and 200 stacked in the vertical direction, the cylindrical bodies 701 correspond to the wells 101 and 201, respectively. , 201 are set so as to be located between each other. Therefore, as will be described in detail later, the liquid that has flowed out from the bottom surface 103 side through the through-hole 104 provided in each well 101 is directed to each well 201 that is arranged to correspond to each well 101. Then, it is selectively supplied via the cylindrical body 701.

  Further, the cylindrical body 701 has an outer diameter that is smaller than the size of the well 201 included in the multi-well plate 200. That is, the cylindrical body 701 is set to a size that can be inserted into the well 201. As a result, when the spacer 700 is disposed between the multiwell filter plate 100 and the multiwell plate 200, the cylindrical body 701 is placed in the thickness direction (vertical direction) of the multiwell plate 200 and the upper end of the well 201. It becomes possible to position it below.

  In addition, as a constituent material of the spacer 700, the thing similar to what was mentioned as a constituent material of each part except the filter 105 of the multiwell filter plate 100 is mentioned, for example.

  The first support base 400 is a member for supporting the multiwell filter plate 100 and the spacer 700, has a flat plate shape, and includes a bottom portion 401 having an opening 402 that opens at the center thereof, and an outer edge of the bottom portion 401. And an upper outer wall 403 provided so as to protrude upward along the lower edge 401 and a lower outer wall 404 provided so as to protrude downward along the outer edge of the bottom portion 401.

  A recess 405 is formed by being surrounded by the upper outer wall 403, and the multiwell filter plate 100 and the spacer 700 are placed in the recess 405 so that the multiwell filter plate 100 is on the upper side and the spacer 700 is on the lower side. The multiwell filter plate 100 and the spacer 700 are supported by the first support base 400 by being inserted in a state where they are overlapped with each other.

  Further, a groove is provided along the edge of the opening 402 of the bottom 401, and a packing (seal member) 407 is disposed so as to correspond to the groove. Accordingly, when the multiwell filter plate 100 and the spacer 700 are inserted into the recess 405, communication between the opening 402 and the outside of the purification apparatus 1 is not allowed.

  In addition, depending on the usage method (first usage method and second usage method) of the purification apparatus 1 to be described later, when both the multiwell filter plate 100 and the spacer 700 are inserted into the recess 405 (the first usage method). There is a case where the multiwell filter plate 100 is inserted alone (second usage method) in addition to the method of use), but this case is also the same as when both the multiwell filter plate 100 and the spacer 700 are inserted. In addition, communication between the opening 402 and the outside of the purification apparatus 1 is not allowed.

  Further, in the present embodiment, as shown in FIG. 3A, the support 705 is fixed in the recess 405 by the edge of the support 705 being locked by the edge of the opening 402 of the bottom 401. . Thereby, since the positional deviation of the spacer 700 in the recessed part 405 is reliably prevented, the cylindrical body 701 can be reliably arranged at the position as described above.

  Further, the upper outer wall 403 has a configuration having an opening 408 in a direction perpendicular to the thickness direction (vertical direction) at the center portion of the two opposing long sides, thereby being inserted into the recess 405 ( The mounted multiwell filter plate 100 can be easily taken in and out.

  Furthermore, a recess 406 is formed by being surrounded by the lower outer wall 404, and a second support base 500, which will be described later, is inserted along the inner peripheral surface of the recess 406 to thereby insert the second protrusion 502. The first support base 400 is fixed on the support base 500.

  In the present embodiment, the outer peripheral surface of the upper outer wall 403 and the outer peripheral surface of the lower outer wall 404 are integrally formed to form a single flat surface.

  In addition, although it does not specifically limit as a constituent material of the 1st support stand 400, For example, resin materials, such as a polycarbonate, an acrylic resin, an acrylonitrile butadiene styrene copolymer (ABS resin), Fe, such as stainless steel Metal alloys such as Al alloys, Cu or Cu alloys, Al or Al alloys, ceramic materials such as alumina, apatite, aluminum nitride, etc., and one or more of these may be combined Can be used.

  The multiwell plate 200 is used by being inserted into a recess 505 provided in the second support base 500.

  Similar to the multiwell filter plate 100, the multiwell plate 200 has an overall shape of a flat plate, and a plurality of (in this embodiment, 96) wells (96 in this embodiment) are provided in the thickness direction (vertical direction). Hole part) 201 is provided.

  These wells (second wells) 201 are provided with a bottom surface 203 on the lower side thereof, and do not penetrate on the bottom surface 203 side, whereby the liquid supplied (stored) to the well 101 is contained therein. It is stored in.

  In addition, each well 101 with which the multiwell filter plate 100 is equipped, and the well 201 with which the multiwell plate 200 is provided are each arrange | positioned corresponding to an up-down direction, when each plate 100,200 is piled up in the up-down direction. Is set to Therefore, as will be described in detail later, the liquid that has flowed out from the bottom surface 103 side through the through-hole 104 provided in each well 101 is directed to each well 201 that is arranged to correspond to each well 101. , Will be selectively supplied.

  The multiwell plate 200 includes a well forming portion 202 in which a plurality of wells 201 are formed, and a frame portion 204 that is disposed so as to surround the periphery of the well forming portion 202. The thickness of the portion 202 is thicker than the thickness of the frame portion 204. As a result, the upper surface of the well forming portion 202 protrudes from the upper surface of the frame portion 204. The shape of the well forming portion 202 in plan view is set to a size that can be inserted into the opening 402 of the first support base 400. Accordingly, when the first support base 400 is placed on the second support base 500 in a state where the multiwell plate 200 is disposed in the recess 505 of the second support base 500, as shown in FIG. In addition, the well forming part 202 can be inserted into the opening 402.

  As shown in FIG. 3A, the lower opening (lower end) 703 of the cylindrical body 701 is formed in the thickness direction (vertical direction) of the multiwell plate 200 more than the upper end of the well 201 of the multiwell plate 200. As long as it can be positioned on the lower side, the thickness of the well forming portion 202 can be reduced, and further, the upper surface of the well forming portion 202 does not protrude from the upper surface of the frame portion 204 and the well forming portion 202 The thickness and the thickness of the frame portion 204 may be the same.

  In addition, examples of the constituent material of the multiwell plate 200 include the same materials as those described as the constituent materials of each part except the filter 105 of the multiwell filter plate 100.

  Similar to the multiwell plate 200, the tray 300 is used by being inserted into a recess 505 provided in the second support base 500. That is, the tray 300 or the multiwell plate 200 is inserted into the recess 505 according to the type of treatment performed by the sugar chain purification method using the purification apparatus 1.

  The tray 300 has the same configuration except that the concave portion 301 is provided at a position corresponding to the above-described multi-well plate 200 and the well forming portion 202 in which the plurality of wells 201 are formed. .

  By configuring the tray 300 to include such a recess 301, as shown in FIG. 3 (b), the second support is provided with the tray 300 disposed in the recess 505 of the second support base 500. When the first support base 400 is placed on the base 500, the liquid that has flowed out from each well 101 of the multiwell filter plate 100 through the through hole 104 is collectively stored in the recess 301 of the tray 300. Will be able to.

  As shown in FIG. 1 and the like, the tray 300 is not limited to a configuration having a convex portion protruding at a position corresponding to the frame portion 204 of the multiwell plate 200, and a concave portion 301 is formed on the center side thereof. As long as the formation of the convex portion is omitted, it may be omitted.

  In addition, examples of the constituent material of the tray 300 include the same materials as those described as the constituent materials of each part excluding the filter 105 of the multiwell filter plate 100.

  Prior to the tray 300 or the multiwell plate 200, the adjustment plate 600 is used by being inserted into the recess 505 provided in the second support base 500.

  That is, the adjustment plate 600 is used by being inserted into the recess 505 in a state where the tray 300 or the multiwell plate 200 and the adjustment plate 600 are overlapped so that the adjustment plate 600 is on the lower side. .

  The adjustment plate 600 has a flat plate shape, and a plurality of plates having different thicknesses are prepared.

  Then, by selecting an appropriate thickness according to the type of the multiwell plate 200 to be inserted into the recess 505, the upper surface of the multiwell plate 200 (well forming portion 202), the multiwell filter plate 100 The separation distance from the bottom surface 103 can be set to a desired size.

  In addition, as a constituent material of the adjustment plate 600, the thing similar to what was mentioned as a constituent material of each part except the filter 105 of the multiwell filter plate 100 is mentioned, for example.

  The second support base 500 is a member for supporting the multi-well plate 200 or the tray 300, and has a flat bottom 501 and an outer wall 503 provided so as to protrude upward along the outer edge of the bottom 501. And have.

  By being surrounded by such an outer wall 503, a recess 505 is formed inside thereof, and the multiwell plate 200 or tray 300 is inserted into the recess 505, so that the multiwell plate 200 or tray 300 is second supported. Supported by a base 500.

  The outer wall 503 has a protruding portion 502 that protrudes upward along the inner edge of the outer wall 503. The projecting portion 502 is set so as to be inserted along the inner peripheral surface of the recess 406 provided in the first support base 400, whereby the first support base is placed on the second support base 500. When the 400 is placed, the protrusion 502 is inserted into the recess 406 of the first support base 400, so that the first support base 400 is fixed on the second support base 500.

  A groove is provided on the upper surface of the outer wall 503 so as to surround the protruding portion 502, and a packing (seal member) 507 is disposed so as to correspond to the groove. Accordingly, when the first support base 400 is placed on the second support base 500, communication between the support bases 400 and 500 is not allowed.

  Furthermore, the outer wall 503 has a through hole 506 that penetrates the recess 505 on the side surface of the outer wall 503. A suction pump (not shown) can be connected to the through hole 506, and the inside of the recess 505 can be decompressed by operating the suction pump.

  In addition, as a constituent material of the 2nd support stand 500, the thing similar to what was mentioned as a constituent material of the 1st support stand 400 is mentioned, for example.

  As shown in FIG. 3 (a), the purification apparatus 1 composed of each member as described above inserts the multiwell filter plate 100 and the spacer 700 into the recess 405 of the first support base 400, A first use method in which the multiwell plate 200 is inserted into the recess 505 of the second support base 500 and a multiwell filter plate in the recess 405 of the first support base 400 as shown in FIG. 100, and the second usage method is used by inserting the tray 300 into the concave portion 505 of the second support base 500, and these usage methods will be described below.

  First, in the first usage method, the multiwell filter plate 100 and the spacer 700 are inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the multiwell plate 200 are inserted into the recess 505 of the second support base 500. In this state, the first support base 400 is placed on the second support base 500 (see FIGS. 2 and 3A).

  As a result, the multiwell filter plate 100 and the spacer 700 are inserted into the recess 405, the multiwell plate 200 is inserted into the recess 505, and the well forming part 202 of the multiwell plate 200 is opened to the first support base 400. Further, the protrusion 502 of the second support base 500 is inserted into the recess 406 of the first support base 400. As a result, the first support base 400 is fixed on the second support base 500 in a state where the wells 201 of the multiwell plate 200 are arranged corresponding to the wells 101 of the multiwell filter plate 100. The

  In such a state, the packing 507 does not allow communication between the support bases 400 and 500, and the packing 407 allows the first support base 400 and the multiwell filter plate 100 to communicate with each other. Therefore, the internal space formed by the multiwell filter plate 100, the first support base 400, and the second support base 500 is a closed space that is closed to the outside. be able to.

  Therefore, by operating the suction pump connected to the through hole 506 and depressurizing the concave portion 505, the closed space can be made negative with respect to the outside. Therefore, a pressure difference is generated between the upper and lower sides of the well 101 of the multi-well filter plate 100 via the filter 105, so that the liquid in the well 101 passes through the filter 105 based on the pressure difference. Therefore, the liquid can flow out from each well 101 through the through hole 104, and as a result, the liquid is supplied into the well 201 of the multi-well plate 200 corresponding to the well 101.

  Furthermore, in this first method of use, a spacer 700 is inserted (arranged) between the plates 100 and 200 stacked in the vertical direction, whereby each cylindrical body 701 is located between the wells 101 and 201. Placed in.

  At this time, in this embodiment, as shown in FIG. 3A, the lower opening (lower end) 703 of the cylindrical body 701 is in the thickness direction (vertical direction) of the multiwell plate 200, and The protrusion 107 provided on the bottom surface 103 of the multiwell filter plate 100 that is located below the upper end of the well 201 and that the cylindrical body 701 faces is in the thickness direction (vertical direction) of the multiwell filter plate 100. The cylindrical body 701 is positioned below the upper opening (upper end) 702.

  Therefore, when the liquid is supplied from the well 101 of the multiwell filter plate 100 to the well 201 of the multiwell plate 200 based on the pressure difference generated above and below the well 101 of the multiwell filter plate 100, the through hole 104 is formed. Thus, it is possible to reliably suppress or prevent the liquid that has flowed out through the region from being scattered in the region other than the well 201. That is, the spacer 700 functions as a liquid guiding unit that guides liquid from the well 101 to the well 201. Therefore, the liquid can be supplied into the well 201 corresponding to the well 101 with high efficiency.

  As described above, the outer diameter of the cylindrical body 701 is smaller than the size of the well 201 included in the multi-well plate 200 and is set to a size that can be inserted into the well 201. As a result, when the spacer 700 is disposed between the multiwell filter plate 100 and the multiwell plate 200, the cylindrical body 701 is placed in the thickness direction (vertical direction) of the multiwell plate 200 and the upper end of the well 201. In addition, a space is formed between the outer peripheral surface of the cylindrical body 701 and the inner peripheral surface of the well 201. Since such a space is formed, when the concave portion 505 is depressurized to make the closed space negative with respect to the outside, the upper and lower sides of the well 101 of the multiwell filter plate 100 through the filter 105 are moved up and down. The pressure difference will surely occur.

  In the present embodiment, the lower opening 703 of the cylindrical body 701 is positioned below the upper end of the well 201 in the thickness direction (vertical direction) of the multi-well plate 200. However, the present invention is limited to this case. Instead, the lower opening 703 of the cylindrical body 701 may coincide with the upper end of the well 201 in the thickness direction. That is, the lower opening 703 of the cylindrical body 701 only needs to coincide with the upper end of the well 201 or be positioned below the upper end of the well 201 in the thickness direction.

  Further, the protruding portion 107 provided on the bottom surface 103 is not positioned below the upper opening (upper end) 702 of the cylindrical body 701 in the thickness direction (vertical direction) of the multi-well filter plate 100, but in the thickness direction. Thus, it may coincide with the upper opening (upper end) 702 of the cylindrical body 701. Further, when the bottom surface 103 is configured as a flat surface without the protrusion 107, the bottom surface 103 only needs to coincide with the upper opening (upper end) 702 of the cylindrical body 701 in the thickness direction. In other words, the bottom surface 103 of the multi-well filter plate 100 only needs to coincide with the upper opening 702 of the cylinder 701 or be positioned below the upper opening 702 of the cylinder 701 in the thickness direction.

  By setting the position of the spacer 700 with respect to the plates 100 and 200 as described above, the spacer 700 can function as a liquid guiding unit that guides the liquid from the well 101 to the well 201, and thus corresponds to the well 101. The liquid can be supplied into the well 201 with high efficiency.

  Next, in the second usage method, the multiwell filter plate 100 is inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the tray 300 are inserted into the recess 505 of the second support base 500, In this state, the first support base 400 is placed on the second support base 500 (see FIGS. 2 and 3B).

  In the second usage method, the multiwell filter plate 100 is inserted alone into the recess 405 of the first support base 400, and the adjustment plate 600 and the tray 300 are inserted into the recess 505 of the second support base 500. Except for this, it is the same as the first usage method.

  Also in such a second usage method, the internal space formed by the multiwell filter plate 100, the first support base 400, and the second support base 500 is closed with respect to the outside. It can be.

  Therefore, as in the first method of use, the suction pump connected to the through hole 506 is operated to decompress the concave portion 505, so that the liquid can flow out from each well 101 through the through hole 104. . As a result, the liquid flowing out from each well 101 can be stored together in the recess 301 of the tray 300 located below the multiwell filter plate 100.

  In this second method of use, the liquid flowing out from each well 101 may be stored in the recess 301 in a lump, so that the distance between the opening of the recess 301 and the through hole 104 is more than necessary. Since it is not necessary to make it small, the insertion of the adjustment plate 600 into the recess 505 of the second support base 500 may be omitted.

  In the present embodiment, the purification apparatus 1 is configured such that liquid is allowed to pass through the filter 105 by setting the lower side of the filter 105 included in each well 101 of the multiwell filter plate 100 to a negative pressure from the upper side. The purification apparatus is not limited to such a configuration, and may have a configuration in which the liquid is allowed to pass through the filter 105 by setting the upper side of the filter 105 to a positive pressure from the lower side.

  Using the purification apparatus (manifold) 1 as described above, for example, sugar chains are purified from glycoproteins having sugar chains as follows.

<Purification method>
4 to 7 are longitudinal sectional views for explaining a method for purifying a sugar chain for purifying a sugar chain from a glycoprotein. In the following description, the upper side in FIGS. 4 to 7 is referred to as “upper” and the lower side is referred to as “lower”.

  In the following purification method for purifying a sugar chain, a case where a sugar chain is purified from a glycoprotein in a gel separated by electrophoresis will be described.

  In the sugar chain purification method shown below, [1] a step of obtaining a gel retaining glycoprotein, [2] a step of releasing the sugar chain by treating the gel with a sugar chain releasing means, [3] A step of eluting the released sugar chain from the gel to obtain a solution containing the sugar chain; and [4] contacting the solution containing the sugar chain with a capture carrier that specifically binds to the sugar chain, A step of capturing a sugar chain, [5] a step of removing substances other than the sugar chain that did not bind to the capture carrier, [6] a step of deactivating functional groups of the capture carrier, [7] The method includes the step of re-releasing the sugar chain bound to the capture carrier, labeling this sugar chain with another compound, and [8] purifying the labeled sugar chain.

  The steps [1] to [8] can be performed as a series of flows, but the operations can be stopped and stored in the respective steps. Therefore, only step [8] may be performed separately. Further, depending on the purity of the sugar chain used, it is possible to omit the first sugar chain purification step and perform only the operations after step [4].

Hereinafter, each process is explained in full detail.
[1] First, a sample containing a glycoprotein is prepared, and a predetermined treatment is performed on the sample to obtain a gel in which the glycoprotein is retained.

  The sample containing the prepared glycoprotein is not particularly limited, and examples thereof include biological samples such as whole blood, serum, plasma, urine, saliva, cells, and tissues. In addition, glycoproteins that have been purified in advance using a predetermined method or that have not been purified can also be used.

  This sample may be pretreated by a method of degreasing, desalting, or protein fractionation sugar.

  Next, the sample is subjected to a predetermined treatment such as SDS-PAGE (SDS-denaturing polyacrylamide gel electrophoresis) or two-dimensional electrophoresis in which isoelectric focusing and SDS-PAGE are combined, so that the sample in the gel Isolate glycoproteins contained in

  Next, the gel after electrophoresis is stained with a staining agent such as Coomassie Brilliant Blue, for example. Thereby, a gel in which the stained glycoprotein is retained can be obtained.

  [2] Next, a band is confirmed in the gel after electrophoresis provided with the stained glycoprotein, and this band is cut out using a blade or the like. Then, this is cut into pieces with a blade or the like, and the stain is washed and removed, and then the sugar chain is released from the glycoprotein held in the gel using a sugar chain releasing means.

  The means for releasing the sugar chain is not particularly limited, and for example, methods such as glycosidase treatment using N-glycosidase or O-glycosidase, hydrazine decomposition, and β elimination by alkali treatment can be used.

  When N-type sugar chains are analyzed, a method using N-glycosidase is preferable. Prior to the N-glycosidase treatment, protease treatment may be performed using trypsin, chymotrypsin, or the like.

  [3] Next, the gel after the sugar chain releasing treatment is taken out, and the gel is rinsed with a washing solution to elute the released sugar chain from the gel into the washing solution. And after precipitating a gel in a washing | cleaning liquid, the solution containing a sugar chain is obtained by collect | recovering this supernatant.

  Although it does not specifically limit as a washing | cleaning liquid, For example, water, various buffer solutions, various organic solvents, etc. are mentioned, Among these, it can use 1 type or in combination of 2 or more types.

  In addition, you may make it increase the content rate of the sugar chain in a solution by concentrating this solution as needed.

  In the steps [1] to [3], the separation of the glycoprotein from the glycoprotein-containing sample and the release of the sugar chain from the glycoprotein can be carried out by subjecting the However, the present invention is not limited to this case, and the sugar chain may be released from the purified glycoprotein by subjecting the sample to affinity chromatography or ion exchange chromatography. Good. Furthermore, when purification of the glycoprotein from the sample may be omitted, a treatment for releasing the sugar chain directly from the glycoprotein contained in the sample may be performed.

  [4] Next, a solution containing a sugar chain is brought into contact with a capture carrier that specifically binds to the sugar chain, and the sugar chain is captured on the capture carrier.

  Here, the sugar chain is the only substance having an aldehyde group among in-vivo substances. That is, a sugar chain is a substance in which a cyclic hemiacetal type and an acyclic aldehyde type exist in an equilibrium state in an aqueous solution or the like.

  In contrast, in vivo substances other than sugar chains such as proteins, nucleic acids and lipids usually do not contain aldehyde groups.

  From this, it is possible to selectively capture only sugar chains by using a capture carrier having a functional group that reacts specifically with an aldehyde group to form a stable bond.

  As the functional group that specifically reacts with the aldehyde group, for example, a hydrazide group, an oxylamino group, an amino group, a semithiocarbazide group, and derivatives thereof are preferable, and a hydrazide group or an oxylamino group is more preferably used. The oxime bond generated by the reaction between the oxylamino group and the aldehyde group, and the hydrazone bond generated by the reaction between the hydrazide group and the aldehyde group are easily cleaved by acid treatment, etc. It can be easily separated from the carrier.

  In general, amino groups are often used to capture and support physiologically active substances, but bonds (Schiff bases) produced by the reaction of amino groups and aldehyde groups are weak in binding force, so reducing agents are used. Amino groups are not preferred for capturing sugar chains.

  As a carrier for capturing sugar chains, it is preferable to use polymer particles. Further, the polymer particles are preferably solid or gel particles having a functional group that specifically reacts with an aldehyde group of a sugar chain on at least a part of the surface.

  If the polymer particles are solid particles or gel particles, the particles can be easily recovered using the well 101 provided in the multiwell filter plate 100 after the sugar chains are captured by the polymer particles.

  Examples of such polymer particles include those represented by the following general formula (1).

  Hereinafter, a method of capturing sugar chains on the polymer particles 20 using the polymer particles 20 as a capture carrier will be described.

  [4-1] First, the polymer particles 20 are dispersed in pure water 21 to obtain a particle dispersion 22 in which the polymer particles are dispersed.

  The shape of the polymer particle 20 is not particularly limited, but for example, a spherical shape or a similar shape is preferable.

  When the polymer particles 20 are spherical, the average particle size is preferably about 0.05 to 1000 μm, more preferably about 0.05 to 200 μm, still more preferably about 0.1 to 200 μm, and most preferably 0.1 to 100 μm. Set to degree. If the average particle size is less than the lower limit, it may be difficult to collect the polymer particles 20 by centrifugation or filtration. On the other hand, when the average particle diameter exceeds the upper limit, the contact area between the polymer particles 20 and a sample solution described later decreases, and the sugar chain capture efficiency may be reduced.

  [4-2] Next, as shown in FIG. 4A, the multiwell filter plate 100 is inserted into the recess 405 of the first support base 400, and the adjustment plate is further inserted into the recess 505 of the second support base 500. 600 and the tray 300 are inserted, and the first support base 400 is placed on the second support base 500 in this state. That is, in this step, the second usage method of the purification apparatus 1 is applied.

  In this state, after supplying the particle dispersion liquid 22 prepared in advance in the step [4-1] into the well 101 provided in the multiwell filter plate 100, the suction pump of the purification apparatus 1 is operated to The internal space formed by the well filter plate 100, the first support base 400, and the second support base 500 is set to a negative pressure, thereby sucking the particle dispersion 22 in the well 101 (FIG. 4 ( See b).

  As a result, the pure water 21 in the particle dispersion 22 passes through the filter 105. On the other hand, due to the relationship between the pore size of the filter 105 and the particle size of the polymer particle 20, the polymer particle 20 cannot pass through the filter 105. Therefore, the pure water 21 that has passed through the filter 105 is selectively supplied into the tray 300 located below the multiwell filter plate 100 through the through hole 104.

  As a result, as shown in FIG. 4C, the polymer particles 20 are individually filled in the wells 101 of the multiwell filter plate 100.

  [4-3] Next, as shown in FIG. 4D, the solution containing the sugar chain obtained in the step [3] (hereinafter referred to as “sugar chain-containing solution 23”) is polymer particles. 20 is supplied into the well 101 of the multiwell filter plate 100 filled with 20.

  [4-4] Next, by heating the sugar chain-containing liquid 23, the sugar chain-containing liquid 23 is kept in a certain temperature range until the supplied sugar chain-containing liquid 23 is dried (see FIG. 4E). .)

  As a result, the polymer particles 20 react with the sugar chains contained in the sugar chain-containing liquid 23, and the sugar chains are captured on the polymer particles 20.

  The pH of the reaction system at this time is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6. In addition, pH adjustment can be performed by adding various buffer solution or an organic solvent after the said process [4-3], for example.

  The temperature at the time of sugar chain capture is preferably kept at a temperature range of about 4 to 100 ° C., more preferably about 20 to 90 ° C., further preferably about 30 to 80 ° C., and most preferably about 40 to 80 ° C. Set to.

  In addition, the reaction time, that is, the time until the solution is dried, is usually set to about 0.1 to 3 hours, preferably about 0.6 to 2 hours, when set in such a temperature range.

  By reacting the polymer particle 20 and the sugar chain under such conditions, the sugar chain is surely captured on the polymer particle 20.

  In addition, by setting it as the structure heated until the sugar chain containing liquid 23 dries like this embodiment, the improvement of the reaction rate of the polymer particle 20 and a sugar chain can be aimed at reliably.

  The method for heating the sugar chain-containing liquid 23 is not particularly limited. For example, the entire purification apparatus 1 may be heated, or the multiwell filter plate 100 may be selectively heated. Also good.

  Through the steps [4-1] to [4-4] as described above, sugar chains are captured on the polymer particles 20.

  In addition, when the polymer particle 20 has a hydrazide group, the reaction represented by the following formula (2) proceeds between the hydrazide group and the reducing end of the sugar chain. The sugar chain is captured.

  As described above, by using the purification apparatus 1 including the multi-well filter plate 100, the reaction of the supplied liquid can be advanced in the well 101 when the multi-well filter plate 100 is allowed to stand. Furthermore, the fixed component contained in the liquid supplied to the well 101 and the liquid component can be easily separated by suction of the multiwell filter plate 100 using the purification apparatus 1.

[5] Next, substances other than the sugar chain captured by the capture carrier are removed.
Here, when the polymer particle 20 is used as the capture carrier as described above, examples of the substance other than the sugar chain captured by the polymer particle 20 include, for example, a polymer particle other than the sugar chain not captured by the polymer particle 20. The surface of 20 includes impurities (protein, lipid, etc.) other than sugar chains adsorbed nonspecifically.
Therefore, in this step [5], these substances are removed by washing.

  Hereinafter, in the same manner as in the above step [4], a substance other than the sugar chain captured by the polymer particles 20 using the well 101 provided in the multiwell filter plate 100 (hereinafter, this substance may be referred to as “cleaning substance”). .) Will be described.

  [5-1] First, as shown in FIG. 5 (a), the cleaning liquid 24 is applied to the polymer particles 20 obtained in the step [4-4] and dried in the well 101 of the multiwell filter plate 100. Add.

  Although it does not specifically limit as the washing | cleaning liquid 24, Water, various buffer solutions, various organic solvents, etc. are mentioned, These can be used in combination as appropriate.

  [5-2] Next, the suction pump of the purification apparatus 1 is applied while the purification apparatus 1 is maintained in the state described in the step [4-2], that is, the second usage method of the purification apparatus 1 is applied. , The internal space formed by the multi-well filter plate 100, the first support base 400, and the second support base 500 is set to a negative pressure, thereby sucking the cleaning liquid 24 in the well 101. (See FIG. 5 (b)).

  As a result, by passing the cleaning liquid 24 through the filter 105, the polymer particles 20 remain selectively on the multiwell filter plate 100 as shown in FIG. The cleaning liquid 24 can be removed to the tray 300, and the cleaning substance dissolved in the cleaning liquid 24 can be removed.

  The cleaning composed of these steps [5-1] and [5-2] is performed a plurality of times (repeatedly) so that the cleaning substance is dissolved in the cleaning liquid 24 and separated into the tray 300. Therefore, the cleaning substance can be reliably removed from the polymer particles 20 in which the sugar chains are captured.

  That is, in the step [5-1] and the step [5-2], a cleaning solution that removes substances other than sugar chains bound to the polymer particles 20 using the multiwell filter plate 100 having a plurality of wells 101 including the filter 105. 24 is supplied to the polymer particles 20 in the well 101, and then a pressure difference is generated between the upper and lower sides of the well 101 by setting the lower side of the filter 105 to a negative pressure. As a result, the filter 105 causes the cleaning liquid 24 and the polymer particles 20 to Will be separated.

  In this case, first, the polymer particles 20 are sufficiently washed using water or a buffer solution as the washing solution 24, and then further washed with an organic solvent washing solution 24. If necessary, washing with these washing solutions 24 is repeated, and finally It is preferable to wash with an organic solvent cleaning solution 24. Thereby, it becomes possible to more reliably remove the cleaning substance, in particular, impurities adsorbed nonspecifically on the surface of the polymer particle 20.

[6] Next, the functional group included in the capture carrier is deactivated.
In this step, the functional group (functional group that reacts specifically with the aldehyde group of the sugar chain) provided in the capture carrier, which was not used for capturing the sugar chain in the step [4], is deactivated.

  Thereby, in the next step [7], when the sugar chain bound to the capture carrier (polymer particle 20) is re-released, the sugar chain is reliably prevented from being captured again by the capture carrier. Can do.

  The deactivation of the functional group provided in the capture carrier can be performed, for example, by bringing a deactivation liquid having a function of deactivating the functional group into contact with the capture carrier.

  Further, the contact of the deactivation liquid with the capture carrier uses the deactivation liquid in place of the washing liquid 24 in the washing constituted by the steps [5-1] and [5-2], and [5-1] and the step [5-2] may be performed.

  Furthermore, the deactivation liquid is not particularly limited, but when the functional group is a hydrazide group, examples thereof include acid anhydrides such as acetic anhydride and succinic anhydride. Thereby, a functional group can be deactivated easily.

  [7] Next, the sugar chain bound to the capture carrier is re-released, and this sugar chain is replaced with another compound (hereinafter sometimes referred to as “compound A”).

  As this compound A, a labeling reagent including a labeling substance such as a fluorescent substance, a light absorbing substance and a radioactive substance is preferably used.

  Hereinafter, as in the steps [4] and [5], the case where the sugar chain is labeled with the compound A using the well 101 provided in the multiwell filter plate 100 will be described.

  [7-1] First, as shown in FIG. 5 (d), the compound-containing solution 25 containing the compound A is placed in the well 101 of the multiwell filter plate 100 filled with the polymer particles 20 in which the sugar chains are captured. Add to.

  The amount of Compound A added to the well 101 of the multi-well filter plate 100 is preferably excessive with respect to the polymer particles 20 in which the sugar chains are captured. Thereby, the improvement of the substitution rate of the compound A with respect to the sugar_chain | carbohydrate in the following process [7-2] can be aimed at.

  Specifically, the amount of compound A added is preferably 1.5 times or more, more preferably 3 times or more, more preferably 3 times or more the amount of the functional group specifically reacting with the sugar chain of the polymer particle 20. The amount is preferably 5 times or more, and most preferably 10 times or more.

  [7-2] Next, by heating the compound-containing liquid 25, the compound-containing liquid 25 is kept in a certain temperature range until the added compound-containing liquid 25 is dried (see FIG. 5 (e)).

  As a result, the captured sugar chain is separated from the polymer particle 20, and compound A is added to the sugar chain almost simultaneously with it, so that the sugar chain is labeled with compound A.

  The pH of the reaction system at this time is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6. In addition, pH adjustment can be performed by adding various buffer solutions after the said process [7-1], for example.

  The temperature at the time of labeling is preferably kept in a temperature range of about 4 to 100 ° C, more preferably about 20 to 90 ° C, more preferably about 30 to 80 ° C, and most preferably about 40 to 80 ° C. Set.

In addition, the reaction time, that is, the time until the solution is dried, is usually set to about 0.1 to 3 hours, preferably about 0.6 to 2 hours, when set in such a temperature range.
By performing labeling under such conditions, the sugar chain is surely labeled with Compound A.

  In addition, by setting it as the structure heated until the compound containing liquid 25 dries like this embodiment, the improvement of the reaction rate of a sugar_chain | carbohydrate and the compound A can be aimed at reliably.

  The sugar chain is labeled with compound A through the steps [7-1] and [7-2] as described above.

  As the compound A, a compound having an aminooxy group or a hydrazide group is preferably used, and particularly when the polymer particle 20 has a hydrazide group, N-aminooxyacetyl-tryptophanyl represented by the following chemical formula (3) (Arginine methyl ester) is preferably used.

  Moreover, as this compound A, the fluorescent substance comprised with an aromatic amine other than the said compound can be used.

  When such an aromatic amine is used as the compound A, the captured sugar chain is first separated from the polymer particle 20 and re-released, and then labeled with the compound A.

  Hereinafter, a case where an aromatic amine is used as compound A and a sugar chain is labeled with compound A will be described.

  [7-1 '] First, a sugar chain releasing solution for re-releasing sugar chains is added into the well 101 of the multiwell filter plate 100 filled with the polymer particles 20 in which the sugar chains have been captured.

  [7-2 '] Next, by heating the sugar chain free solution, the sugar chain free solution is kept in a certain temperature range until the added sugar chain free solution is dried.

  As a result, the captured sugar chain is separated from the polymer particle 20, whereby the sugar chain is liberated again.

  The pH of the reaction system at this time is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6. This pH adjustment is performed by adding the sugar chain free solution in the above step [7-1 '], and various buffer solutions are used as the sugar chain free solution.

  The temperature at the time of sugar chain release is preferably kept at a temperature range of about 4 to 100 ° C, more preferably about 25 to 90 ° C, more preferably about 30 to 80 ° C, and most preferably about 60 to 80 ° C. Set to.

  In addition, the reaction time, that is, the time until the solution (sugar chain free liquid) is dried is usually about 0.1 to 3 hours, preferably about 0.6 to 2 hours, when set in such a temperature range. Is set.

  By re-releasing the sugar chain under such conditions, the sugar chain is surely separated from the polymer particle 20.

  In addition, by setting it as the structure heated until a sugar chain free liquid dries like this embodiment, the improvement of the release rate of sugar chains can be aimed at reliably.

  [7-3 '] Next, the compound-containing liquid 25 containing an aromatic amine as the compound A is added into the well 101 of the multiwell filter plate 100 in which the sugar chains are released from the polymer particles 20.

  Although it does not specifically limit as an aromatic amine (compound A), For example, 2-Aminobenzoamide, 2-Aminobenzoic acid, 8-Aminopyrene-1,3,6-trisulfonate, 8-Aminonaphthalene-1,3,6-trisulfonate, 2-Amino9 (10H) -acidone, 5-Aminofluorescein, Dansylethylenediamine, 7-Amino-4-methylcoumarin, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3- (Acetylamino) -6-aminoidino-idinoacidominorido-6-idinominorido 6-aminoidinoirido. Among these, 2-aminobenzoamide or 2-aminobenzoic acid is preferable. These compounds are preferably used because of their availability as reagents and the convenience of the reaction.

  In addition, when 2-aminobenzamide or 2-aminobenzoic acid is used as the aromatic amine, it is used at 0.35 mol / L under general conditions, but in this step, the solution in the well 101 after addition of the compound-containing solution 25 is used. That is, the concentration of the aromatic amine in the solution containing the re-released sugar chain is preferably set to 0.5 mol / L or more, more preferably 1.4 mol / L or more. Thereby, it becomes possible to improve the labeling efficiency of the sugar chain by the compound A. However, when the concentration is 3 mol / L or more, in the subsequent step [8], it may be difficult to remove the aromatic amine (compound A) that has not been used in the reaction. It is set to 4 mol / L or more and 3 mol / L or less.

  Further, when the liquid volume is defined by the polymer particles 20 filled in the well 101, the liquid volume is usually set so that the polymer particles 20 are immersed, for example, about 50 μL with respect to 5 mg of the polymer particles 20. The liquid volume (volume) is preferably set to about 100 μL of the double volume. Thereby, it becomes possible to improve the labeling efficiency of the sugar chain by the compound A. However, the liquid volume may exceed 100 μL, but if it exceeds a certain volume, it may be difficult to remove the aromatic amine (compound A) that was not used in the reaction in the post-process [8]. Therefore, the most preferred liquid amount is set between 100 μL and 200 μL.

  More specifically, when 2-aminobenzamide is used as the aromatic amine, it is dissolved in 30% acetic acid / dimethyl sulfoxide (DMSO) in the well 101 so as to have a concentration of 1.4 M 2-aminobenzamide, 1 M sodium cyanoborohydride. 100 μL of the resulting solution is added as compound-containing solution 25.

  [7-4 '] Next, the compound-containing liquid 25 is heated to keep the compound-containing liquid 25 in a certain temperature range.

  Thereby, the sugar chain re-released from the polymer particle 20 reacts with the compound A, and as a result, the sugar chain is labeled with the compound A.

  The pH of the reaction system at this time is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6.

  The temperature at the time of labeling is preferably set so as to be maintained in a temperature range of about 0 to 100 ° C, more preferably about 4 to 95 ° C, and further preferably about 30 to 90 ° C.

  The reaction time is usually set to about 0.1 to 20 hours, preferably about 0.6 to 12 hours when set in such a temperature range.

More specifically, when 2-aminobenzamide is used as the aromatic amine, the compound-containing liquid 25 is heated in a temperature range of 30 to 70 ° C. and reacted for about 1 to 10 hours.
By performing labeling under such conditions, the sugar chain is surely labeled with Compound A.

  In addition, when an aromatic amine is used as the compound A, the drying of the compound-containing liquid 25 as described in the above step [7-2] can be omitted.

  The sugar chain is labeled with the compound A also by the steps [7-1 ′] to [7-4 ′] as described above.

  [8] Next, the sugar chain labeled with Compound A (hereinafter sometimes referred to as “labeled sugar chain”) is purified.

  Here, when the labeled sugar chain is obtained by re-releasing the sugar chain captured by the polymer particle 20 as described above, in addition to the sugar chain labeled with the compound A, in the multiwell filter plate 100. Examples of the substance contained in the polymer include polymer particles 20 from which sugar chains are released and compound A that has not been used for labeling sugar chains (hereinafter also referred to as “unused compound A”).

  Therefore, in this step [8], the labeled sugar chain is purified by removing the polymer particles 20 and the unused compound A.

  Hereinafter, similarly to the steps [4] to [7], the case where the labeled sugar chain is purified using the well 101 provided in the multi-well filter plate 100 will be described.

  [8-1] First, as shown in FIG. 6 (a), a multi-layer in which the dried labeled sugar chains and polymer particles 20 obtained in the step [7-2] are accommodated in the well 101. The well filter plate 100 and the spacer 700 are inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the multiwell plate 200 are inserted into the recess 505 of the second support base 500. The first support base 400 is placed on the support base 500. That is, in this step, the first usage method of the purification apparatus 1 is applied.

  In this state, the lysis solution 26 is added into the well 101 provided in the multiwell filter plate 100.

  The solution 26 is not particularly limited as long as it is a solution capable of dissolving the labeled sugar chain, and examples thereof include water, various buffer solutions, and various organic solvents.

  If the polymer particles 20 are not dried by heating the compound-containing liquid 25 in the step [7-2], the addition of the solution 26 into the well 101 in the step [8-1] is omitted. be able to.

  Furthermore, when a fluorescent substance composed of an aromatic amine is used as the compound A, the sugar chain labeled with the compound A obtained in the above steps [7-1 ′] to [7-4 ′] Since it is not dried, also in this case, the addition of the lysis solution 26 into the well 101 in this step [8-1] can be omitted.

  [8-2] Next, by operating the suction pump of the purification apparatus 1, the internal space formed by the multiwell filter plate 100, the first support base 400, and the second support base 500 is negatively affected. Thus, the solution 26 in the well 101 is sucked (see FIG. 6B).

  As a result, as shown in FIG. 6C, the solution 26 is allowed to pass through the filter 105, so that the polymer particles 20 remain in the wells 101 of the multiwell filter plate 100, and the multiwell filter plate 100. The lysate 26 can be selectively stored in the well 201 provided in the multiwell plate 200 at a position corresponding to the well 101, and the labeled sugar chain dissolved in the lysate 26 can be added to the well 101. It can be moved to the well 201 of the multi-well plate 200. As a result, the labeled sugar chain and the polymer particle 20 are separated.

  That is, in the step [8-2], the lysate 26 is supplied into the well 101 using the multiwell filter plate 100 having a plurality of wells 101 including the filter 105, and then the lower side of the filter 105 is set to a negative pressure. Thus, a pressure difference is generated between the upper and lower sides of the well 101, and as a result, the labeled sugar chain and the polymer particle 20 are separated.

  At this time, since the unused compound A also usually shows solubility in the solution 26, it moves to the well 201 side of the multiwell plate 200 in a state of being dissolved in the solution 26.

  In the first method of using the purification apparatus 1, as described above, the lower opening 703 of the cylindrical body 701 coincides with the upper end of the well 201 in the thickness direction (height direction) or the well 201 The bottom surface 103 of the multi-well filter plate 100 is set to be positioned below the upper end, and the bottom surface 103 of the multiwell filter plate 100 coincides with the upper opening 702 of the cylinder 701 or is below the upper opening 702 of the cylinder 701 in the thickness direction. It is set to be located. Accordingly, it is possible to surely suppress or prevent the solution 26 that has flowed out through the through-hole 104 from being scattered in a region other than the well 201, and therefore, in the well 201 corresponding to the well 101. The dissolving liquid (liquid) 26 can be supplied with high efficiency.

  [8-3] Next, instead of the filter 105, a multi-well filter plate (multi-well silica gel plate) 100 ′ in which silica gel 106 is arranged below the well 101 is prepared. The adjustment plate 600 and the tray 300 are inserted into the concave portion 405 of the first support base 400, and the adjustment plate 600 and the tray 300 are further inserted into the concave portion 505 of the second support base 500. The support base 400 is placed.

  Then, as shown in FIG. 7A, in the step [8-2], the solution 26 containing labeled sugar chains separated in each well 201 of the multi-well plate 200 is mixed with non-water such as acetonitrile. Dilute with solvent and feed into well 101 with silica gel 106 of multiwell filter plate 100 ′.

  As a result, as shown in FIG. 7B, the solution 26 that has passed through the silica gel 106 of the well 101 flows out as a droplet to the concave portion 301 of the tray 300 corresponding to each well 101 through the through hole 104. It becomes.

  At this time, since the solution 26 obtained by diluting the silica gel 106 with a non-aqueous solvent such as acetonitrile permeates, the labeled sugar chain and the unused compound A contained in the solution 26 are adsorbed on the silica gel.

  [8-4] Next, while maintaining the state of FIG. 7B, a nonaqueous solvent such as acetonitrile is further supplied into the well 101 of the multiwell filter plate 100 ′ to wash the silica gel 106.

  Here, the adsorptive power to silica gel is higher for labeled sugar chains than for unused compound A. Therefore, by supplying a non-aqueous solvent into the well 101 and washing the silica gel 106, at least a part of the unused compound A can be removed from the silica gel.

  [8-5] Next, as shown in FIG. 7C, the multiwell filter plate 100 ′ and the spacer 700 are inserted into the recess 405 of the first support base 400, and the second support base 500 In place of the tray 300, the multiwell plate 200 is inserted into the recess 505, and in this state, the first support base 400 is placed on the second support base 500.

  Thereafter, water 27 is added into the multi-well filter plate 100 ′ and the silica gel is permeated to prevent the mixture of unused compound A from being accurately prevented or suppressed, as shown in FIG. 7 (d). The labeled sugar chain is purified (separated) in a state dissolved in the water 27 in the well 201 of the well plate 200.

  In this step [8-3] to [8-5], the labeled sugar chain was purified using the multiwell filter plate 100 ′ having the protrusion 107 formed thereon. The labeled sugar chain can be purified in the same manner as described above also by the multiwell filter plate 100 ′ in which the formation of is omitted.

  As described above, in steps [8-3] to [8-5], by using the multiwell filter plate (multiwell silica gel plate) 100 ′ having a plurality of wells 101 including the silica gel 106, the labeled sugar for the silica gel 106 is used. Based on the difference in adsorption force between the chain and the unused compound A, the labeled sugar chain is separated from the unused compound A.

  Through the above steps, the sugar chain labeled with the compound A is purified using the purification apparatus 1.

  If there is no need to deactivate the functional group of the capture carrier, this step [6] may be omitted.

  In addition, the sugar chain labeled with Compound A can be analyzed by a technique such as mass spectrometry represented by MALDI-TOF MS, and high performance liquid chromatography (HPLC).

  In particular, when the sugar chain is labeled with N-aminooxyacetyl-tryptophanyl (arginine methyl ester) represented by the chemical formula (3) described above, high-sensitivity analysis can be performed using MALDI-TOF MS.

  According to such a method for purifying sugar chains, sugar chains can be separated and purified with excellent accuracy by simple operations. Furthermore, since the processing solutions supplied to the plurality of wells can be processed at once, a large amount of sugar chains can be purified by a single purification process.

  Further, in the sugar chain purification method as described above, a supplemental carrier (polymer particle 20) and the purification apparatus 1 are used, the pressure difference between the upper and lower sides of the well 101 using a suction pump, the filtration using the filter 105, and the like. By using this means, the process can be simplified as compared with the above-described conventional method, so that waste of cost and time can be reduced.

  Although the spacer and the processing apparatus of the present invention have been described based on the illustrated embodiments, the present invention is not limited to these.

  Moreover, the structure of each part of the spacer and the processing apparatus of the present invention can be replaced with an arbitrary one that can exhibit the same function, or an arbitrary structure can be added.

  Furthermore, in the said embodiment, in the refiner | purifier, the edge part of the support body which a spacer has is latched by the edge part in which the opening part of the bottom part which a 1st support stand has is provided, and 1st Although the case where it fixes in the recessed part of a support stand was demonstrated, it is not limited to this, It shall be provided with the engaging part which engages a spacer with a multiwell filter plate, and, thereby, a spacer is a 1st support stand. You may make it fix in a recessed part.

  In the above embodiment, in the purification apparatus, the lower side of the filter of each well of the multi-well filter plate is configured to allow the liquid to permeate the filter by making the lower pressure from the upper side. However, this is not the case. Instead, the permeation of the liquid through the filter may be performed by a centrifugal force applied to the multiwell filter plate. Furthermore, when the liquid has a low viscosity or the like and easily passes through the filter, the liquid may pass through the filter by gravity, that is, free fall.

  In other words, the force difference generated above and below the filter (well) is not limited to the pressure difference in which the lower side of the filter is set to a negative pressure from the upper side, and centrifugal force or gravity is applied to the liquid located above the filter. Can be obtained.

  Furthermore, in the above-described embodiment, a case where a sugar chain is purified from a glycoprotein using a purification apparatus, that is, a case where a sugar chain is purified using a multiwell filter plate, a spacer, and a multiwell plate has been described. Without being limited thereto, an apparatus including a multiwell filter plate, a spacer, and a multiwell plate can be applied to size removal, bead / resin / base assay, sample concentration / desalting / purification, and the like.

DESCRIPTION OF SYMBOLS 1 Purification apparatus 20 Polymer particle 21 Pure water 22 Particle dispersion liquid 23 Sugar chain containing liquid 24 Washing liquid 25 Compound containing liquid 26 Dissolving liquid 27 Water 100, 100 'Multiwell filter plate 101 Well 103 Bottom face 104 Through-hole 105 Filter 106 Silica gel 107 Protrusion Part 200 multiwell plate 201 well 202 well forming part 203 bottom face 204 frame part 300 tray 301 recessed part 400 first support base 401 bottom part 402 opening part 403 upper outer wall 404 lower outer wall 405 recessed part 406 recessed part 407 packing 408 opening part 500 second Support base 501 bottom 502 projection 503 outer wall 505 recess 506 through-hole 507 packing 600 adjustment plate 700 spacer 701 cylinder 702 upper opening 703 lower opening 705 support 706 through-hole

Claims (7)

A multi-well filter plate comprising a plurality of first wells, a filter housed in the bottom of the first well, and a through-hole penetrating the bottom surface of the first well;
A multi-well plate disposed below the multi-well filter plate and having a plurality of second wells corresponding to the plurality of first wells;
A spacer having a plurality of cylinders corresponding to the plurality of first wells,
When arranged between the multi-well filter plate and the multi-well plate, the lower end of the cylinder is vertically aligned with the upper end of the second well or below the upper end of the second well. The bottom surface of the multi-well filter plate facing the cylinder body is set to be aligned with the upper end of the cylinder body or positioned below the upper end of the cylinder body. A spacer characterized by being set.   The cylindrical body is a liquid that has flowed out to the lower side of the first well through the filter and the through hole based on a force difference generated above and below the first well of the multi-well filter plate. The spacer according to claim 1, wherein the spacer functions as a liquid guiding unit that guides the liquid to the second well of the multi-well plate. The multi-well filter plate includes a projecting portion projecting downward on the bottom surface facing the cylindrical body,
The spacer according to claim 1 or 2, wherein a lower end of the projecting portion is vertically aligned with an upper end of the cylindrical body or positioned below the upper end of the cylindrical body.   The spacer according to claim 3, wherein the protruding portion is set to a size that allows the protruding portion to be inserted into the cylindrical body.   The spacer according to any one of claims 1 to 4, wherein the cylindrical body is set to a size that can be inserted into the second well.   The spacer according to claim 1, wherein the force difference is a pressure difference. A multi-well filter plate comprising a plurality of first wells, a filter housed in the bottom of the first well, and a through-hole penetrating the bottom surface of the first well;
A multi-well plate disposed under the multi-well filter plate and having a plurality of second wells corresponding to the plurality of first wells;
A processing apparatus comprising: the multi-well filter plate; and a spacer having a plurality of cylindrical bodies disposed between the multi-well plate and corresponding to the plurality of first wells,
When the spacer is disposed between the multiwell filter plate and the multiwell plate, the lower end of the cylindrical body coincides with the upper end of the second well in the vertical direction, or the second spacer The bottom surface of the multi-well filter plate, which is set to be positioned below the upper end of the well and faces the cylindrical body, coincides with the upper end of the cylindrical body in the vertical direction, or from the upper end of the cylindrical body A processing apparatus which is set to be positioned on the lower side.

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