WO2013047707A1 - Sugar chain refinement device and sugar chain refinement method - Google Patents

Sugar chain refinement device and sugar chain refinement method Download PDF

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Publication number
WO2013047707A1
WO2013047707A1 PCT/JP2012/074983 JP2012074983W WO2013047707A1 WO 2013047707 A1 WO2013047707 A1 WO 2013047707A1 JP 2012074983 W JP2012074983 W JP 2012074983W WO 2013047707 A1 WO2013047707 A1 WO 2013047707A1
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WIPO (PCT)
Prior art keywords
sugar chain
plate
lower plate
upper plate
state
Prior art date
Application number
PCT/JP2012/074983
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French (fr)
Japanese (ja)
Inventor
秀行 島岡
碧 阪口
Original Assignee
住友ベークライト株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2012213077A external-priority patent/JP2013081456A/en
Priority claimed from JP2012213078A external-priority patent/JP6123206B2/en
Application filed by 住友ベークライト株式会社 filed Critical 住友ベークライト株式会社
Publication of WO2013047707A1 publication Critical patent/WO2013047707A1/en

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    • C—CHEMISTRY; METALLURGY
    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K13/00—Sugars not otherwise provided for in this class
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds

Definitions

  • the present invention relates to an apparatus and method for 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, sialic acid and their derivatives are linked in a chain by glycosidic bonds. .
  • Sugar chains are very diverse and are substances 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 function and interaction regulation.
  • biopolymers having sugar chains include proteoglycans on the cell walls of plant cells that contribute to cell stabilization, cell differentiation, cell proliferation, cell adhesion, cell migration, and the like, and Examples thereof include glycoproteins involved in cell-cell interaction and cell recognition.
  • the mechanisms by which these high-molecular sugar chains control sophisticated and precise biological reactions while substituting, assisting, amplifying, regulating, or inhibiting each other's functions are gradually being 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).
  • sugar chains present on the cell surface play an important role as scaffolds for various biological reactions.
  • sugar chains are said to be involved in the occurrence of diseases due to abnormal interaction with receptors, infection with AIDS virus, influenza virus, etc., and the entry of pathogenic E. coli O157 toxin and cholera toxin into cells.
  • specific sugar chains appear on the cell surface.
  • sugar chains on the cell surface are considered to be important molecules that give the cells individuality.
  • sugar chain structure analysis techniques In order to analyze the occurrence of these diseases, sugar chain structure analysis techniques have been developed. These techniques combine sugar chain separation and purification, sugar chain labeling, and the like, by cutting out sugar chains from complex carbohydrates. However, these processes are extremely complicated.
  • gel filtration is performed using the difference in molecular weight between the labeled sugar chain and the contaminants to remove the contaminants.
  • Patent Documents 1, 2, and 3 various methods for applying fluorescent labels to sugar chains have been developed (for example, Patent Documents 1, 2, and 3).
  • the labeling efficiency was not 100%, and a labeled sugar chain and an unlabeled sugar chain were mixed in one sample. This situation does not pose a major problem when the sugar chain is detected by fluorescence using high performance liquid chromatography (HPLC) or capillary electrophoresis (CE method).
  • HPLC high performance liquid chromatography
  • CE method capillary electrophoresis
  • mass spectrometry there is a problem that the peaks of mass spectra and mass chromatograms become complicated.
  • the labeling efficiency to the sugar chain is poor, there is a possibility that the sensitivity for detecting the sugar chain may be lowered in the analysis by HPLC or CE.
  • An object of the present invention is to provide a sugar chain purification apparatus capable of separating and purifying a large amount of sugar chains easily and with excellent accuracy, and a sugar chain purification method using such an apparatus.
  • a sugar chain purification apparatus comprising: a dispensing means having a nozzle that sucks and discharges; and a moving means for relatively moving the plate assembly on the stage and the nozzle,
  • the plate assembly includes a plurality of first wells that are plate-shaped and configured by a first recess opening on an upper surface of the plate assembly, and a first through hole penetrating the bottom of the first recess.
  • the plate assembly includes a first state in which the first upper plate is supported by the support and the first lower plate is supported; A second state in which the first lower plate and the second lower plate are exchanged in the first state, and the second lower plate is supported by the support; In the second state, the first upper plate and the second upper plate are exchanged, the second upper plate is supported by the support, the second lower plate and the first plate A third state in which the lower plate is replaced and the first lower plate is supported by the support; In the third state, the first lower plate and the second lower plate are exchanged, and the second lower plate can take a fourth state supported by the support.
  • the sugar chain purification apparatus according to (2).
  • each of the first wells communicates with the first reservoir through the first through hole.
  • each of the first wells communicates with each of the second storage portions via the first through hole,
  • each of the second wells communicates with the first reservoir through the second through hole, and
  • each of the second wells communicates with each of the second reservoirs through the second through hole, respectively, and the sugar chain purification apparatus according to (4) above .
  • the sugar chain purification apparatus includes a first step of supplying a capture carrier that specifically binds to the sugar chain to each of the first wells; A second step of dispensing the solution into the first wells from the nozzle, bringing the solution into contact with the capture carrier, and capturing the sugar chain on the capture carrier; A third step of removing substances other than sugar chains bound to the capture carrier from the well; A fourth step of re-releasing the sugar chain bound to the capture carrier; A fifth step of separating the purified sugar chain from the capture carrier and purifying the sugar chain in sequence, In the first step, the second step, the third step, and the fourth step, the plate assembly is set to the first state, and in the fifth step, the plate assembly is sequentially changed.
  • the sugar chain purification apparatus according to (5), wherein the second state, the third state, and the fourth state are set.
  • the heating means may be the plate assembly or the first step in each of the first step, the second step, the third step, the fourth step, and the fifth step.
  • the support body includes an upper support member that supports the one of the first upper plate and the second upper plate, and a separate body from the upper support member.
  • a lower support member that supports the one of the side plate and the second lower plate;
  • the upper support member and the lower support member are such that the upper support member supports the one of the first upper plate and the second upper plate, and the lower support member is the first support member.
  • the sugar chain purification apparatus according to (1) wherein the sugar chain purification apparatus is configured to be disassembleable and assembleable in a state where the one of the lower plate and the second lower plate is supported.
  • the replacement of the first upper plate and the second upper plate and the replacement of the first lower plate and the second lower plate are respectively the upper support member and the second upper plate.
  • the sugar chain purification apparatus according to (1), further including a washing tank for washing the nozzle.
  • the fluorescent substances include 2-Aminobenzoamide, 2-Aminobenzoic acid, 8-Aminopyrene-1,3,6-trisulfonate, 8-Aminophathalene-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-aminoacidine described in at least one of the above (22) Sugar chain purification method.
  • the present invention it is possible to easily separate and purify sugar chains with excellent accuracy, and to fluorescently label sugar chains with high yield. Furthermore, since liquids (solutions) respectively supplied to a plurality of first wells and a plurality of second wells can be processed at once, a large amount of sugar chains can be purified and labeled in a single process. Will be able to.
  • FIG. 1 is a perspective view showing a first embodiment of the sugar chain purification apparatus of the present invention.
  • FIG. 2 is a perspective view showing a plate assembly provided in the sugar chain purification apparatus shown in FIG.
  • FIG. 3 is an exploded perspective view of the plate assembly shown in FIG. 4 is a cross-sectional view taken along line BB in FIG. 2 (a cross-sectional view showing a first state of the plate assembly).
  • 5 is a cross-sectional view taken along line BB in FIG. 2 (a cross-sectional view showing a second state of the plate assembly).
  • 6 is a cross-sectional view taken along the line BB in FIG. 2 (a cross-sectional view showing a third state of the plate assembly).
  • 7 is a cross-sectional view taken along the line BB in FIG.
  • FIG. 8 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 9 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 10 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying a sugar chain with the sugar chain purification apparatus shown in FIG.
  • FIG. 11 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 12 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG.
  • FIG. 13 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG.
  • FIG. 14 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG.
  • FIG. 15 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG.
  • FIG. 16 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying a sugar chain with the sugar chain purification apparatus shown in FIG.
  • FIG. 17 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG.
  • FIG. 18 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG.
  • FIG. 19 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG.
  • FIG. 20 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG.
  • FIG. 21 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG.
  • FIG. 22 is a perspective view showing a second embodiment of the sugar chain purification apparatus of the present invention.
  • FIG. 23 is a perspective view showing a third embodiment of the sugar chain purification apparatus of the present invention.
  • FIG. 1 is a perspective view showing a first embodiment of the sugar chain purification apparatus of the present invention
  • FIG. 2 is a perspective view showing a plate assembly provided in the sugar chain purification apparatus shown in FIG. 1
  • FIG. 4 to 7 are sectional views taken along the line BB in FIG. 2 (FIG. 4 shows a first state of the plate assembly, and FIG. FIG. 6 shows a third state of the plate assembly, FIG. 7 shows a fourth state of the plate assembly)
  • FIGS. FIG. 2 is a cross-sectional view (a cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying a sugar chain with the sugar chain purification apparatus shown in FIG.
  • the upper side in FIGS. 1 to 21 is referred to as “upper” or “upper”, and the lower side is referred to as “lower” or “lower”.
  • the horizontal direction is the x-axis direction in the horizontal direction (front-rear direction) of the apparatus
  • the y-axis direction is the direction perpendicular to the x-axis direction (horizontal direction of the apparatus)
  • the vertical direction up-down direction
  • a sugar chain purification apparatus (hereinafter simply referred to as “purification apparatus”) 1 shown in FIG. 1 is a method for purifying sugar chains from a sugar chain-containing solution (hereinafter referred to as “sugar chain-containing solution”) 23. It is a device used for.
  • the purification apparatus 1 includes a stage 3, a plate assembly 10 placed on the stage 3, a dispensing means 4 having a function of dispensing a liquid to the plate assembly 10 on the stage 3, and a stage 3
  • the moving means 5 for moving the nozzle (pipettor) 41 of the dispensing means 4 with respect to the plate assembly 10, the heating means 6 for heating the plate assembly 10 on the stage 3 together with the stage 3, and the nozzle 41 are washed.
  • a cleaning tank 7 and a control panel 11 having a function of controlling operations of the dispensing means 4, the moving means 5 and the heating means 6 are provided.
  • the configuration of each unit will be described.
  • the plate assembly 10 includes a first upper plate (multiwell filter plate) 100, a second upper plate (cleanup plate) 100 ′, and a first lower plate. (Waste liquid tray) 300, second lower plate (microwell plate) 200, adjustment plate (spacer) 600, and support 700 are provided. That is, the plate assembly 10 includes a plurality of first upper plates 100, a plurality of second upper plates 100 ′, a plurality of first lower plates 300, A second lower plate 200. In the plate assembly 10, these members can be disassembled and assembled. And the plate assembly 10 can take four assembly states by selection of each plate. As the four assembled states, there are a first state shown in FIG. 4, a second state shown in FIG. 5, a third state shown in FIG. 6, and a fourth state shown in FIG. FIG. 2 shows a set of plate assemblies 10 (hereinafter also simply referred to as “plate assemblies 10”) mounted on the plate assembly mounting portion 31 of the stage 3.
  • plate assemblies 10 hereinafter also simply referred to as “plate assemblies 10” mounted on the plate assembly
  • the first upper plate 100 of the first upper plate 100 and the second upper plate 100 ′ is selected, and the first lower plate 300 and the second lower plate 200 are selected.
  • the first lower plate 300 is selected.
  • the selected first upper plate 100 and first lower plate 300 are assembled so as to be arranged vertically and supported by the support 700.
  • the first upper plate 100 of the first upper plate 100 and the second upper plate 100 ′ is selected, and the first lower plate 300 and the second lower plate 200 are selected.
  • the second lower plate 200 is selected.
  • the selected first upper plate 100 and second lower plate 200 are assembled so as to be arranged vertically and supported by the support 700.
  • the second upper plate 100 ′ of the first upper plate 100 and the second upper plate 100 ′ is selected, and the first lower plate 300 and the second lower plate 200 are selected.
  • the first lower plate 300 is selected.
  • the selected second upper plate 100 ′ and first lower plate 300 are assembled so as to be arranged vertically and supported by the support 700.
  • the second upper plate 100 ′ of the first upper plate 100 and the second upper plate 100 ′ is selected, and the first lower plate 300 and the second lower plate 200 are selected.
  • the second lower plate 200 is selected.
  • the selected second upper plate 100 ′ and second lower plate 200 are assembled so as to be arranged one above the other and supported by the support 700.
  • the stage 3 has a flat plate shape that is long in the y-axis direction of FIG. 1, and its upper surface is divided into a plurality of regions. As shown in FIG. 1, the stage 3 includes a plate assembly mounting unit 31, a first upper plate mounting unit 32, a second upper plate mounting unit 33, and a first lower plate mounting. It is divided into a part 34, a second lower plate placing part 35, a cleaning tank placing part 36, and a processing liquid placing part 37.
  • the plate assembly placement portion 31 is an area where the set of plate assemblies 10 is placed, which is located at a substantially central portion in the longitudinal direction of the stage 3.
  • the sugar chain is purified from the sugar chain-containing liquid 23 contained in the plate assembly 10 placed on the plate assembly placement unit 31.
  • the first upper plate placement section 32 is an area where one or more first upper plates 100 are placed on top of each other, positioned in front of the right side of the stage 3 toward the purification apparatus 1 of FIG. Each first upper plate 100 placed on the first upper plate placement portion 32 is unused and used for replacement.
  • the second upper plate placing portion 33 is located further to the right than the first upper plate placing portion 32 toward the purification device 1 in FIG. 1, and one or more second upper plates 100 ′ are stacked. It is an area to be placed. Each second upper plate 100 ′ placed on the second upper plate placing portion 33 is unused and used for replacement.
  • the first lower plate mounting portion 34 is located further back than the second upper plate mounting portion 33 toward the purification device 1 in FIG. 1, and one or more first lower plates 300 are provided. It is an area to be placed in an overlapping manner. Each first lower plate 300 placed on the first lower plate placing portion 34 is unused and used for replacement.
  • the second lower plate mounting portion 35 is located further back than the first upper plate mounting portion 32 toward the purification device 1 in FIG. 1, and one or more second lower plates 200 are provided. It is an area to be placed in an overlapping manner. Each second lower plate 200 placed on the second lower plate placing portion 35 is unused and used for replacement.
  • each plate is exchanged manually by an operator who operates the purification apparatus 1.
  • the washing tank mounting part 36 is located in front of the left side of the stage 3 toward the purification apparatus 1 in FIG. 1 and is an area where the cleaning tank 7 is placed.
  • the cleaning tank 7 mounted on the cleaning tank mounting part 36 is fixed by a fixing mechanism (not shown).
  • the processing liquid mounting unit 37 is located further to the left of the cleaning tank mounting unit 36 toward the purification apparatus 1 in FIG. 1, and includes a multiwell plate 81 in which various liquids (processing liquids) are stored, tanks 82 to 87 is an area on which to be placed.
  • the constituent material of the stage 3 is not particularly limited, and various metal materials such as stainless steel, aluminum, and aluminum alloy can be used.
  • the dispensing unit 4 includes a nozzle 41, a nozzle head 44 that supports and fixes the nozzle 41, a pump 42, and a tube 43 that connects the nozzle 41 and the pump 42.
  • the nozzle 41 is composed of a tubular member having an open end (lower end).
  • the nozzle 41 can suck and discharge the liquid through the tip opening 411 by the operation of the pump 42.
  • the number of nozzles 41 is one in the configuration shown in FIG. 1, but is not limited to this. For example, a plurality of nozzles 41 may be arranged.
  • the pump 42 is, for example, a gear pump or a vane pump, and is disposed on the back side of the wall portion 12 erected from the stage 3.
  • the tube 43 connects the nozzle 41 and the pump 42. Thereby, the nozzle 41 and the pump 42 communicate with each other through the tube 43.
  • the tube 43 has flexibility, and can follow the movement when the nozzle 41 is moved by the operation of the moving means 5.
  • the moving means 5 includes an x-axis direction moving mechanism (horizontal direction moving mechanism) 51, a y-axis direction moving mechanism (horizontal direction moving mechanism) 52, and a z-axis direction moving mechanism (vertical direction moving mechanism) 53.
  • the x-axis direction moving mechanism 51, the y-axis direction moving mechanism 52, and the z-axis direction moving mechanism 53 are, for example, a motor (not shown), a ball screw (not shown) connected to the motor, and a ball screw. And a linear guide (not shown).
  • the nozzle 41 is supported by the z-axis direction moving mechanism 53 via the nozzle head 44 and can be moved in the z-axis direction by the z-axis direction moving mechanism 53.
  • the x-axis direction moving mechanism 51 supports the nozzle 41 together with the z-axis direction moving mechanism 53. Thereby, the nozzle 41 can move in the x-axis direction together with the z-axis direction moving mechanism 53.
  • the y-axis direction moving mechanism 52 supports the nozzle 41 together with the z-axis direction moving mechanism via the x-axis direction moving mechanism 51. Accordingly, the nozzle 41 can move in the y-axis direction together with the x-axis direction moving mechanism 51 and the z-axis direction moving mechanism 53.
  • the moving means 5 having such a configuration allows the nozzle 41 to reciprocate between the plate assembly 10, the multiwell plate 81, and the tanks 82 to 87.
  • the heating means 6 has a heater 61 embedded immediately below the plate assembly mounting portion 31 of the stage 3.
  • the heater 61 is configured by winding a heating wire such as a nichrome wire that generates heat when energized in a ring shape. Further, the area of the heater 61 is larger than the area of the plate assembly 10 in plan view. That is, the plate assembly 10 is disposed inside the heater 61 in plan view.
  • the heater 61 is embedded in the stage 3, it is possible to reliably prevent the operator of the refining apparatus 1 from touching the heater 61 by mistake. Thereby, even if the heater 61 is generating heat, it is possible to prevent or suppress the operator from being burned.
  • the heating means 6 detects the temperature on the plate assembly mounting portion 31 of the stage 3. And according to the detection result, the voltage applied to the heater 61 can be controlled. Thereby, the heating temperature for the plate assembly 10 is changed in each step (first step, second step, third step, fourth step and fifth step) performed in the purification apparatus 1. Therefore, the temperature can be adjusted to an optimum temperature, which contributes to the purification of sugar chains with excellent accuracy.
  • the washing tank 7 is composed of a member having a bottomed cylindrical shape, and a washing liquid 71 for washing the nozzle 41 can be stored inside the washing tank 7.
  • the nozzle 41 is inserted into the cleaning tank 7 each time on the way from the plate assembly 10 to the multiwell plate 81 and the tanks 82 to 87, and is cleaned with the cleaning liquid 71 in the cleaning tank 7. Thereby, for example, contamination in the plate assembly 10 can be prevented.
  • the cleaning liquid 71 is replaced with an unused one every time the nozzle 41 is cleaned, that is, every time it is used. Thereby, the said contamination can be prevented reliably.
  • the cleaning liquid 71 is not particularly limited, and for example, alcohols represented by purified water (distilled water), methanol, and ethanol can be used. It is also possible to install two or more cleaning tanks 7 on the stage 3 and perform continuous cleaning with two or more cleaning liquids 71 stored in each of the two or more cleaning tanks 7.
  • the control panel 11 is disposed in the vicinity of the first upper plate placing portion 32 on the stage 3.
  • the control panel 11 is provided with a liquid crystal screen 111 as a display unit and an operation unit (input unit).
  • the liquid crystal screen 111 displays, for example, an input screen for setting various operating conditions of the refining device 1.
  • the operator can set various operating conditions by touching the liquid crystal screen 111 with a finger. By this setting, the operations of the dispensing means 4, the moving means 5 and the heating means 6 are controlled.
  • the plate assembly 10 includes a first upper plate 100, a second upper plate 100 ′, a first lower plate 300, and a second lower plate 200. And an adjustment plate 600 and a support 700.
  • the plate assembly 10 has four states: a first state shown in FIG. 4, a second state shown in FIG. 5, a third state shown in FIG. 6, and a fourth state shown in FIG. The assembled state can be taken.
  • the support 700 supports the first upper plate 100 or the second upper plate 100 ′ with the first support base 400 and the second support base 500 according to the four assembled states.
  • the first lower plate 300 or the second lower plate 200 is supported.
  • the first upper plate 100 has a flat plate shape as a whole and is provided in the thickness direction (vertical direction).
  • a plurality (96 in this embodiment) of wells (first wells) 101 constituted by concave portions are provided. These wells 101 are arranged in a matrix (12 rows in the x-axis direction and 8 columns in the y-axis direction), and are used when purifying sugar chains from the sugar chain-containing liquid 23.
  • each of these wells 101 has a bottom portion 103 on the lower side thereof, and further, a through hole (first through hole) penetrating the bottom portion 103 at a substantially central portion of the bottom portion 103. Hole) 104.
  • the liquid supplied (stored) to the well 101 flows out from the bottom 103 to the outside of the well 101 through the through hole 104.
  • the hole diameter of the through hole 104 is preferably set to about 0.1 to 1 mm, more preferably about 0.2 to 0.7 mm.
  • a membrane-like filter (first filter) 105 is disposed on the lower side (bottom 103 side) of each well 101 so as to close (cover) 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 inhibited from flowing out to the outside through the through hole 104 when the first upper plate 100 is left standing. (Prevented) On the other hand, as will be described later, when the first upper plate 100 is attached to the first support base 400 and the inside of the concave portion 505 of the second support base 500 is sucked using the suction pump 13. In this case, permeation (passage) of the liquid filter 105 is allowed. Therefore, the liquid flows out from the bottom 103 side to the outside of the well 101 through the through hole 104.
  • a raw material of the filter 105 For example, a porous film, a nonwoven fabric, etc. are mentioned.
  • 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.
  • transmittance of the said liquid can be aimed at.
  • this hydrophilic treatment for example, surface modification represented by plasma treatment, corona discharge, graft treatment, acid treatment, or the like, or imparting (coating) a surfactant, water-soluble silicon, polypropylene glycol, or the like to the filter 105 is performed. It can be done by processing.
  • the pore diameter of the pores provided in the filter 105 is preferably set to about 0.1 to 50 ⁇ m, more preferably about 0.2 to 20 ⁇ m, and further preferably 0.4 to 10 ⁇ m.
  • the second upper plate 100 ′ has a flat plate shape as a whole and is provided in the thickness direction (vertical direction). That is, the second upper plate 100 ′ is configured by a recess (second recess) opened in the upper surface 108 ′. A plurality (96 in this embodiment) of wells (second wells) 101 ′ are provided. These wells 101 ′ are arranged in a matrix (12 rows in the x-axis direction and 8 columns in the y-axis direction), similar to the wells 101 of the first upper plate 100, and from the sugar chain-containing liquid 23. Used when purifying sugar chains.
  • each of these wells 101 ′ has a bottom 103 ′ on the lower side, and further, a through hole (through the bottom 103 ′ substantially at the center of the bottom 103 ′). 2nd through-hole) 104 '. Accordingly, the liquid supplied (stored) to the well 101 ′ flows out from the bottom 103 ′ to the outside of the well 101 ′ through the through-hole 104 ′.
  • a filter (second filter) 106 is disposed on the lower side (bottom 103 side) of each well 101 so as to close (cover) the through hole 104.
  • the liquid supplied to the well 101 ′ is prevented (blocked) from flowing out to the outside through the through hole 104 ′.
  • the suction pump 13 is used to suck the inside of the recess 505 of the second support base 500. Is allowed to pass through (pass through) the liquid filter 106. Therefore, the liquid flows out of the well 101 'from the bottom 103' side through the through hole 104 '.
  • the filter 106 is made of a material different from that of the filter 105, and has a different function of capturing an object such as a sugar chain, that is, an object to be captured.
  • the filter 106 is made of silica gel, for example.
  • the first upper plate 100 and the second upper plate 100 ′ as described above are mounted on the first support base 400 of the support 700.
  • the first support base 400 has a flat plate shape, and includes a bottom portion 401 having an opening 402 that opens at the center thereof, and protrudes upward along the outer edge of the bottom portion 401.
  • An upper outer wall 403 provided on the bottom portion 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 in the first support base 400 by the upper outer wall 403.
  • the inserted first upper plate 100 or the second upper plate 100 ′ becomes the first support base 400. Is supported by
  • a groove is provided along the edge of the opening 402 of the bottom 401.
  • a packing (seal member) 407 is disposed so as to correspond to the groove. Accordingly, when the first upper plate 100 or the second upper plate 100 ′ is inserted into the recess 405, the communication between the opening 402 and the outside thereof is blocked.
  • the upper outer wall 403 has an opening 408 in a direction perpendicular to the thickness direction (vertical direction) at the center of two opposing long sides.
  • a recess 406 is formed in the first support base 400 by the lower outer wall 404.
  • the first support base 400 is fixed on the second support base 500 by inserting a protrusion 502 included in the second support base 500 described later along the inner peripheral surface of the recess 406.
  • the outer peripheral surface of the upper outer wall 403 and the outer peripheral surface of the lower outer wall 404 are formed as a single flat surface by being integrally formed.
  • the first lower plate 300 has a flat plate shape as a whole, and has a single recess (third recess) that opens on the upper surface 302 (first reservoir). 301.
  • the first lower plate 300 includes a concave portion forming portion 303 in which a concave portion 301 is formed, and a frame portion 304 arranged so as to surround the concave portion forming portion 303.
  • the thickness of the recess forming portion 303 is larger than the thickness of the frame portion 304.
  • the shape of the recess forming portion 303 in plan view is set to a size that can be inserted into the opening 402 of the first support base 400.
  • the first support base 400 is mounted on the second support base 500 in a state where the first lower plate 300 is disposed in the recess 505 of the second support base 500 (see FIGS. 4 and 6).
  • the recess forming portion 303 can be inserted into the opening 402 of the first support base 400.
  • the upper surface 302 of the recessed part formation part 303 protrudes from the upper surface of the frame part 304 as mentioned above, it is not limited to this, The same height as the upper surface of the frame part 304 may be sufficient. Also in this case, the sugar chain can be reliably separated and purified.
  • the second lower plate 200 has a flat plate shape as in the first lower plate 300 and is provided in the thickness direction (vertical direction).
  • a plurality (96 in the present embodiment) of wells (second reservoirs) 201 each including a recess (fourth recess) opened on the upper surface 204 is provided.
  • These wells 201 are arranged in a matrix (12 rows in the x-axis direction and 8 columns in the y-axis direction).
  • Each of these wells 201 has a bottom 203 on the lower side thereof. Thereby, the liquid supplied (stored) to the well 201 is stored therein.
  • the second lower plate 200 includes a well forming portion 202 in which a plurality of wells 201 are formed, and a frame portion 205 disposed so as to surround the well forming portion 202.
  • the thickness of the well forming portion 202 is thicker than the thickness of the frame portion 205.
  • the well forming portion 202 is set to a size that can be inserted into the opening 402 of the first support base 400 in a plan view. Accordingly, the first support base 400 is mounted on the second support base 500 in a state where the second lower plate 200 is disposed in the recess 505 of the second support base 500 (see FIGS. 5 and 7). Then, the well forming part 202 can be inserted into the opening 402 of the first support base 400.
  • the adjustment plate 600 Prior to the first lower plate 300 or the second lower plate 200, the adjustment plate 600 is used by being inserted into the recess 505 provided in the second support base 500.
  • the adjusting plate 600 has a flat plate shape, and a plurality of plates having different thicknesses are prepared.
  • the adjustment plate 600 having an appropriate thickness according to the type of the first lower plate 300 and the second lower plate 200 inserted into the recess 505, the following effects can be obtained.
  • Obtainable In the first state, the first upper plate 100 and the first lower plate 300 can be brought as close as possible. In the second state, the first upper plate 100 and the second lower plate 200 can be brought as close as possible. In the third state, the second upper plate 100 ′ and the first lower plate 300 can be as close as possible. In the fourth state, the second upper plate 100 ′ and the second lower plate 200 can be brought as close as possible. In particular, the first upper plate 100 and the second lower plate 200 can be brought as close as possible in the second state, and the second upper plate 100 ′ and the second lower plate in the fourth state. It is effective in the process described later that 200 can be brought as close as possible.
  • the second support base 500 is a member for supporting the first lower plate 300 or the second lower plate 200. As shown in FIGS. 4 to 7, the second support base 500 is configured separately from the first support base 400, and protrudes upward along the flat bottom 501 and the outer edge of the bottom 501.
  • the outer wall 503 is provided.
  • a recess 505 is formed inside thereof.
  • the first lower plate 300 or the second lower plate 200 is supported by the second support base 500 by inserting the first lower plate 300 or the second lower plate 200 into the recess 505. Is done.
  • the outer wall 503 has a protruding portion 502 that protrudes upward along the inner edge of the outer wall 503.
  • the protrusion 502 is set to be inserted along the inner peripheral surface of the recess 406 provided in the first support base 400. As a result, when the first support base 400 is placed on the second support base 500, the protrusion 502 is inserted into the concave portion 406 of the first support base 400, whereby the second support base 500.
  • the first support base 400 is fixed on the top.
  • a groove is provided on the upper surface of the outer wall 503 so as to surround the protruding portion 502.
  • a packing (seal member) 507 is disposed so as to correspond to the groove.
  • the outer wall 503 has a through hole 506 penetrating the outer wall 503 on the side surface of the outer wall 503.
  • the through-hole 506 is disposed adjacent to the pump 42 on the back side of the wall portion 12 erected from the stage 3, and a suction pump 13 constituted by a vacuum pump is connected via the tube 14.
  • a suction pump 13 constituted by a vacuum pump is connected via the tube 14.
  • the constituent materials of the first upper plate 100, the second upper plate 100 ′, the first lower plate 300, and the second lower plate 200 are not particularly limited.
  • polypropylene, polyethylene, polystyrene And resin materials such as polyvinyl chloride and polytetrafluoroethylene can be used, and one or more of them can be used in combination.
  • the resin material can be used as the constituent material of the first support base 400 and the second support base 500.
  • the constituent materials include Fe-based alloys such as stainless steel, Cu- or Cu-based alloys, metal-based materials such as Al or Al-based alloys, and ceramic-based materials such as alumina, apatite, and aluminum nitride. 1 type or 2 types or more can be used in combination.
  • the first state, the second state, the third state, and the fourth state in the plate assembly 10 will be described. Whether the plate assembly 10 is in the first state, the second state, the third state, or the fourth state depends on whether the plate assembly 10 purifies sugar chains with the purification device 1 described later. It is selected according to which process among the processes to be performed.
  • the pitch between the wells 101 of the first upper plate 100, the pitch between the wells 101 ′ of the second upper plate 100 ′, and the wells 201 of the second lower plate 200 is set to the same size.
  • the first upper plate 100 is inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the first plate are further inserted into the recess 505 of the second support base 500.
  • a lower plate 300 is inserted.
  • the first support base 400 is placed on the second support base 500.
  • the recessed portion forming portion 303 of the first lower plate 300 is inserted into the opening 402 of the first support base 400, and the protruding portion 502 of the second support base 500 is further inserted into the first support base 400. It is inserted into the recess 406.
  • each well 101 of the first upper plate 100 communicates with the recess 301 of the first lower plate 300 through the through-hole 104 in a lump.
  • the first support base 400 is fixed on the second support base 500.
  • the packing 507 blocks communication between the inside and the outside of the plate assembly 10 between the first support base 400 and the second support base 500. Further, the packing 407 blocks communication between the first support base 400 and the first upper plate 100. Accordingly, the internal space 9a formed (defined) by the first upper plate 100, the first support base 400, and the second support base 500 is closed with respect to the outside. It can be.
  • the suction pump 13 connected to the through-hole 506 to depressurize the internal space 9a (recessed portion 505), the internal space 9a can be made negative with respect to the outside. Therefore, in each well 101 of the first upper plate 100, a pressure difference is generated between the upper and lower sides of the well 105 through the filter 105. Therefore, the liquid in the well 101 passes through the filter 105 due to the pressure difference. Therefore, the liquid can flow out from each well 101 through the through-hole 104 and can be stored in the recess 301 of the first lower plate 300 in a lump.
  • a pressure difference in which the lower side of the filter 105 is negative from the upper side thereof is used.
  • Centrifugal force applied to the liquid located above the filter 105 or gravity can be used.
  • the viscosity of the liquid in the well 101 is relatively low and the liquid easily passes through the filter 105, the liquid can pass through the filter 105 by gravity, that is, free fall.
  • the first upper plate 100 is inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the second plate are further inserted into the recess 505 of the second support base 500.
  • the lower plate 200 is inserted.
  • the first support base 400 is placed on the second support base 500.
  • the well forming portion 202 of the second lower plate 200 is inserted into the opening 402 of the first support base 400, and the protrusion 502 of the second support base 500 is further inserted into the first support base 400. It is inserted into the recess 406.
  • each well 101 of the first upper plate 100 communicates with the well 201 of the second lower plate 200 through the through hole 104.
  • the first support base 400 is fixed on the second support base 500.
  • the internal space 9a can be a closed space closed to the outside. Then, by operating the suction pump 13 connected to the through-hole 506 and depressurizing the internal space 9 a (recess 505), 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 independently in each well 201 of the second lower plate 200.
  • the second upper plate 100 ′ is inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the first plate are further inserted into the recess 505 of the second support base 500.
  • 1 lower plate 300 is inserted.
  • the first support base 400 is placed on the second support base 500.
  • the recess forming portion 303 of the first lower plate 300 is inserted into the opening 402 of the first support base 400, and the protrusion 502 of the second support base 500 is further inserted. Is inserted into the recess 406 of the first support base 400.
  • each well 101 ′ of the second upper plate 100 ′ is in communication with the recess 301 of the first lower plate 300 through the through hole 104 ′.
  • the first support base 400 is fixed on the second support base 500.
  • the packing 507 blocks communication between the inside and the outside of the plate assembly 10 between the first support base 400 and the second support base 500. Further, the packing 407 blocks communication between the first support base 400 and the second upper plate 100 ′. Accordingly, the internal space 9b formed (defined) by the second upper plate 100 ′, the first support base 400, and the second support base 500 is closed with respect to the outside. It can be a space.
  • the suction pump 13 connected to the through hole 506 to depressurize the internal space 9b (recessed portion 505), the internal space 9b can be made negative with respect to the outside. Therefore, in each well 101 ′ of the second upper plate 100 ′, a pressure difference is generated between the upper and lower sides through the filter 106, so that the liquid in the well 101 ′ causes the filter 106 to pass through the pressure difference. To Penetrate. Accordingly, the liquid can flow out from each well 101 ′ through the through hole 104 ′ and can be stored in the recess 301 of the first lower plate 300 in a lump.
  • the second upper plate 100 ′ is inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the first plate are further inserted into the recess 505 of the second support base 500.
  • Two lower plates 200 are inserted.
  • the first support base 400 is placed on the second support base 500.
  • the well forming portion 202 of the second lower plate 200 is inserted into the opening 402 of the first support base 400, and the protrusion 502 of the second support base 500 is further inserted into the first support base 400. It is inserted into the recess 406.
  • each well 101 'of the second upper plate 100' communicates with the well 201 of the second lower plate 200 through the through hole 104 '.
  • the first support base 400 is fixed on the second support base 500.
  • the internal space 9b can be a closed space closed to the outside. Then, by operating the suction pump 13 connected to the through hole 506 and depressurizing the internal space 9b (recessed portion 505), 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 independently in each well 201 of the second lower plate 200.
  • the adjustment plate 600 brings the lower surface 109 of the first upper plate 100 and the upper surface 302 of the first lower plate 300 as close as possible to each other in the first state. it can be set as small as possible a distance d 1.
  • it can be a first lower surface 109 of the upper plate 100 is brought closer as possible and the upper surface 204 of the second lower plate 200, set as small as possible these the distance d 2.
  • the lower surface 109 ′ of the second upper plate 100 ′ and the upper surface 302 of the first lower plate 300 can be as close as possible, and the separation distance d 3 can be set as small as possible. it can.
  • the fourth state it is brought closer as possible to the 'lower surface 109' of the second upper plate 100 and the upper surface 204 of the second lower plate 200, smaller is as much as possible these separation distance d 4 it can.
  • the liquid is supplied into the well 201 of the second lower plate 200 corresponding to each well 101 with high efficiency. (Can be recovered).
  • the liquid is placed in the well 201 of the second lower plate 200 corresponding to each well 101 ′. Can be supplied with efficiency.
  • the separation distances d 1 to d 4 are not particularly limited, and for example, are preferably set to 10 mm or less, more preferably 0.5 mm to 10 mm, and further preferably 0.5 mm to 3 mm. By setting the separation distances d 1 to d 4 within such a range, the above effects can be exhibited more remarkably.
  • a ring-shaped protrusion 107 protrudes from the bottom surface (lower surface 109 ′) of the second upper plate 100 ′ so as to surround each through hole 104 ′. It is preferable. Since the protrusion 107 is formed, the liquid that has passed through the through hole 104 ′ can be guided into the well 201 of the second lower plate 200, and thus the liquid can be recovered more efficiently. be able to.
  • the sugar chain is obtained from a glycoprotein provided with the sugar chain will be described as an example. Although it does not specifically limit as a sample containing a glycoprotein, For example, biological samples, such as whole blood, serum, plasma, urine, saliva, a cell, a structure
  • the sample is then 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.
  • 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.
  • SDS-PAGE SDS-denaturing polyacrylamide gel electrophoresis
  • two-dimensional electrophoresis in which isoelectric focusing and SDS-PAGE are combined.
  • the glycoprotein contained in the sample is separated in the gel.
  • the sugar chain is released from the glycoprotein retained in the gel using a sugar chain releasing method.
  • the method for releasing the sugar chain is not particularly limited, and for example, methods such as glycosidase treatment using N-glycosidase or O-glycosidase,
  • the gel after the sugar chain release treatment is taken out, and the gel is rinsed using a cleaning solution such as water. By doing so, the released sugar chain is eluted from the gel into the washing solution. And after precipitating a gel in a washing
  • the sugar chain-containing liquid 23 is stored in advance in a multi-well plate 81 having a plurality (96 in this embodiment) of wells 811.
  • electrophoresis treatment is used to release sugar chains.
  • the present invention is not limited to this.
  • affinity chromatography treatment or ion exchange chromatography treatment may be used.
  • the glycoprotein contained in the sample may be subjected to a treatment for directly releasing the sugar chain.
  • sugar chains are the only substances in the living body that have an aldehyde group. That is, a sugar chain is a substance in which a cyclic hemiacetal structure and an acyclic aldehyde structure exist in an equilibrium state in an aqueous solution or the like.
  • 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.
  • the sugar chain can be easily detached from the carrier.
  • amino groups are frequently used for capturing and supporting physiologically active substances.
  • the bond (Schiff base) generated by the reaction of the amino group and the aldehyde group has a weak binding force, a secondary treatment using a reducing agent or the like is necessary. For this reason, amino groups are not preferred for capturing sugar chains.
  • polymer particles are preferably solid particles 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.
  • the particles can be easily recovered using the well 101 provided in the first upper 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).
  • polymer particles 20 are used as a capture carrier that specifically binds to sugar chains, and sugar chains are captured on the polymer particles 20.
  • the polymer particles 20 are stored in advance in a tank 82 as a particle dispersion 22 in which the polymer particles 20 are dispersed in pure water 21.
  • the shape of the polymer particle 20 is not particularly limited, but for example, a spherical shape or a similar shape is preferable.
  • the average particle size is preferably about 0.05 to 1000 ⁇ m, more preferably about 0.05 to 200 ⁇ m, still more preferably about 0.1 to 200 ⁇ m, and most preferably 0.1 to 100 ⁇ m. 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.
  • the capture carrier supply step is a step of supplying capture carriers (polymer particles 20) that specifically bind to sugar chains to the wells 101 of the first upper plate 100, respectively.
  • the plate assembly 10 is used as the first state.
  • the sugar chain-containing liquid 23 is dispensed to each well 101 of the first upper plate 100 with the nozzle 41, and the sugar chain-containing liquid 23 and the polymer particles 20 are brought into contact with each other.
  • the plate assembly 10 is used as the first state following the capturing carrier supplying step.
  • the substance removing step is a step of removing substances other than the sugar chains bonded to the polymer particles 20.
  • the plate assembly 10 is used as the first state.
  • the re-releasing step is a step for re-releasing the sugar chains bound to the polymer particles 20.
  • the plate assembly 10 is used as the first state following the substance removing process.
  • the sugar chain purification step is a step in which the re-released sugar chain is separated from the polymer particles 20 and purified.
  • the plate assembly 10 is used by sequentially changing to the second state, the third state, and the fourth state.
  • the second state on the stage 3, the first lower plate 300 and the second lower plate 200 are exchanged in the plate assembly 10 in the first state, and the second lower plate 200 is replaced. Is supported by the second support base 500 (support 700).
  • the third state the first upper plate 100 ′ and the second upper plate 100 ′ are exchanged in the plate assembly 10 in the second state, and the second upper plate 100 ′ becomes the first support base.
  • the second lower plate 200 and the first lower plate 300 are exchanged, and the first lower plate 300 is replaced with the second support base 500 (support 700).
  • the first lower plate 300 and the second lower plate 200 are exchanged in the plate assembly 10 in the third state, and the second lower plate 200 is changed to the second state. This is achieved by supporting the support table 500 (support 700).
  • the moving means 5 is operated to move the nozzle 41 that sucked the particle dispersion 22 onto the plate assembly 10. Further, along with this movement, the particle dispersion liquid 22 (polymer particles 20) is supplied (dispensed) to each well 101 of the first upper plate 100 of the plate assembly 10 respectively.
  • the pure water 21 in the particle dispersion liquid 22 passes through the filter 105.
  • the polymer particles 20 cannot pass through the filter 105 due to the relationship between the pore diameter of the filter 105 and the particle diameter of the polymer particles 20. Therefore, the pure water 21 that has passed through the filter 105 is selectively supplied into the first lower plate 300 (recessed portion 301) through the through hole 104.
  • the moving means 5 is operated and the nozzle 41 is moved to the well of the multi-well plate 81 in which the sugar chain-containing liquid 23 is stored. It inserts in 811 and the pump 42 is operated in that state. As a result, the sugar chain-containing liquid 23 is sucked into the nozzle 41 through the tip opening 411 of the nozzle 41.
  • the sugar chain-containing liquid 23 may contain a volatile organic solvent typified by acetonitrile and an acid (such as acetic acid) as a pH adjuster.
  • 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 solution is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6.
  • pH adjustment can be performed by adding various buffer solution or organic solvent to the sugar_chain
  • the temperature of the reaction solution at the time of sugar chain capture is preferably kept in the temperature range of about 4 to 100 ° C, more preferably about 25 to 90 ° C, still more preferably about 30 to 80 ° C, and most preferably about 60 to 80 ° C. Set to sag.
  • reaction time that is, the time until the sugar chain-containing liquid 23 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. Is done.
  • reaction rate of the polymer particle 20 and the sugar chain can be reliably improved by heating the sugar chain-containing liquid 23 until the sugar chain-containing liquid 23 is dried as in the present embodiment.
  • 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.
  • the reaction of the supplied liquid can proceed in each well 101 of the first upper plate 100. 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 internal space 9 a of the plate assembly 10.
  • these substances are removed by washing with the washing liquid 24.
  • cleaning liquid 24 Water, various buffer solutions, various organic solvents, etc. are mentioned, These can be used in combination as appropriate.
  • the cleaning liquid 24 can pass through the filter 105. As shown in FIG. 11 (12), with the polymer particles 20 remaining on the first upper plate 100, the cleaning liquid 24 is selectively passed from the first upper plate 100 into the first lower plate 300. Can be removed. Moreover, the cleaning substance dissolved in the cleaning liquid 24 can be removed.
  • FIGS. 11 (10) to (12) are repeated a plurality of times, so that the cleaning substance can be separated into the first lower plate 300 in a state where the cleaning substance is dissolved in the cleaning liquid 24. it can. Therefore, it is possible to reliably remove the cleaning substance from the polymer particles 20 in which the sugar chains are captured.
  • the polymer particles 20 are sufficiently washed with water or a buffer solution as the washing solution 24, and then the polymer particles 20 are washed with the washing solution 24 of an organic solvent, and the washing with the washing solution 24 is performed as necessary. It is preferable that the polymer particles 20 be washed repeatedly with an organic solvent washing solution 24 lastly. Thereby, it becomes possible to more reliably remove the cleaning substance, in particular, impurities adsorbed nonspecifically on the surface of the polymer particle 20.
  • Re-release step Here, the sugar chain bonded to the polymer particle 20 is re-released. Further, this sugar chain is substituted with another compound (hereinafter sometimes referred to as “compound A”), that is, the sugar chain is labeled with compound A.
  • compound A a labeling reagent comprising a fluorescent substance, a light-absorbing substance, a radioactive substance and the like is preferably used.
  • the amount of compound A added to the well 101 of the first upper plate 100 is preferably excessive with respect to the polymer particles 20 in which the sugar chains are captured. Thereby, when the compound containing liquid 25 is heated next, the substitution rate of the compound A with respect to a sugar chain can be improved.
  • 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.
  • the sugar chain labeled with the compound A may be referred to as “labeled sugar chain”.
  • the pH of the reaction solution (compound-containing solution 25) at this time is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6.
  • the pH adjustment can be performed, for example, by adding various buffer solutions to the well 101 after supplying the compound-containing solution 25 to each well 101 of the first upper plate 100.
  • the temperature of the reaction solution at the time of labeling is preferably kept in a temperature range of about 4 to 100 ° C., more preferably about 25 to 90 ° C., further preferably about 30 to 80 ° C., and most preferably about 60 to 80 ° C.
  • reaction time that is, the time until the solution dries
  • the reaction time is usually set to about 0.1 to 3 hours, preferably about 0.6 to 2 hours when set in such a temperature range.
  • reaction rate of a sugar chain and the compound A can be improved reliably by heating the compound-containing liquid 25 until the compound-containing liquid 25 is dried as in this embodiment.
  • a compound having an aminooxy group or a hydrazide group is preferably used.
  • N-aminooxyacetyl-tryptophanyl (arginineargmethyl ester) represented by the following chemical formula (3) is particularly preferably used.
  • [12] Sugar chain purification step when the labeled sugar chain is obtained by re-releasing the sugar chain captured by the polymer particles 20 as described above, in addition to the sugar chain labeled with the compound A, The substances contained in the well 101 of the first upper plate 100 include polymer particles 20 from which sugar chains are released and compound A that has not been used for labeling sugar chains (hereinafter referred to as “unused compound A”). Sometimes).
  • the labeled sugar chain is purified by removing the polymer particles 20 and the unused compound A.
  • the moving means 5 is operated, and the nozzle 41 is inserted into the tank 85 in which the solution 26 that is a solution capable of dissolving the dried labeled sugar chain is stored.
  • the pump 42 is operated.
  • the solution 26 is sucked into the nozzle 41 through the tip opening 411 of the nozzle 41.
  • the solution 26 is not particularly limited, and examples thereof include water, various buffer solutions, and various organic solvents.
  • the polymer particles 20 remain in each well 101 of the first upper plate 100, and the well 201 of the second lower plate 200 corresponding to the well 101. Then, the solution 26 is stored. At that time, the labeled sugar chain dissolved in the solution 26 can also be moved to the well 201 of the second lower plate 200. As a result, the labeled sugar chain and the polymer particle 20 are separated. In addition, the unused compound A also usually shows solubility in the solution 26, and thus moves to the well 201 of the second lower plate 200 in a state dissolved in the solution 26.
  • the filter 106 made of silica gel. Adsorb.
  • the adsorptive power to the filter 106 is higher in the labeled sugar chain than in the unused compound A. Accordingly, as shown in FIG. 18 (31), a part of the unused compound A flows (is stored) together with the solution 26 into the first lower plate 300 (recessed portion 301).
  • the filter 106 has a labeling sugar chain and the remaining unused compounds A excluding the unused compounds A flowing into the first lower plate 300 out of the unused compounds A contained in the solution 26.
  • the used compound A is in an adsorbed state.
  • the moving means 5 is operated to move the nozzle 41 that sucked the nonaqueous solvent 27 onto the plate assembly 10. Further, along with this movement, the non-aqueous solvent 27 is supplied to each well 101 ′ of the second upper plate 100 ′ of the plate assembly 10.
  • the amount thereof is such that there is substantially no influence when measuring the amount of the unused compound A using an HPLC or mass spectrometer described later. is there. Further, the labeled sugar chain is still adsorbed on the filter 106.
  • the operator of the refining apparatus 1 mounts the support 700 of the plate assembly 10 on the stage 3 and the second upper plate 100 ′. Is divided into a first support base 400 that supports the first lower plate 300 in which a mixed liquid of the solution 26 and the non-aqueous solvent 27 is stored. This disassembly operation is performed by lifting the first support base 400 together with the second upper plate 100 '.
  • the sugar chain labeled with the compound A is purified using the plate assembly 10.
  • the sugar chain labeled with Compound A can be analyzed by mass spectrometry represented by MALDI-TOFTOMS, and further by a technique such as high performance liquid chromatography (HPLC).
  • mass spectrometry represented by MALDI-TOFTOMS
  • HPLC high performance liquid chromatography
  • N-aminooxyacetyl-tryptophanyl arginine methyl ester
  • HPLC high performance liquid chromatography
  • the first upper plate 100 and the second upper plate 100 ′ can be exchanged with respect to the plate assembly 10 on the stage 3, and the first lower plate 300 and the second lower plate 200 can be exchanged.
  • the first support base 400 and the second support base 500 are configured such that the first support base 400 supports the first upper plate 100 or the second upper plate 100 ′,
  • the support base 500 is configured to be disassembleable and assembleable in a state where the first lower plate 300 or the second lower plate 200 is supported. Then, in a state in which the first support base 400 and the second support base 500 are disassembled, the first upper plate 100 and the second upper plate 100 ′ are exchanged, and the first lower plate 300 Exchange with the second lower plate 200 can be performed. Thereafter, the assembly plate 10 can be easily assembled.
  • the purification apparatus 1 is configured so that the plate can be exchanged easily and quickly on the stage 3.
  • the plate assembly 10 on the stage 3 can be easily and surely set in any one of the first state to the fourth state so as to be suitable for use in each process. Therefore, a large amount of sugar chains can be separated and purified easily and with excellent accuracy.
  • the step of deactivating the functional group included in the capture carrier (polymer particle 20) is performed immediately after the [7] substance removing step or just before the [9] re-releasing step.
  • the functional group functional group that reacts specifically with the aldehyde group of the sugar chain
  • the capture carrier that was not used for capturing the sugar chain in the step [4] is deactivated.
  • 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 and allowing it to stand.
  • a deactivation liquid having a function of deactivating the functional group into contact with the capture carrier and allowing it to stand.
  • acid anhydrides such as an acetic anhydride and a succinic anhydride
  • a functional group can be deactivated easily.
  • the reaction conditions are preferably a quenching solution: 10% acetic anhydride / methanol, temperature: normal temperature (room temperature), and standing time: 30 minutes, but are not limited thereto. Further, the plate assembly 10 is used as the first state.
  • non-aqueous solvent supply is performed between [12-4 (nozzle cleaning)] and [12-5 (plate assembly disassembly)].
  • This non-aqueous solvent supply means that acetonitrile is supplied to each well 101 of the first upper plate 100 and the acetonitrile is sucked.
  • the chain solution can be changed to an acetonitrile solution.
  • the hydrophobicity of a solution increases by adding acetonitrile.
  • the labeled sugar chain (hydrophilic) is easily held by the filter 106 (hydrophilic) made of silica gel.
  • the filter 106 hydrophilic
  • an acetonitrile solution is prepared so that the acetonitrile content is 90% or more (preferably 95%).
  • FIG. 22 is a perspective view showing a second embodiment of the sugar chain purification apparatus of the present invention.
  • This embodiment is the same as the first embodiment except that the configuration of the heating means is different.
  • the heater 61 (heating means 6) is omitted from the stage 3.
  • the heating means 6 ⁇ / b> A is a thermostatic chamber including a chamber 62 disposed at a position different from the stage 3 and a heater 63 that heats the inside of the chamber 62.
  • the chamber 62 has a box shape, and includes a chamber main body 64 having a mouth portion 641 through which the first upper plate 100 is taken in and out, and a door 65 for opening and closing the mouth portion 641.
  • the heater 63 is disposed in the chamber 62 and is configured by a heating wire such as a nichrome wire that generates heat when energized.
  • the operator of the purification apparatus 1 moves the first upper plate 100 into the chamber 62. Thereafter, the heater 63 is operated to heat the liquid.
  • FIG. 23 is a perspective view showing a third embodiment of the sugar chain purification apparatus of the present invention.
  • This embodiment is the same as the first embodiment except that the configuration of the heating means is different.
  • the heater 61 (heating means 6) is omitted from the stage 3 in the purification apparatus 1 of the present embodiment shown in FIG. Instead, the heating means 6B is disposed adjacent to the plate assembly mounting portion 31 of the stage 3 and has a heating device body 66 having a recess 661 filled with a heat medium, and a heater 67 for heating the inside of the recess 661. And a thermostat (heat block).
  • a heat medium For example, liquids, such as water, can be used.
  • the heating device main body 66 has a recess 661 that opens upward in the drawing.
  • the recess 661 can be filled with a heat medium, and the first upper plate 100 can be accommodated in the heating apparatus main body 6 in the filled state.
  • the heater 67 is arranged on the outside of the recess 661, and is configured by a heating wire such as a nichrome wire that generates heat when energized. The heat of the heater 67 is transmitted to the first upper plate 100 through the heat medium.
  • the operator of the purification apparatus 1 moves the first upper plate 100 to the recess 661 filled with the heat medium. Thereafter, the heater 67 is operated to heat the liquid.
  • the timing of the re-releasing of the sugar chain and the labeling of the sugar chain are shifted, that is, the first except that the sugar chain is labeled after the re-releasing of the sugar chain. This is the same as the embodiment.
  • a fluorescent material composed of an aromatic amine is used as compound A.
  • the sugar chain captured on the polymer particle 20 is first separated from the polymer particle 20 and re-released, and then labeled with the compound A. .
  • the sugar chain free solution is heated by operating the heater 61. By doing so, 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 solution 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 into the well 101 in the step [9-1 '], and various buffer solutions are used as the sugar chain free solution.
  • the temperature at the time of sugar chain release is preferably kept in a temperature range of about 4 to 100 ° C., more preferably about 25 to 90 ° C., further preferably about 30 to 80 ° C., and most preferably about 60 to 80 ° C. Set to.
  • reaction time that is, the time until the solution (sugar chain free solution) 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.
  • the sugar chain is reliably separated from the polymer particle 20 by re-releasing the sugar chain.
  • the aromatic amine is not particularly limited, and examples thereof include 2-aminobenzoamide, 2-aminobenzoic acid, 8-aminopyrene-1,3,6-trisulfonate, 8-aminophenylene-1,3,6-trisulfonate, 2-Amino9 (10H) -acridone, 5-Aminofluorescein, dansylethylenediamine, 7-Amino-4-methylcoumarin, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3- (Acetylamino) -6-acidolamino-6-acidolamino-6-acidolamino Of 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.
  • the concentration of the aromatic amine in the solution in the well 101 after the addition of the compound-containing solution 25, that is, the solution containing the re-released sugar chain is preferably 0.5 mol / L or more, more preferably 1 It is set to 4 mol / L or more.
  • the concentration of the aromatic amine is 3 mol / L or more, it becomes difficult to remove the aromatic amine (compound A) that has not been used for the sugar chain labeling reaction in the subsequent sugar chain purification step. There is a fear. Therefore, the most preferable aromatic amine concentration is set to 1.4 mol / L or more and 3 mol / L or less.
  • the amount of the compound-containing solution 25 when the amount of the compound-containing solution 25 is defined by the polymer particles 20 contained in the well 101, the amount of the solution is usually such that the polymer particles 20 are immersed, For example, it is set to about 50 ⁇ L with respect to 5 mg of polymer particles 20. However, in the present embodiment, it is preferable to set the liquid volume (volume) of the compound-containing liquid 25 to about 100 ⁇ L that is twice the normal liquid volume. Thereby, it becomes possible to improve the labeling efficiency of the sugar chain by the compound A.
  • the liquid volume may exceed 100 ⁇ L, but if it exceeds a certain amount, the aromatic amine (compound A) that has not been used for the sugar chain labeling reaction is removed in the subsequent sugar chain purification step. May be difficult. Therefore, the most preferable amount of liquid is set between 100 ⁇ L and 200 ⁇ L.
  • the well 101 is dissolved in 30% acetic acid / dimethyl sulfoxide (DMSO) so as to have a concentration of 1.4 M 2-Aminobenzamid, 1 M sodium cyanoborohydride. 100 ⁇ L of the prepared solution is added as the compound-containing solution 25.
  • DMSO dimethyl sulfoxide
  • the compound-containing liquid 25 is kept in a certain temperature range by operating the heater 61 to heat the compound-containing liquid 25.
  • 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 solution is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6.
  • the temperature of the compound-containing liquid 25 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.
  • the compound-containing liquid 25 is heated in the temperature range of 30 to 70 ° C. and reacted for about 1 to 10 hours.
  • the sugar chain is labeled with the compound A also by the steps [9-1 ′] to [9-4 ′] as described above.
  • the sugar chain purification apparatus and the sugar chain purification method of the present invention have been described with reference to the illustrated embodiment.
  • the present invention is not limited to this, and each part constituting the sugar chain purification apparatus has the same function.
  • the nozzle of the dispensing means is fixed to the nozzle head in each of the above embodiments, but is not limited to this, and may be detachably attached to the nozzle head.
  • the moving means is configured to move the nozzle of the dispensing means with respect to the plate assembly on the stage in each of the above embodiments. It may be configured to move a solid.
  • the sugar chain purification apparatus may omit the heating means.
  • the sugar chain purification apparatus is configured such that, in the plate assembly, the lower side of the filter of each well of the upper plate (first upper plate, second upper plate) has a negative pressure from the upper side.
  • the liquid is allowed to pass through the filter, but the present invention is not limited to this, and the liquid may pass through the filter by setting the upper side of the filter to a positive pressure from the lower side.
  • the exchange of the first upper plate and the second upper plate and the exchange of the first lower plate and the second lower plate of the plate assembly may not be performed on the stage.
  • the movement of the plate, the disassembly of the plate assembly, and the attachment / detachment of the members are performed manually by the operator (operator) of the refining apparatus in each of the above embodiments. You may automate using the means of.
  • the entire sugar chain purification apparatus may be stored in a chamber in which the inside can be heated.
  • the present invention provides a sugar chain purification apparatus.
  • the sugar chain purification apparatus includes a plate assembly used when purifying the sugar chain from a solution containing sugar chains, a stage on which the plate assembly is mounted, and the plate assembly on the stage.
  • a dispensing means having a nozzle for sucking and discharging a liquid and a moving means for relatively moving the plate assembly and the nozzle on the stage are provided.
  • the plate assembly includes a plurality of first wells that are plate-shaped and configured by a first recess opening on an upper surface of the plate assembly, and a first through hole penetrating the bottom of the first recess.
  • a first upper plate having a first filter installed at the bottom of the first recess so as to cover the first through holes of the first wells, and a plate shape;
  • a plurality of second wells each having a second through hole formed with a second recess opening on the upper surface thereof and penetrating the bottom of the second recess, and the second well of each second well.
  • the first upper plate and the second upper plate are exchanged with respect to the one support body, and the first lower plate and the second lower plate are exchanged.
  • the plate is configured to be exchanged. This makes it possible to separate and purify sugar chains easily and with excellent accuracy, and to fluorescently label sugar chains with high yield. Therefore, the present invention has industrial applicability.

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Abstract

Provided is a sugar chain refinement device and a sugar chain refinement method capable of separating and refining large volumes of sugar chains simply and with outstanding precision. A sugar chain refinement device (1) refines sugar chains from a liquid containing sugar chains, and is provided with: a plate assembly (10) formed from a first upper plate (100), a second upper plate (100'), a first lower plate (300), a second lower plate (200), and a support body (700) that supports either the first upper plate (100) or the second upper plate (100') and either the first lower plate (300) or the second lower plate (200) while assembled in a vertical arrangement; and a stage (3) onto which the plate assembly (10) is mounted. In the plate assembly (10), on the stage (3), the first upper plate (100) and the second upper plate (100') are interchanged and the first lower plate (300) and the second lower plate (200) are interchanged.

Description

糖鎖精製装置および糖鎖精製方法Sugar chain purification apparatus and sugar chain purification method
　本発明は、糖タンパク質が有する糖鎖を精製する装置および方法に関する。 The present invention relates to an apparatus and method for purifying a sugar chain possessed by a glycoprotein.
　糖鎖とは、グルコース、ガラクトース、マンノース、フコース、キシロース、Ｎ－アセチルグルコサミン、Ｎ－アセチルガラクトサミン、シアル酸等の単糖およびこれらの誘導体がグリコシド結合によって鎖状に複数結合した分子の総称である。 A sugar chain is a general term for molecules in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid and their derivatives are linked in a chain by glycosidic bonds. .
　糖鎖は、非常に多様性に富んでおり、天然に存在する生物が有する様々な機能に関与する物質である。糖鎖は、生体内でタンパク質や脂質等に結合した複合糖質として存在することが多く、生体内の重要な構成成分の一つである。生体内の糖鎖は、細胞間情報伝達、タンパク質の機能や相互作用の調整等に深く関わっていることが明らかになりつつある。 Sugar chains are very diverse and are substances 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 function and interaction regulation.
　糖鎖を有する生体高分子としては、例えば、細胞の安定化に寄与する植物細胞の細胞壁のプロテオグリカン、細胞の分化、細胞の増殖、細胞の接着、細胞の移動等に影響を与える糖脂質、および細胞間相互作用や細胞認識に関与している糖タンパク質等が挙げられる。これらの高分子の糖鎖が、互いに機能を代行、補助、増幅、調節、あるいは阻害し合いながら、高度で精密な生体反応を制御する機構が次第に明らかにされつつある。さらに、このような糖鎖と細胞の分化増殖、細胞接着、免疫、および細胞の癌化との関係が明確にされれば、この糖鎖工学と、医学、細胞工学、あるいは臓器工学とを密接に関連させて新たな展開を図ることが期待できる（非特許文献１）。 Examples of biopolymers having sugar chains include proteoglycans on the cell walls of plant cells that contribute to cell stabilization, cell differentiation, cell proliferation, cell adhesion, cell migration, and the like, and Examples thereof include glycoproteins involved in cell-cell interaction and cell recognition. The mechanisms by which these high-molecular sugar chains control sophisticated and precise biological reactions while substituting, assisting, amplifying, regulating, or inhibiting each other's functions are gradually being clarified. 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).
　特に細胞表面に存在する糖鎖は、様々な生体反応の足場として重要な役割をしている事が明らかとなってきた。例えば、糖鎖は、レセプターとの相互作用異常による疾病の発生、あるいはエイズウイルスやインフルエンザウイルス等の感染、病原性大腸菌Ｏ１５７の毒素やコレラ毒素の細胞への侵入に関わるとされている。また、ある種の癌細胞では、特異的な糖鎖が細胞表面に現れる。このように、細胞表面の糖鎖は、細胞に個性をあたえる重要な分子とされている。 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, sugar chains are said to be involved in the occurrence of diseases due to abnormal interaction with receptors, infection with AIDS virus, influenza virus, etc., and the entry of pathogenic E. coli O157 toxin and cholera toxin into cells. In some cancer cells, specific sugar chains appear on the cell surface. In this way, sugar chains on the cell surface are considered to be important molecules that give the cells individuality.
　これら疾病の発生などの解析のため、糖鎖構造解析の技術が開発されている。これらの技術は、複合糖質からの糖鎖を切り出し、糖鎖の分離精製、糖鎖の標識化等の工程を組み合わせたものである。しかしながら、これらの工程はきわめて煩雑である。 In order to analyze the occurrence of these diseases, sugar chain structure analysis techniques have been developed. These techniques combine sugar chain separation and purification, sugar chain labeling, and the like, by cutting out sugar chains from complex carbohydrates. However, these processes are extremely complicated.
　さらに、糖鎖の分離精製には、例えば、イオン交換樹脂を用いる方法、逆相クロマトグラフィ、活性炭を用いる方法、ゲル濾過クロマトグラフィ等の手法が用いられる。しかしながら、これらの分離手法は糖を特異的に認識する方法ではないので、莢雑物（ペプチド、タンパク質等）の混入が避けられない。また、糖鎖の構造によって、糖鎖の回収率に差異が生じることが多い。 Furthermore, methods such as a method using an ion exchange resin, a reverse phase chromatography, a method using activated carbon, and a gel filtration chromatography are used for separation and purification of sugar chains. However, since these separation methods are not methods for specifically recognizing sugars, contamination of impurities (peptides, proteins, etc.) is inevitable. In addition, the sugar chain recovery often varies depending on the structure of the sugar chain.
　また、クロマトグラフィで糖鎖を高精度に分離する場合には、糖鎖にピリジルアミノ化等の蛍光標識を施す必要があり、煩雑な操作が必要となる。そして、蛍光標識した糖鎖を分析するには、標識後の反応液中より未反応の２－アミノピリジン等の夾雑物を除き、この標識化糖鎖を精製することが必要である。 In addition, when a sugar chain is separated with high accuracy by chromatography, it is necessary to apply a fluorescent label such as pyridylamination to the sugar chain, which requires complicated operations. In order to analyze the fluorescently labeled sugar chain, it is necessary to remove unreacted impurities such as 2-aminopyridine from the labeled reaction solution and purify the labeled sugar chain.
　一般には、標識化糖鎖と夾雑物の分子量の差を利用してゲルろ過を行い、夾雑物を除去する。しかしながら、この方法は器具を多く用いる点と、操作に多くの時間を要する点から、多数の試料を短時間に処理するのは困難である。 Generally, gel filtration is performed using the difference in molecular weight between the labeled sugar chain and the contaminants to remove the contaminants. However, it is difficult to process a large number of samples in a short time because this method uses many instruments and requires a lot of time for operation.
　また、簡易な方法として共沸により夾雑物を留去する方法も試みられているが、十分に夾雑物を除去するのは難しい。 Also, as a simple method, a method of distilling off impurities by azeotropy has been tried, but it is difficult to sufficiently remove the impurities.
　さらに、糖鎖構造と各種疾患の関係を調べるためには、その関係の統計的処理ができるように、多数の検体の糖鎖構造を調べる必要がある。 Furthermore, in order to investigate the relationship between the sugar chain structure and various diseases, it is necessary to examine the sugar chain structure of a large number of specimens so that the relationship can be statistically processed.
　上述したような従来法では、糖鎖を分離するために、煩雑な工程を経る必要があり、膨大なコストと時間が必要となる。そのため、簡単にかつ優れた精度で多量の糖鎖を分離精製する手法が求められていた。 In the conventional method as described above, it is necessary to go through complicated steps to separate sugar chains, and enormous costs and time are required. Therefore, there has been a demand for a method for separating and purifying a large amount of sugar chains easily and with excellent accuracy.
　さらに、糖鎖に蛍光標識を施す方法は、種々開発されている（例えば、特許文献１、２、３）。しかしながら、その標識化効率は１００％ではなく、１つのサンプルに、標識された糖鎖と、標識されていない糖鎖とが混在していた。この状況は、高速液体クロマトグラフィ(ＨＰＬＣ)や、キャピラリー電気泳動法（ＣＥ法）を用いて糖鎖を蛍光検出している際には大きな問題とならない。しかしながら、質量分析法により糖鎖を分析する際に、マススペクトルやマスクロマトグラムのピークが複雑化するという問題点があった。また、糖鎖への標識化効率が悪い場合、ＨＰＬＣやＣＥによる分析においても、糖鎖を検出する感度が低下するという問題が生じる可能性があった。 Furthermore, various methods for applying fluorescent labels to sugar chains have been developed (for example, Patent Documents 1, 2, and 3). However, the labeling efficiency was not 100%, and a labeled sugar chain and an unlabeled sugar chain were mixed in one sample. This situation does not pose a major problem when the sugar chain is detected by fluorescence using high performance liquid chromatography (HPLC) or capillary electrophoresis (CE method). However, when analyzing sugar chains by mass spectrometry, there is a problem that the peaks of mass spectra and mass chromatograms become complicated. Moreover, when the labeling efficiency to the sugar chain is poor, there is a possibility that the sensitivity for detecting the sugar chain may be lowered in the analysis by HPLC or CE.
特開平７－２０１３１号公報Japanese Patent Laid-Open No. 7-20131 特開２００６－０９８３６７号公報JP 2006-098367 A 特開２００８－３０９５０１号公報JP 2008-309501 A
　本発明の目的は、簡単にかつ優れた精度で多量の糖鎖を分離精製することができる糖鎖精製装置、および、かかる装置を用いた糖鎖精製方法を提供することにある。 An object of the present invention is to provide a sugar chain purification apparatus capable of separating and purifying a large amount of sugar chains easily and with excellent accuracy, and a sugar chain purification method using such an apparatus.
　このような目的は、下記（１）～（２５）の本発明により達成される。 Such an object is achieved by the present invention described in (1) to (25) below.
　（１）　糖鎖を含有する溶液中から前記糖鎖を精製する際に用いられるプレート組立体と、該プレート組立体が載置されるステージと、該ステージ上の前記プレート組立体に対し、液体を吸引し、吐出するノズルを有する分注手段と、前記ステージ上の前記プレート組立体と前記ノズルとを相対的に移動させる移動手段とを備える糖鎖精製装置であって、
　前記プレート組立体は、板状をなし、その上面に開口する第１の凹部で構成され、該第１の凹部の底部を貫通する第１の貫通孔が形成された複数の第１のウェルと、該各第１のウェルの前記第１の貫通孔をそれぞれ覆うように前記第１の凹部の前記底部に設置された第１のフィルタとを有する第１の上側プレートと、
　板状をなし、その上面に開口する第２の凹部で構成され、該第２の凹部の底部を貫通する第２の貫通孔が形成された複数の第２のウェルと、該各第２のウェルの前記第２の貫通孔をそれぞれ覆うように前記第２の凹部の前記底部に設置され、前記第１のフィルタと異なる第２のフィルタとを有する第２の上側プレートと、
　板状をなし、その上面に開口する第３の凹部で構成された１つの第１の貯留部を有する第１の下側プレートと、
　板状をなし、その上面に開口する第４の凹部で構成された複数の第２の貯留部を有する第２の下側プレートと、
　前記第１の上側プレートおよび前記第２の上側プレートのうちの一方と、前記第１の下側プレートおよび前記第２の下側プレートのうちの一方とを上下に配置して組み立てた状態で支持する１つの支持体とを備え、
　前記プレート組立体は、前記ステージ上で、前記１つの支持体に対して、前記第１の上側プレートと前記第２の上側プレートとが交換されるとともに、前記第１の下側プレートと前記第２の下側プレートとが交換されるよう構成されていることを特徴とする糖鎖精製装置。 (1) A plate assembly used when purifying the sugar chain from a solution containing a sugar chain, a stage on which the plate assembly is placed, and a liquid for the plate assembly on the stage. A sugar chain purification apparatus comprising: a dispensing means having a nozzle that sucks and discharges; and a moving means for relatively moving the plate assembly on the stage and the nozzle,
The plate assembly includes a plurality of first wells that are plate-shaped and configured by a first recess opening on an upper surface of the plate assembly, and a first through hole penetrating the bottom of the first recess. A first upper plate having a first filter installed at the bottom of the first recess so as to cover the first through hole of each first well,
A plurality of second wells each having a plate-like shape and configured by a second recess opening on an upper surface thereof and having a second through-hole penetrating the bottom of the second recess; A second upper plate installed at the bottom of the second recess so as to cover the second through-holes of the wells and having a second filter different from the first filter;
A first lower plate having a first storage portion formed of a third recess that is plate-shaped and opens on an upper surface thereof;
A second lower plate having a plurality of second storage portions each having a plate shape and configured by a fourth recess opening on an upper surface thereof;
One of the first upper plate and the second upper plate and one of the first lower plate and the second lower plate are vertically arranged and supported in an assembled state And a single support that
In the plate assembly, the first upper plate and the second upper plate are exchanged with respect to the one support on the stage, and the first lower plate and the first plate are exchanged. 2. A sugar chain purification apparatus, wherein the lower plate is exchanged.
　（２）　前記プレート組立体を加熱する加熱手段さらを備える上記（１）に記載の糖鎖精製装置。 (2) The sugar chain purification apparatus according to (1) above, further comprising a heating means for heating the plate assembly.
　（３）　前記第１の下側プレートと前記第２の下側プレートとの交換が行われるときには、前記加熱手段の作動が停止する上記（２）に記載の糖鎖精製装置。 (3) The sugar chain purification apparatus according to (2), wherein when the first lower plate and the second lower plate are exchanged, the operation of the heating unit is stopped.
　（４）　前記プレート組立体は、前記支持体に前記第１の上側プレートが支持され、前記第１の下側プレートが支持された第１の状態と、
　前記第１の状態において前記第１の下側プレートと前記第２の下側プレートとが交換され、該第２の下側プレートが前記支持体に支持された第２の状態と、
　前記第２の状態において前記第１の上側プレートと前記第２の上側プレートとが交換され、該第２の上側プレートが前記支持体に支持され、前記第２の下側プレートと前記第１の下側プレートとが交換され、該第１の下側プレートが前記支持体に支持された第３の状態と、
　前記第３の状態において前記第１の下側プレートと前記第２の下側プレートとが交換され、該第２の下側プレートが前記支持体に支持された第４の状態とを取り得る上記（２）に記載の糖鎖精製装置。 (4) The plate assembly includes a first state in which the first upper plate is supported by the support and the first lower plate is supported;
A second state in which the first lower plate and the second lower plate are exchanged in the first state, and the second lower plate is supported by the support;
In the second state, the first upper plate and the second upper plate are exchanged, the second upper plate is supported by the support, the second lower plate and the first plate A third state in which the lower plate is replaced and the first lower plate is supported by the support;
In the third state, the first lower plate and the second lower plate are exchanged, and the second lower plate can take a fourth state supported by the support. The sugar chain purification apparatus according to (2).
　（５）　前記第１の状態では、前記各第１のウェルは、その前記第１の貫通孔を介して、前記第１の貯留部と一括して連通しており、
　前記第２の状態では、前記各第１のウェルは、その前記第１の貫通孔を介して、前記各第２の貯留部とそれぞれ連通しており、
　前記第３の状態では、前記各第２のウェルは、その前記第２の貫通孔を介して、前記第１の貯留部と一括して連通しており、
　前記第４の状態では、前記各第２のウェルは、その前記第２の貫通孔を介して、前記各第２の貯留部とそれぞれ連通している上記（４）に記載の糖鎖精製装置。 (5) In the first state, each of the first wells communicates with the first reservoir through the first through hole.
In the second state, each of the first wells communicates with each of the second storage portions via the first through hole,
In the third state, each of the second wells communicates with the first reservoir through the second through hole, and
In the fourth state, each of the second wells communicates with each of the second reservoirs through the second through hole, respectively, and the sugar chain purification apparatus according to (4) above .
　（６）　当該糖鎖精製装置は、前記各第１のウェルにそれぞれ前記糖鎖と特異的に結合する捕捉担体を供給する第１の工程と、
　前記各第１のウェルにそれぞれ前記溶液を前記ノズルから分注して、該溶液と前記捕捉担体とを接触させ、該捕捉担体上に前記糖鎖を捕捉する第２の工程と、
　前記捕捉担体に結合した糖鎖以外の物質を前記ウェルから除去する第３の工程と、
　前記捕捉担体に結合した糖鎖を再遊離させる第４の工程と、
　再遊離した前記糖鎖を前記捕捉担体と分離して精製する第５の工程とを順に行なうよう構成され、
　前記第１の工程、前記第２の工程、前記第３の工程および前記第４の工程では、前記プレート組立体を前記第１の状態とし、前記第５の工程では、前記プレート組立体を順に前記第２の状態、前記第３の状態、前記第４の状態とする上記（５）に記載の糖鎖精製装置。 (6) The sugar chain purification apparatus includes a first step of supplying a capture carrier that specifically binds to the sugar chain to each of the first wells;
A second step of dispensing the solution into the first wells from the nozzle, bringing the solution into contact with the capture carrier, and capturing the sugar chain on the capture carrier;
A third step of removing substances other than sugar chains bound to the capture carrier from the well;
A fourth step of re-releasing the sugar chain bound to the capture carrier;
A fifth step of separating the purified sugar chain from the capture carrier and purifying the sugar chain in sequence,
In the first step, the second step, the third step, and the fourth step, the plate assembly is set to the first state, and in the fifth step, the plate assembly is sequentially changed. The sugar chain purification apparatus according to (5), wherein the second state, the third state, and the fourth state are set.
　（７）　前記加熱手段は、前記第１の工程、前記第２の工程、前記第３の工程、前記第４の工程および前記第５の工程の各工程で、前記プレート組立体または前記第１の上側プレートに対する加熱温度を変更可能に構成されている上記（６）に記載の糖鎖精製装置。 (7) The heating means may be the plate assembly or the first step in each of the first step, the second step, the third step, the fourth step, and the fifth step. The sugar chain refining device according to (6), wherein the heating temperature for the upper plate is changeable.
　（８）　前記加熱手段は、前記ステージに埋設されたヒータを有する上記（２）に記載の糖鎖精製装置。 (8) The sugar chain purification apparatus according to (2), wherein the heating means includes a heater embedded in the stage.
　（９）　前記加熱手段は、ヒータを有する恒温槽である上記（２）に記載の糖鎖精製装置。 (9) The sugar chain purification apparatus according to (2), wherein the heating means is a thermostatic bath having a heater.
　（１０）　前記支持体は、前記第１の上側プレートおよび前記第２の上側プレートのうちの前記一方を支持する上側支持部材と、該上側支持部材と別体で構成され、前記第１の下側プレートおよび前記第２の下側プレートのうちの前記一方を支持する下側支持部材とを有し、
　前記上側支持部材と前記下側支持部材とは、前記上側支持部材が前記第１の上側プレートおよび前記第２の上側プレートのうちの前記一方を支持し、前記下側支持部材が前記第１の下側プレートおよび前記第２の下側プレートのうちの前記一方を支持した状態で、分解可能、組立可能に構成されている上記（１）に記載の糖鎖精製装置。 (10) The support body includes an upper support member that supports the one of the first upper plate and the second upper plate, and a separate body from the upper support member. A lower support member that supports the one of the side plate and the second lower plate;
The upper support member and the lower support member are such that the upper support member supports the one of the first upper plate and the second upper plate, and the lower support member is the first support member. The sugar chain purification apparatus according to (1), wherein the sugar chain purification apparatus is configured to be disassembleable and assembleable in a state where the one of the lower plate and the second lower plate is supported.
　（１１）　前記第１の上側プレートと前記第２の上側プレートとの交換と、前記第１の下側プレートと前記第２の下側プレートとの交換とは、それぞれ、前記上側支持部材と前記下側支持部材とが分解された状態で行なわれる上記（１０）に記載の糖鎖精製装置。 (11) The replacement of the first upper plate and the second upper plate and the replacement of the first lower plate and the second lower plate are respectively the upper support member and the second upper plate. The sugar chain purification apparatus according to (10), which is performed in a state where the lower support member is disassembled.
　（１２）　前記プレート組立体では、前記第１の上側プレートおよび前記第２の上側プレートのうちの前記一方の下面と、前記第１の下側プレートおよび前記第２の下側プレートのうちの前記一方の上面とが離間して配置され、その離間距離が０．５ｍｍ以上１０ｍｍ以下である上記（１）に記載の糖鎖精製装置。 (12) In the plate assembly, the one lower surface of the first upper plate and the second upper plate, the first lower plate and the second lower plate of the first upper plate and the second lower plate. The sugar chain refining device according to (1), wherein the upper surface is spaced apart from each other, and the separation distance is from 0.5 mm to 10 mm.
　（１３）　前記第１のフィルタは、多孔性フィルムまたは不織布で構成されている上記（１）に記載の糖鎖精製装置。 (13) The sugar chain purification apparatus according to (1), wherein the first filter is formed of a porous film or a nonwoven fabric.
　（１４）　前記第１のフィルタの目開きは、０．１～５０μｍである上記（１３）に記載の糖鎖精製装置。 (14) The sugar chain purification apparatus according to (13), wherein the opening of the first filter is 0.1 to 50 μm.
　（１５）　前記第２のフィルタは、シリカゲルで構成されている上記（１）に記載の糖鎖精製装置。 (15) The sugar chain purification apparatus according to (1), wherein the second filter is made of silica gel.
　（１６）　前記分注手段は、ポンプと、該ポンプと前記ノズルとを連結するチューブとを有する上記（１）に記載の糖鎖精製装置。 (16) The sugar chain purification apparatus according to (1), wherein the dispensing unit includes a pump and a tube connecting the pump and the nozzle.
　（１７）　前記移動手段は、前記ノズルを水平方向に移動させる水平方向移動機構と、前記ノズルを鉛直方向に移動させる鉛直方向移動機構とを有する上記（１）に記載の糖鎖精製装置。 (17) The sugar chain purification apparatus according to (1), wherein the moving means includes a horizontal movement mechanism that moves the nozzle in the horizontal direction and a vertical movement mechanism that moves the nozzle in the vertical direction.
　（１８）　前記ステージには、前記第１の上側プレートが複数枚重ねて載置される第１の上側プレート載置部と、前記第２の上側プレートが複数枚重ねて載置される第２の上側プレート載置部と、前記第１の下側プレートが複数枚重ねて載置される第１の下側プレート載置部と、前記第２の下側プレートが複数枚重ねて載置される第２の下側プレート載置部とが設けられている上記（１）に記載の糖鎖精製装置。 (18) On the stage, a first upper plate placing portion on which a plurality of the first upper plates are placed and a second on which a plurality of the second upper plates are placed. An upper plate mounting portion, a first lower plate mounting portion on which a plurality of the first lower plates are stacked, and a plurality of the second lower plates are stacked. The sugar chain purification apparatus according to (1), wherein a second lower plate mounting portion is provided.
　（１９）　前記ノズルを洗浄する洗浄槽をさらに備える上記（１）に記載の糖鎖精製装置。 (19) The sugar chain purification apparatus according to (1), further including a washing tank for washing the nozzle.
　（２０）　上記（６）に記載の糖鎖精製装置を用いて、前記糖鎖を精製する方法であって、
　前記第４の工程において、前記捕捉担体に結合した糖鎖を再遊離させるとともに、該糖鎖をラベル化試薬でラベル化することを特徴とする糖鎖精製方法。 (20) A method for purifying the sugar chain using the sugar chain purification apparatus according to (6) above,
In the fourth step, a sugar chain purification method, wherein the sugar chain bound to the capture carrier is re-released and the sugar chain is labeled with a labeling reagent.
　（２１）　前記第４の工程で、前記捕捉担体からの前記糖鎖の再遊離の後に、前記糖鎖を前記ラベル化試薬でラベル化する上記（２０）に記載の糖鎖精製方法。 (21) The sugar chain purification method according to (20), wherein in the fourth step, the sugar chain is labeled with the labeling reagent after the sugar chain is re-released from the capture carrier.
　（２２）　前記ラベル化試薬は、芳香族アミンで構成される蛍光物質である上記（２１）に記載の糖鎖精製方法。 (22) The sugar chain purification method according to (21), wherein the labeling reagent is a fluorescent substance composed of an aromatic amine.
　（２３）　再遊離した前記糖鎖を含む前記溶液における前記蛍光物質の濃度は、０．５ｍｏｌ／Ｌ以上である上記（２２）に記載の糖鎖精製方法。 (23) The sugar chain purification method according to (22), wherein the concentration of the fluorescent substance in the solution containing the re-released sugar chain is 0.5 mol / L or more.
　（２４）　前記蛍光物質は、２－Ａｍｉｎｏｂｅｎｚａｍｉｄｅ、２－Ａｍｉｎｏｂｅｎｚｏｉｃ  ａｃｉｄ、８－Ａｍｉｎｏｐｙｒｅｎｅ－１，３，６－ｔｒｉｓｕｌｆｏｎａｔｅ、８－Ａｍｉｎｏｎａｐｈｔｈａｌｅｎｅ－１，３，６－ｔｒｉｓｕｌｐｈｏｎａｔｅ、２－Ａｍｉｎｏ９（１０Ｈ）－ａｃｒｉｄｏｎｅ、５－Ａｍｉｎｏｆｌｕｏｒｅｓｃｅｉｎ、Ｄａｎｓｙｌｅｔｈｙｌｅｎｅｄｉａｍｉｎｅ、７－Ａｍｉｎｏ－４－ｍｅｔｈｙｌｃｏｕｍａｒｉｎｅ、３－Ａｍｉｎｏｂｅｎｚｏｉｃ  ａｃｉｄ、７－Ａｍｉｎｏ－１－ｎａｐｈｔｈｏｌ、３－（Ａｃｅｔｙｌａｍｉｎｏ）－６－ａｍｉｎｏａｃｒｉｄｉｎｅから選ばれる少なくとも１種である上記（２２）に記載の糖鎖精製方法。 (24) The fluorescent substances include 2-Aminobenzoamide, 2-Aminobenzoic acid, 8-Aminopyrene-1,3,6-trisulfonate, 8-Aminophathalene-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-aminoacidine described in at least one of the above (22) Sugar chain purification method.
　（２５）　前記捕捉担体は、ヒドラジド基またはオキシルアミノ基を有するポリマー粒子である上記（２０）に記載の糖鎖精製方法。 (25) The sugar chain purification method according to (20), wherein the capture carrier is polymer particles having a hydrazide group or an oxylamino group.
　本発明によれば、糖鎖を簡便にかつ優れた精度で分離精製し、収率よく糖鎖を蛍光標識することが可能となる。さらに、複数の第１のウェルや複数の第２のウェルにそれぞれ供給された液体（溶液）を一括して処理することができるため、一度の処理で、多量の糖鎖を精製、標識することができるようになる。 According to the present invention, it is possible to easily separate and purify sugar chains with excellent accuracy, and to fluorescently label sugar chains with high yield. Furthermore, since liquids (solutions) respectively supplied to a plurality of first wells and a plurality of second wells can be processed at once, a large amount of sugar chains can be purified and labeled in a single process. Will be able to.
図１は、本発明の糖鎖精製装置の第１実施形態を示す斜視図である。FIG. 1 is a perspective view showing a first embodiment of the sugar chain purification apparatus of the present invention. 図２は、図１に示す糖鎖精製装置が備えるプレート組立体を示す斜視図である。FIG. 2 is a perspective view showing a plate assembly provided in the sugar chain purification apparatus shown in FIG. 図３は、図２に示すプレート組立体の分解斜視図である。FIG. 3 is an exploded perspective view of the plate assembly shown in FIG. 図４は、図２中のＢ－Ｂ線断面図（プレート組立体の第１の状態を示す断面図）である。4 is a cross-sectional view taken along line BB in FIG. 2 (a cross-sectional view showing a first state of the plate assembly). 図５は、図２中のＢ－Ｂ線断面図（プレート組立体の第２の状態を示す断面図）である。5 is a cross-sectional view taken along line BB in FIG. 2 (a cross-sectional view showing a second state of the plate assembly). 図６は、図２中のＢ－Ｂ線断面図（プレート組立体の第３の状態を示す断面図）である。6 is a cross-sectional view taken along the line BB in FIG. 2 (a cross-sectional view showing a third state of the plate assembly). 図７は、図２中のＢ－Ｂ線断面図（プレート組立体の第４の状態を示す断面図）である。7 is a cross-sectional view taken along the line BB in FIG. 2 (a cross-sectional view showing a fourth state of the plate assembly). 図８は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。FIG. 8 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図９は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。9 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図１０は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。10 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying a sugar chain with the sugar chain purification apparatus shown in FIG. 図１１は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。11 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図１２は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。12 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図１３は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。FIG. 13 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図１４は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。FIG. 14 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図１５は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。FIG. 15 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図１６は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。16 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying a sugar chain with the sugar chain purification apparatus shown in FIG. 図１７は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。FIG. 17 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図１８は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。18 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図１９は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。FIG. 19 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図２０は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。20 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying the sugar chain with the sugar chain purification apparatus shown in FIG. 図２１は、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。FIG. 21 is a cross-sectional view (cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying a sugar chain with the sugar chain purification apparatus shown in FIG. 図２２は、本発明の糖鎖精製装置の第２実施形態を示す斜視図である。FIG. 22 is a perspective view showing a second embodiment of the sugar chain purification apparatus of the present invention. 図２３は、本発明の糖鎖精製装置の第３実施形態を示す斜視図である。FIG. 23 is a perspective view showing a third embodiment of the sugar chain purification apparatus of the present invention.
　以下、本発明の糖鎖精製装置および糖鎖精製方法を添付図面に示す好適な実施形態に基づいて詳細に説明する。 Hereinafter, the sugar chain purification apparatus and the sugar chain purification method of the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings.
　＜第１実施形態＞
　図１は、本発明の糖鎖精製装置の第１実施形態を示す斜視図、図２は、図１に示す糖鎖精製装置が備えるプレート組立体を示す斜視図、図３は、図２に示すプレート組立体の分解斜視図、図４～図７は、それぞれ、図２中のＢ－Ｂ線断面図（図４は、プレート組立体の第１の状態を示し、図５は、プレート組立体の第２の状態を示し、図６は、プレート組立体の第３の状態を示し、図７は、プレート組立体の第４の状態を示す）、図８～図２１は、それぞれ、図１に示す糖鎖精製装置で糖鎖を精製する工程を順に示す断面図（図１中の矢印Ａ方向から見た断面図）である。なお、以下では、説明の都合上、図１～図２１中の上側を「上」または「上方」、下側を「下」または「下方」と言う。また、水平方向であって装置の横方向（前後方向）をｘ軸方向、水平方向であって前記ｘ軸方向に対し垂直な方向（装置の左右方向）をｙ軸方向、鉛直方向（上下方向）をｚ軸方向と言う。 <First Embodiment>
1 is a perspective view showing a first embodiment of the sugar chain purification apparatus of the present invention, FIG. 2 is a perspective view showing a plate assembly provided in the sugar chain purification apparatus shown in FIG. 1, and FIG. 4 to 7 are sectional views taken along the line BB in FIG. 2 (FIG. 4 shows a first state of the plate assembly, and FIG. FIG. 6 shows a third state of the plate assembly, FIG. 7 shows a fourth state of the plate assembly), and FIGS. FIG. 2 is a cross-sectional view (a cross-sectional view seen from the direction of arrow A in FIG. 1) for sequentially illustrating the steps of purifying a sugar chain with the sugar chain purification apparatus shown in FIG. In the following, for convenience of explanation, the upper side in FIGS. 1 to 21 is referred to as “upper” or “upper”, and the lower side is referred to as “lower” or “lower”. Further, the horizontal direction is the x-axis direction in the horizontal direction (front-rear direction) of the apparatus, the y-axis direction is the direction perpendicular to the x-axis direction (horizontal direction of the apparatus), and the vertical direction (up-down direction). ) Is called the z-axis direction.
　図１に示す糖鎖精製装置（以下単に「精製装置」と言う）１は、糖鎖を含有する溶液（以下「糖鎖含有液」と言う）２３中から糖鎖を精製する糖鎖精製方法に用いられる装置である。精製装置１は、ステージ３と、ステージ３に載置されるプレート組立体１０と、ステージ３上のプレート組立体１０に対して液体を分注する機能を有する分注手段４と、ステージ３上のプレート組立体１０に対して分注手段４のノズル（ピペッタ）４１を移動する移動手段５と、ステージ３上のプレート組立体１０をステージ３ごと加熱する加熱手段６と、ノズル４１を洗浄する洗浄槽７と、分注手段４、移動手段５および加熱手段６の作動を制御する機能を有するコントロールパネル１１とを備えている。以下、各部の構成について説明する。 A sugar chain purification apparatus (hereinafter simply referred to as “purification apparatus”) 1 shown in FIG. 1 is a method for purifying sugar chains from a sugar chain-containing solution (hereinafter referred to as “sugar chain-containing solution”) 23. It is a device used for. The purification apparatus 1 includes a stage 3, a plate assembly 10 placed on the stage 3, a dispensing means 4 having a function of dispensing a liquid to the plate assembly 10 on the stage 3, and a stage 3 The moving means 5 for moving the nozzle (pipettor) 41 of the dispensing means 4 with respect to the plate assembly 10, the heating means 6 for heating the plate assembly 10 on the stage 3 together with the stage 3, and the nozzle 41 are washed. A cleaning tank 7 and a control panel 11 having a function of controlling operations of the dispensing means 4, the moving means 5 and the heating means 6 are provided. Hereinafter, the configuration of each unit will be described.
　図２、図３に示すように、プレート組立体１０は、第１の上側プレート（マルチウェルフィルタプレート）１００と、第２の上側プレート（クリーンアッププレート）１００’と、第１の下側プレート（廃液トレイ）３００と、第２の下側プレート（マイクロウェルプレート）２００と、調整板（スペーサ）６００と、支持体７００とを備えている。すなわち、プレート組立体１０は、図１のステージに示される、複数の第１の上側プレート１００と、複数の第２の上側プレート１００’と、複数の第１の下側プレート３００と、複数の第２の下側プレート２００とを含んでいる。プレート組立体１０において、これら部材同士は、分解可能および組立可能なものである。そして、プレート組立体１０は、各プレートの選択により、４つの組立状態を取り得る。４つの組立状態としては、図４に示す第１の状態と、図５に示す第２の状態と、図６に示す第３の状態と、図７に示す第４の状態とがある。なお、図２は、ステージ３のプレート組立体載置部３１に載置される１組のプレート組立体１０（以下、単に「プレート組立体１０」ともいう）を示している。 As shown in FIGS. 2 and 3, the plate assembly 10 includes a first upper plate (multiwell filter plate) 100, a second upper plate (cleanup plate) 100 ′, and a first lower plate. (Waste liquid tray) 300, second lower plate (microwell plate) 200, adjustment plate (spacer) 600, and support 700 are provided. That is, the plate assembly 10 includes a plurality of first upper plates 100, a plurality of second upper plates 100 ′, a plurality of first lower plates 300, A second lower plate 200. In the plate assembly 10, these members can be disassembled and assembled. And the plate assembly 10 can take four assembly states by selection of each plate. As the four assembled states, there are a first state shown in FIG. 4, a second state shown in FIG. 5, a third state shown in FIG. 6, and a fourth state shown in FIG. FIG. 2 shows a set of plate assemblies 10 (hereinafter also simply referred to as “plate assemblies 10”) mounted on the plate assembly mounting portion 31 of the stage 3.
　第１の状態では、第１の上側プレート１００および第２の上側プレート１００’のうちの第１の上側プレート１００が選択され、第１の下側プレート３００および第２の下側プレート２００のうちの第１の下側プレート３００が選択されている。そして、選択された第１の上側プレート１００および第１の下側プレート３００は、上下に配置されるように組み立てられ、支持体７００に支持される。 In the first state, the first upper plate 100 of the first upper plate 100 and the second upper plate 100 ′ is selected, and the first lower plate 300 and the second lower plate 200 are selected. The first lower plate 300 is selected. The selected first upper plate 100 and first lower plate 300 are assembled so as to be arranged vertically and supported by the support 700.
　第２の状態では、第１の上側プレート１００および第２の上側プレート１００’のうちの第１の上側プレート１００が選択され、第１の下側プレート３００および第２の下側プレート２００のうちの第２の下側プレート２００が選択されている。そして、選択された第１の上側プレート１００および第２の下側プレート２００は、上下に配置されるように組み立てられ、支持体７００に支持される。 In the second state, the first upper plate 100 of the first upper plate 100 and the second upper plate 100 ′ is selected, and the first lower plate 300 and the second lower plate 200 are selected. The second lower plate 200 is selected. Then, the selected first upper plate 100 and second lower plate 200 are assembled so as to be arranged vertically and supported by the support 700.
　第３の状態では、第１の上側プレート１００および第２の上側プレート１００’のうちの第２の上側プレート１００’が選択され、第１の下側プレート３００および第２の下側プレート２００のうちの第１の下側プレート３００が選択されている。そして、選択された第２の上側プレート１００’および第１の下側プレート３００は、上下に配置されるように組み立てられ、支持体７００に支持される。 In the third state, the second upper plate 100 ′ of the first upper plate 100 and the second upper plate 100 ′ is selected, and the first lower plate 300 and the second lower plate 200 are selected. The first lower plate 300 is selected. Then, the selected second upper plate 100 ′ and first lower plate 300 are assembled so as to be arranged vertically and supported by the support 700.
　第４の状態では、第１の上側プレート１００および第２の上側プレート１００’のうちの第２の上側プレート１００’が選択され、第１の下側プレート３００および第２の下側プレート２００のうちの第２の下側プレート２００が選択されている。そして、選択された第２の上側プレート１００’および第２の下側プレート２００は、上下に配置されるように組み立てられ、支持体７００に支持される。 In the fourth state, the second upper plate 100 ′ of the first upper plate 100 and the second upper plate 100 ′ is selected, and the first lower plate 300 and the second lower plate 200 are selected. The second lower plate 200 is selected. Then, the selected second upper plate 100 ′ and second lower plate 200 are assembled so as to be arranged one above the other and supported by the support 700.
　ステージ３は、図１のｙ軸方向に長い平板状をなすものであり、その上面が複数の領域に分けられている。図１に示すように、ステージ３は、プレート組立体載置部３１と、第１の上側プレート載置部３２と、第２の上側プレート載置部３３と、第１の下側プレート載置部３４と、第２の下側プレート載置部３５と、洗浄槽載置部３６と、処理液載置部３７とに分けられている。 The stage 3 has a flat plate shape that is long in the y-axis direction of FIG. 1, and its upper surface is divided into a plurality of regions. As shown in FIG. 1, the stage 3 includes a plate assembly mounting unit 31, a first upper plate mounting unit 32, a second upper plate mounting unit 33, and a first lower plate mounting. It is divided into a part 34, a second lower plate placing part 35, a cleaning tank placing part 36, and a processing liquid placing part 37.
　プレート組立体載置部３１は、ステージ３の長手方向のほぼ中央部に位置し、１組のプレート組立体１０が載置される領域である。プレート組立体載置部３１に載置されたプレート組立体１０に含まれる糖鎖含有液２３から糖鎖が精製される。 The plate assembly placement portion 31 is an area where the set of plate assemblies 10 is placed, which is located at a substantially central portion in the longitudinal direction of the stage 3. The sugar chain is purified from the sugar chain-containing liquid 23 contained in the plate assembly 10 placed on the plate assembly placement unit 31.
　第１の上側プレート載置部３２は、図１の精製装置１に向かってステージ３の右側手前に位置し、第１の上側プレート１００が１枚以上重ねられて載置される領域である。第１の上側プレート載置部３２に載置された各第１の上側プレート１００は、それぞれ、未使用のものであり、交換に用いられる。 The first upper plate placement section 32 is an area where one or more first upper plates 100 are placed on top of each other, positioned in front of the right side of the stage 3 toward the purification apparatus 1 of FIG. Each first upper plate 100 placed on the first upper plate placement portion 32 is unused and used for replacement.
　第２の上側プレート載置部３３は、図１の精製装置１に向かって第１の上側プレート載置部３２よりもさらに右側に位置し、第２の上側プレート１００’が１枚以上重ねられて載置される領域である。第２の上側プレート載置部３３に載置された各第２の上側プレート１００’は、それぞれ、未使用のものであり、交換に用いられる。 The second upper plate placing portion 33 is located further to the right than the first upper plate placing portion 32 toward the purification device 1 in FIG. 1, and one or more second upper plates 100 ′ are stacked. It is an area to be placed. Each second upper plate 100 ′ placed on the second upper plate placing portion 33 is unused and used for replacement.
　第１の下側プレート載置部３４は、図１の精製装置１に向かって第２の上側プレート載置部３３よりもさらに奥側に位置し、第１の下側プレート３００が１枚以上重ねられて載置される領域である。第１の下側プレート載置部３４に載置された各第１の下側プレート３００は、それぞれ、未使用のものであり、交換に用いられる。 The first lower plate mounting portion 34 is located further back than the second upper plate mounting portion 33 toward the purification device 1 in FIG. 1, and one or more first lower plates 300 are provided. It is an area to be placed in an overlapping manner. Each first lower plate 300 placed on the first lower plate placing portion 34 is unused and used for replacement.
　第２の下側プレート載置部３５は、図１の精製装置１に向かって第１の上側プレート載置部３２よりもさらに奥側に位置し、第２の下側プレート２００が１枚以上重ねられて載置される領域である。第２の下側プレート載置部３５に載置された各第２の下側プレート２００は、それぞれ、未使用のものであり、交換に用いられる。 The second lower plate mounting portion 35 is located further back than the first upper plate mounting portion 32 toward the purification device 1 in FIG. 1, and one or more second lower plates 200 are provided. It is an area to be placed in an overlapping manner. Each second lower plate 200 placed on the second lower plate placing portion 35 is unused and used for replacement.
　なお、精製装置１では、各プレートの交換は、精製装置１を操作する操作者の手作業により行なわれる。 In the purification apparatus 1, each plate is exchanged manually by an operator who operates the purification apparatus 1.
　また、プレート組立体１０の詳細な構成については、後述する。 The detailed structure of the plate assembly 10 will be described later.
　洗浄槽載置部３６は、図１の精製装置１に向かってステージ３の左側手前に位置し、洗浄槽７が載置される領域である。洗浄槽載置部３６に載置された洗浄槽７は、図示しない固定機構により固定される。 The washing tank mounting part 36 is located in front of the left side of the stage 3 toward the purification apparatus 1 in FIG. 1 and is an area where the cleaning tank 7 is placed. The cleaning tank 7 mounted on the cleaning tank mounting part 36 is fixed by a fixing mechanism (not shown).
　処理液載置部３７は、図１の精製装置１に向かって洗浄槽載置部３６よりもさらに左側に位置し、各種液体（処理液）がそれぞれ貯留されたマルチウェルプレート８１、タンク８２～８７が載置される領域である。 The processing liquid mounting unit 37 is located further to the left of the cleaning tank mounting unit 36 toward the purification apparatus 1 in FIG. 1, and includes a multiwell plate 81 in which various liquids (processing liquids) are stored, tanks 82 to 87 is an area on which to be placed.
　ステージ３の構成材料としては、特に限定されず、例えば、ステンレス鋼、アルミニウムやアルミニウム合金などの各種金属材料を用いることができる。 The constituent material of the stage 3 is not particularly limited, and various metal materials such as stainless steel, aluminum, and aluminum alloy can be used.
　分注手段４は、ノズル４１と、ノズル４１を支持、固定するノズルヘッド４４と、ポンプ４２と、ノズル４１とポンプ４２とを連結するチューブ４３と有している。 The dispensing unit 4 includes a nozzle 41, a nozzle head 44 that supports and fixes the nozzle 41, a pump 42, and a tube 43 that connects the nozzle 41 and the pump 42.
　ノズル４１は、先端（下端）が開口した管状部材で構成されている。このノズル４１は、ポンプ４２の作動により、先端開口部４１１を介して液体を吸引し、吐出することができる。なお、ノズル４１は、図１に示す構成では１本であるが、これに限定されず、例えば、複数本配置されていてもよい。 The nozzle 41 is composed of a tubular member having an open end (lower end). The nozzle 41 can suck and discharge the liquid through the tip opening 411 by the operation of the pump 42. The number of nozzles 41 is one in the configuration shown in FIG. 1, but is not limited to this. For example, a plurality of nozzles 41 may be arranged.
　ポンプ４２は、例えば歯車ポンプやベーンポンプであり、ステージ３から立設する壁部１２の裏側に配置されている。 The pump 42 is, for example, a gear pump or a vane pump, and is disposed on the back side of the wall portion 12 erected from the stage 3.
　チューブ４３は、ノズル４１とポンプ４２とを連結している。これにより、ノズル４１とポンプ４２とがチューブ４３を介して連通する。また、チューブ４３は、可撓性を有し、移動手段５の作動によってノズル４１が移動した際、その移動に追従することができる。 The tube 43 connects the nozzle 41 and the pump 42. Thereby, the nozzle 41 and the pump 42 communicate with each other through the tube 43. The tube 43 has flexibility, and can follow the movement when the nozzle 41 is moved by the operation of the moving means 5.
　移動手段５は、ｘ軸方向移動機構（水平方向移動機構）５１と、ｙ軸方向移動機構（水平方向移動機構）５２と、ｚ軸方向移動機構（鉛直方向移動機構）５３とを有している。
ｘ軸方向移動機構５１、ｙ軸方向移動機構５２、ｚ軸方向移動機構５３は、それぞれ、例えば、モータ（図示せず）と、モータに連結されたボールネジ（図示せず）と、ボールネジに連結されたリニアガイド（図示せず）とで構成されている。 The moving means 5 includes an x-axis direction moving mechanism (horizontal direction moving mechanism) 51, a y-axis direction moving mechanism (horizontal direction moving mechanism) 52, and a z-axis direction moving mechanism (vertical direction moving mechanism) 53. Yes.
The x-axis direction moving mechanism 51, the y-axis direction moving mechanism 52, and the z-axis direction moving mechanism 53 are, for example, a motor (not shown), a ball screw (not shown) connected to the motor, and a ball screw. And a linear guide (not shown).
　そして、ノズル４１は、ノズルヘッド４４を介してｚ軸方向移動機構５３に支持されており、当該ｚ軸方向移動機構５３によってｚ軸方向に移動することができる。 The nozzle 41 is supported by the z-axis direction moving mechanism 53 via the nozzle head 44 and can be moved in the z-axis direction by the z-axis direction moving mechanism 53.
　また、ｘ軸方向移動機構５１は、ｚ軸方向移動機構５３ごとノズル４１を支持している。これにより、ノズル４１は、ｚ軸方向移動機構５３とともにｘ軸方向に移動することができる。 The x-axis direction moving mechanism 51 supports the nozzle 41 together with the z-axis direction moving mechanism 53. Thereby, the nozzle 41 can move in the x-axis direction together with the z-axis direction moving mechanism 53.
　さらに、ｙ軸方向移動機構５２は、ｘ軸方向移動機構５１を介して、ｚ軸方向移動機構ごとノズル４１を支持している。これにより、ノズル４１は、ｘ軸方向移動機構５１、ｚ軸方向移動機構５３とともにｙ軸方向に移動することができる。 Furthermore, the y-axis direction moving mechanism 52 supports the nozzle 41 together with the z-axis direction moving mechanism via the x-axis direction moving mechanism 51. Accordingly, the nozzle 41 can move in the y-axis direction together with the x-axis direction moving mechanism 51 and the z-axis direction moving mechanism 53.
　このような構成の移動手段５により、ノズル４１がプレート組立体１０とマルチウェルプレート８１、タンク８２～８７との間を往復することができる。 The moving means 5 having such a configuration allows the nozzle 41 to reciprocate between the plate assembly 10, the multiwell plate 81, and the tanks 82 to 87.
　加熱手段６は、ステージ３のプレート組立体載置部３１の直下に埋設されたヒータ６１を有している。ヒータ６１は、通電により発熱するニクロム線等のような電熱線をリング状に巻回して構成されたものである。また、ヒータ６１の面積は、平面視でプレート組立体１０の面積よりも大きい。すなわち、平面視でヒータ６１の内側にプレート組立体１０が配置される。 The heating means 6 has a heater 61 embedded immediately below the plate assembly mounting portion 31 of the stage 3. The heater 61 is configured by winding a heating wire such as a nichrome wire that generates heat when energized in a ring shape. Further, the area of the heater 61 is larger than the area of the plate assembly 10 in plan view. That is, the plate assembly 10 is disposed inside the heater 61 in plan view.
　このようなヒータ６１が作動、すなわち、発熱することにより、その熱がステージ３、プレート組立体１０の支持体７００（第２の支持台（下側支持部材）５００、第１の支持台（上側支持部材）４００）に順に伝わる。これにより、第１の上側プレート１００上の液体を加熱して蒸発させることができる（図９（５）、図１２（１４）参照）。 When such a heater 61 is operated, that is, generates heat, the heat is applied to the stage 3, the support 700 of the plate assembly 10 (second support (lower support member) 500, first support (upper). To the support member 400) in turn. Thereby, the liquid on the 1st upper side plate 100 can be heated and evaporated (refer FIG. 9 (5) and FIG. 12 (14)).
　また、ヒータ６１がステージ３に埋設されていることにより、精製装置１の操作者が誤ってヒータ６１に触れるのを確実に防止することができる。これにより、ヒータ６１が発熱していたとしても、操作者が火傷を負うのを防止または抑止することができる。 Further, since the heater 61 is embedded in the stage 3, it is possible to reliably prevent the operator of the refining apparatus 1 from touching the heater 61 by mistake. Thereby, even if the heater 61 is generating heat, it is possible to prevent or suppress the operator from being burned.
　また、加熱手段６は、ステージ３のプレート組立体載置部３１上の温度を検出する。そして、その検出結果に応じて、ヒータ６１に印加される電圧を制御することができる。これにより、精製装置１で行なわれる各工程（第１の工程、第２の工程、第３の工程、第４の工程および第５の工程）で、プレート組立体１０に対する加熱温度を変更して、最適な温度とすることができ、優れた精度で糖鎖を精製するのに寄与する。 Also, the heating means 6 detects the temperature on the plate assembly mounting portion 31 of the stage 3. And according to the detection result, the voltage applied to the heater 61 can be controlled. Thereby, the heating temperature for the plate assembly 10 is changed in each step (first step, second step, third step, fourth step and fifth step) performed in the purification apparatus 1. Therefore, the temperature can be adjusted to an optimum temperature, which contributes to the purification of sugar chains with excellent accuracy.
　洗浄槽７は、有底筒状をなす部材で構成され、その内側に、ノズル４１を洗浄する洗浄液７１を貯留することができる。ノズル４１は、プレート組立体１０からマルチウェルプレート８１やタンク８２～８７に向かう途中で、毎回、洗浄槽７内に挿入され、当該洗浄槽７中の洗浄液７１で洗浄される。これにより、例えばプレート組立体１０でのコンタミネーションを防止することができる。 The washing tank 7 is composed of a member having a bottomed cylindrical shape, and a washing liquid 71 for washing the nozzle 41 can be stored inside the washing tank 7. The nozzle 41 is inserted into the cleaning tank 7 each time on the way from the plate assembly 10 to the multiwell plate 81 and the tanks 82 to 87, and is cleaned with the cleaning liquid 71 in the cleaning tank 7. Thereby, for example, contamination in the plate assembly 10 can be prevented.
　また、精製装置１では、洗浄液７１は、ノズル４１を洗浄するごとに、すなわち、使用されるごとに、未使用ものに交換される。これにより、前記コンタミネーションを確実に防止することができる。 In the refining device 1, the cleaning liquid 71 is replaced with an unused one every time the nozzle 41 is cleaned, that is, every time it is used. Thereby, the said contamination can be prevented reliably.
　洗浄液７１としては、特に限定されず、例えば、精製水（蒸留水）、メタノール、エタノールに代表されるアルコール類を用いることができる。また、２つ以上の洗浄槽７をステージ３上に設置しておき、２以上の洗浄槽７のそれぞれに貯留される２種類以上の洗浄液７１で連続的に洗浄することもできる。 The cleaning liquid 71 is not particularly limited, and for example, alcohols represented by purified water (distilled water), methanol, and ethanol can be used. It is also possible to install two or more cleaning tanks 7 on the stage 3 and perform continuous cleaning with two or more cleaning liquids 71 stored in each of the two or more cleaning tanks 7.
　コントロールパネル１１は、ステージ３上の第１の上側プレート載置部３２の近傍に配置されている。コントロールパネル１１には、表示部および操作部（入力部）としての液晶画面１１１が設置されている。そして、液晶画面１１１には、例えば精製装置１の各種動作条件を設定する入力画面等が表示される。操作者は、液晶画面１１１を指で触れることにより、各種動作条件の設定等を行なうことができる。この設定により、分注手段４、移動手段５および加熱手段６の作動がそれぞれ制御される。 The control panel 11 is disposed in the vicinity of the first upper plate placing portion 32 on the stage 3. The control panel 11 is provided with a liquid crystal screen 111 as a display unit and an operation unit (input unit). The liquid crystal screen 111 displays, for example, an input screen for setting various operating conditions of the refining device 1. The operator can set various operating conditions by touching the liquid crystal screen 111 with a finger. By this setting, the operations of the dispensing means 4, the moving means 5 and the heating means 6 are controlled.
　前述したように、ステージ３のプレート組立体載置部３１には、１組のプレート組立体１０が載置される。図２、図３に示すように、このプレート組立体１０は、第１の上側プレート１００と、第２の上側プレート１００’と、第１の下側プレート３００と、第２の下側プレート２００と、調整板６００と、支持体７００とを備えている。また、プレート組立体１０は、図４に示す第１の状態と、図５に示す第２の状態と、図６に示す第３の状態と、図７に示す第４の状態との４つの組立状態を取り得る。そして、前記４つの組立状態に応じて、支持体７００は、第１の支持台４００で第１の上側プレート１００または第２の上側プレート１００’を支持するとともに、第２の支持台５００で第１の下側プレート３００または第２の下側プレート２００を支持する。 As described above, one set of plate assemblies 10 is mounted on the plate assembly mounting portion 31 of the stage 3. As shown in FIGS. 2 and 3, the plate assembly 10 includes a first upper plate 100, a second upper plate 100 ′, a first lower plate 300, and a second lower plate 200. And an adjustment plate 600 and a support 700. In addition, the plate assembly 10 has four states: a first state shown in FIG. 4, a second state shown in FIG. 5, a third state shown in FIG. 6, and a fourth state shown in FIG. The assembled state can be taken. The support 700 supports the first upper plate 100 or the second upper plate 100 ′ with the first support base 400 and the second support base 500 according to the four assembled states. The first lower plate 300 or the second lower plate 200 is supported.
　図３に示すように、第１の上側プレート１００は、その全体形状が平板状をなし、その厚さ方向（上下方向）に設けられた、すなわち、その上面１０８に開口する凹部（第１の凹部）で構成された複数（本実施形態では９６個）のウェル（第１のウェル）１０１を備えている。これらのウェル１０１は、行列状（ｘ軸方向に１２行、ｙ軸方向に８列）に配置されており、糖鎖含有液２３中から糖鎖を精製する際に用いられる。 As shown in FIG. 3, the first upper plate 100 has a flat plate shape as a whole and is provided in the thickness direction (vertical direction). A plurality (96 in this embodiment) of wells (first wells) 101 constituted by concave portions are provided. These wells 101 are arranged in a matrix (12 rows in the x-axis direction and 8 columns in the y-axis direction), and are used when purifying sugar chains from the sugar chain-containing liquid 23.
　図３、図４に示すように、これらのウェル１０１は、それぞれ、その下側に底部１０３を備え、さらに、この底部１０３のほぼ中心部に当該底部１０３を貫通する貫通孔（第１の貫通孔）１０４を備えている。これにより、この貫通孔１０４を介して、ウェル１０１に供給（収納）された液体が底部１０３からウェル１０１の外部へ流出される。 As shown in FIGS. 3 and 4, each of these wells 101 has a bottom portion 103 on the lower side thereof, and further, a through hole (first through hole) penetrating the bottom portion 103 at a substantially central portion of the bottom portion 103. Hole) 104. As a result, the liquid supplied (stored) to the well 101 flows out from the bottom 103 to the outside of the well 101 through the through hole 104.
　貫通孔１０４の孔径は、好ましくは０．１～１ｍｍ程度、より好ましくは０．２～０．７ｍｍ程度に設定される。 The hole diameter of the through hole 104 is preferably set to about 0.1 to 1 mm, more preferably about 0.2 to 0.7 mm.
　さらに、各ウェル１０１の下側（底部１０３側）には、それぞれ、その貫通孔１０４を塞ぐ（覆う）ように、膜状のフィルタ（第１のフィルタ）１０５が配置されている。このフィルタ１０５と貫通孔１０４の孔径との関係により、第１の上側プレート１００の静置時には、ウェル１０１に供給された液体が貫通孔１０４を介して、その外部へ流出することが阻害される（阻止される）。これに対して、後述するように、第１の上側プレート１００を第１の支持台４００に装着して、吸引ポンプ１３を用いて、第２の支持台５００が有する凹部５０５内を吸引した際には、前記液体のフィルタ１０５の透過（通過）が許容される。そのため、前記液体が貫通孔１０４を介して、底部１０３側からウェル１０１の外側に流出される。 Furthermore, a membrane-like filter (first filter) 105 is disposed on the lower side (bottom 103 side) of each well 101 so as to close (cover) 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 inhibited from flowing out to the outside through the through hole 104 when the first upper plate 100 is left standing. (Prevented) On the other hand, as will be described later, when the first upper plate 100 is attached to the first support base 400 and the inside of the concave portion 505 of the second support base 500 is sucked using the suction pump 13. In this case, permeation (passage) of the liquid filter 105 is allowed. Therefore, the liquid flows out from the bottom 103 side to the outside of the well 101 through the through hole 104.
　フィルタ１０５の素材としては、特に限定されないが、例えば、多孔性フィルムおよび不織布等が挙げられる。また、その構成材料としては、ポリテトラフルオロエチレン、セルロースエステル、フッ化ビニリデン、ポリカーボネート、ポリエチレン、ポリプロピレンおよびナイロン等が挙げられ、これらのうちの１種または２種以上を組み合わせて用いることができる。 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.
　なお、このフィルタ１０５には、親水処理が施されているのが好ましい。これにより、前記液体の透過性の向上を図ることができる。この親水処理は、例えば、プラズマ処理、コロナ放電、グラフト処理、酸処理などに代表される表面改質、または、界面活性剤、水溶性シリコン、ポリプロピレングリコール等をフィルタ１０５に付与（塗布）等する処理により行われることができる。 Note that the filter 105 is preferably subjected to a hydrophilic treatment. Thereby, the permeability | transmittance of the said liquid can be aimed at. In this hydrophilic treatment, for example, surface modification represented by plasma treatment, corona discharge, graft treatment, acid treatment, or the like, or imparting (coating) a surfactant, water-soluble silicon, polypropylene glycol, or the like to the filter 105 is performed. It can be done by processing.
　フィルタ１０５が備える細孔の孔径、すなわち、フィルタ１０５の目開きは、好ましくは０．１～５０μｍ程度、より好ましくは０．２～２０μｍ程度、さらに好ましくは０．４～１０μｍに設定される。これにより、前記液体に含まれる液状成分の透過は許容されるとともに、後述するようなポリマー粒子２０の透過は確実に許容されなくなる。 The pore diameter of the pores provided in the filter 105, that is, the opening of 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.
　第２の上側プレート１００’は、その全体形状が平板状をなし、その厚さ方向（上下方向）に設けられた、すなわち、その上面１０８’に開口する凹部（第２の凹部）で構成された複数（本実施形態では９６個）のウェル（第２のウェル）１０１’を備えている。これらのウェル１０１’は、第１の上側プレート１００のウェル１０１と同様に、行列状（ｘ軸方向に１２行、ｙ軸方向に８列）に配置されており、糖鎖含有液２３中から糖鎖を精製する際に用いられる。 The second upper plate 100 ′ has a flat plate shape as a whole and is provided in the thickness direction (vertical direction). That is, the second upper plate 100 ′ is configured by a recess (second recess) opened in the upper surface 108 ′. A plurality (96 in this embodiment) of wells (second wells) 101 ′ are provided. These wells 101 ′ are arranged in a matrix (12 rows in the x-axis direction and 8 columns in the y-axis direction), similar to the wells 101 of the first upper plate 100, and from the sugar chain-containing liquid 23. Used when purifying sugar chains.
　図６、図７に示すように、これらのウェル１０１’は、それぞれ、その下側に底部１０３’を備える、さらに、この底部１０３’のほぼ中心部に当該底部１０３’を貫通する貫通孔（第２の貫通孔）１０４’を備える。これにより、この貫通孔１０４’を介して、ウェル１０１’に供給（収納）された液体が底部１０３’側からウェル１０１’の外部へ流出される。 As shown in FIGS. 6 and 7, each of these wells 101 ′ has a bottom 103 ′ on the lower side, and further, a through hole (through the bottom 103 ′ substantially at the center of the bottom 103 ′). 2nd through-hole) 104 '. Accordingly, the liquid supplied (stored) to the well 101 ′ flows out from the bottom 103 ′ to the outside of the well 101 ′ through the through-hole 104 ′.
　さらに、各ウェル１０１の下側（底部１０３側）には、それぞれ、その貫通孔１０４を塞ぐ（覆う）ようにフィルタ（第２のフィルタ）１０６が配置されている。これにより、第２の上側プレート１００’の静置時には、ウェル１０１’に供給された液体が貫通孔１０４’を介して、その外部へ流出することが阻害される（阻止される）。これに対して、後述するように、第２の上側プレート１００’を第１の支持台４００に装着して、吸引ポンプ１３を用いて、第２の支持台５００の凹部５０５内を吸引した際には、前記液体のフィルタ１０６の透過（通過）が許容される。そのため、前記液体が貫通孔１０４’を介して、底部１０３’側からウェル１０１’の外部に流出される。 Furthermore, a filter (second filter) 106 is disposed on the lower side (bottom 103 side) of each well 101 so as to close (cover) the through hole 104. Thereby, when the second upper plate 100 ′ is allowed to stand, the liquid supplied to the well 101 ′ is prevented (blocked) from flowing out to the outside through the through hole 104 ′. On the other hand, as described later, when the second upper plate 100 ′ is mounted on the first support base 400 and the suction pump 13 is used to suck the inside of the recess 505 of the second support base 500. Is allowed to pass through (pass through) the liquid filter 106. Therefore, the liquid flows out of the well 101 'from the bottom 103' side through the through hole 104 '.
　フィルタ１０６は、フィルタ１０５と異なる構成材料で構成され、糖鎖などの対象物の捕捉機能、すなわち、捕捉対象物が異なる。フィルタ１０６は、例えば、シリカゲルで構成されている。 The filter 106 is made of a material different from that of the filter 105, and has a different function of capturing an object such as a sugar chain, that is, an object to be captured. The filter 106 is made of silica gel, for example.
　以上のような第１の上側プレート１００、第２の上側プレート１００’は、支持体７００の第１の支持台４００に装着される。 The first upper plate 100 and the second upper plate 100 ′ as described above are mounted on the first support base 400 of the support 700.
　図３～図７に示すように、第１の支持台４００は、平板状をなし、その中央部で開口する開口部４０２を備える底部４０１と、底部４０１の外縁に沿って上方に突出するように設けられた上側外壁４０３と、底部４０１の外縁に沿って下方に突出するように設けられた下側外壁４０４とを有している。 As shown in FIGS. 3 to 7, the first support base 400 has a flat plate shape, and includes a bottom portion 401 having an opening 402 that opens at the center thereof, and protrudes upward along the outer edge of the bottom portion 401. An upper outer wall 403 provided on the bottom portion 401, and a lower outer wall 404 provided so as to protrude downward along the outer edge of the bottom portion 401.
　そして、上側外壁４０３によって、第１の支持台４００に凹部４０５が形成される。この凹部４０５内に第１の上側プレート１００または第２の上側プレート１００’を挿入することで、当該挿入された第１の上側プレート１００または第２の上側プレート１００’が第１の支持台４００により支持される。 Then, a recess 405 is formed in the first support base 400 by the upper outer wall 403. By inserting the first upper plate 100 or the second upper plate 100 ′ into the recess 405, the inserted first upper plate 100 or the second upper plate 100 ′ becomes the first support base 400. Is supported by
　また、底部４０１の開口部４０２の縁部に沿って溝が設けられている。この溝に対応するようにパッキン（シール部材）４０７が配置されている。これにより、凹部４０５内に第１の上側プレート１００または第２の上側プレート１００’を挿入した際に、開口部４０２とその外部との連通が遮断される。 Further, a groove is provided along the edge of the opening 402 of the bottom 401. A packing (seal member) 407 is disposed so as to correspond to the groove. Accordingly, when the first upper plate 100 or the second upper plate 100 ′ is inserted into the recess 405, the communication between the opening 402 and the outside thereof is blocked.
　なお、上側外壁４０３は、対向する２つの長辺の中央部において、厚さ方向（上下方向）に直交する方向に開口部４０８を有する。これにより、凹部４０５内に挿入（載置）された第１の上側プレート１００または第２の上側プレート１００’を取り出す際に、開口部４０８に指を入れて、当該凹部４０５内に挿入された上側プレートを持ち上げることができ、その取り出し作業を容易に行なうことができる。 Note that the upper outer wall 403 has an opening 408 in a direction perpendicular to the thickness direction (vertical direction) at the center of two opposing long sides. Thereby, when taking out the first upper plate 100 or the second upper plate 100 ′ inserted (placed) in the recess 405, a finger is put into the opening 408 and inserted into the recess 405. The upper plate can be lifted, and the removal work can be easily performed.
　さらに、下側外壁４０４によって、第１の支持台４００に凹部４０６が形成される。この凹部４０６の内周面に沿って、後述する第２の支持台５００が有する突出部５０２を挿入することで、第２の支持台５００上に第１の支持台４００が固定される。 Furthermore, a recess 406 is formed in the first support base 400 by the lower outer wall 404. The first support base 400 is fixed on the second support base 500 by inserting a protrusion 502 included in the second support base 500 described later along the inner peripheral surface of the recess 406.
　なお、本実施形態では、上側外壁４０３の外周面と下側外壁４０４の外周面とが、一体的に形成されていることで、１つの平坦面により構成されている。 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 formed as a single flat surface by being integrally formed.
　図４、図６に示すように、第１の下側プレート３００は、その全体形状が平板状をなし、その上面３０２に開口する１つの凹部（第３の凹部）（第１の貯留部）３０１を有する。 As shown in FIGS. 4 and 6, the first lower plate 300 has a flat plate shape as a whole, and has a single recess (third recess) that opens on the upper surface 302 (first reservoir). 301.
　図３に示すように、第１の下側プレート３００は、凹部３０１が形成された凹部形成部３０３と、この凹部形成部３０３の周囲を取り囲むように配置されたフレーム部３０４とを有する。凹部形成部３０３の厚さは、フレーム部３０４の厚さより厚くなっている。これにより、凹部形成部３０３の上面３０２は、フレーム部３０４の上面から突出する。この凹部形成部３０３の平面視形状は、第１の支持台４００の開口部４０２に挿入可能な大きさに設定されている。従って、第２の支持台５００の凹部５０５内に第１の下側プレート３００を配置した状態（図４、図６参照）で、第２の支持台５００上に第１の支持台４００を載置すると、凹部形成部３０３を、第１の支持台４００の開口部４０２内に挿入させることができる。 As shown in FIG. 3, the first lower plate 300 includes a concave portion forming portion 303 in which a concave portion 301 is formed, and a frame portion 304 arranged so as to surround the concave portion forming portion 303. The thickness of the recess forming portion 303 is larger than the thickness of the frame portion 304. Thereby, the upper surface 302 of the recess forming part 303 protrudes from the upper surface of the frame part 304. The shape of the recess forming portion 303 in plan view is set to a size that can be inserted into the opening 402 of the first support base 400. Accordingly, the first support base 400 is mounted on the second support base 500 in a state where the first lower plate 300 is disposed in the recess 505 of the second support base 500 (see FIGS. 4 and 6). When placed, the recess forming portion 303 can be inserted into the opening 402 of the first support base 400.
　なお、凹部形成部３０３の上面３０２は、前述したようにフレーム部３０４の上面から突出しているが、これに限定されず、フレーム部３０４の上面と同じ高さであってもよい。この場合も、糖鎖を確実に分離精製することができる。 In addition, although the upper surface 302 of the recessed part formation part 303 protrudes from the upper surface of the frame part 304 as mentioned above, it is not limited to this, The same height as the upper surface of the frame part 304 may be sufficient. Also in this case, the sugar chain can be reliably separated and purified.
　図５、図７に示すように、第２の下側プレート２００は、第１の下側プレート３００と同様に、その全体形状が平板状をなし、その厚さ方向（上下方向）に設けられた、その上面２０４に開口する凹部（第４の凹部）で構成された複数（本実施形態では９６個）のウェル（第２の貯留部）２０１を備えている。これらのウェル２０１は、行列状（ｘ軸方向に１２行、ｙ軸方向に８列）に配置されている。 As shown in FIG. 5 and FIG. 7, the second lower plate 200 has a flat plate shape as in the first lower plate 300 and is provided in the thickness direction (vertical direction). In addition, a plurality (96 in the present embodiment) of wells (second reservoirs) 201 each including a recess (fourth recess) opened on the upper surface 204 is provided. These wells 201 are arranged in a matrix (12 rows in the x-axis direction and 8 columns in the y-axis direction).
　これらのウェル２０１は、それぞれ、その下側に底部２０３を備えている。これにより、ウェル２０１に供給（収納）された液体がその内部に貯留される。 Each of these wells 201 has a bottom 203 on the lower side thereof. Thereby, the liquid supplied (stored) to the well 201 is stored therein.
　また、図３に示すように、第２の下側プレート２００は、複数のウェル２０１が形成されたウェル形成部２０２と、このウェル形成部２０２の周囲を取り囲むように配置されたフレーム部２０５とを有する。ウェル形成部２０２の厚さは、フレーム部２０５の厚さより厚くなっている。これにより、ウェル形成部２０２の上面は、フレーム部２０５の上面から突出する。このウェル形成部２０２は、その平面視形状で、第１の支持台４００の開口部４０２に挿入可能な大きさに設定されている。従って、第２の支持台５００の凹部５０５内に第２の下側プレート２００を配置した状態（図５、図７参照）で、第２の支持台５００上に第１の支持台４００を載置すると、ウェル形成部２０２を、第１の支持台４００の開口部４０２内に挿入させることができる。 As shown in FIG. 3, the second lower plate 200 includes a well forming portion 202 in which a plurality of wells 201 are formed, and a frame portion 205 disposed so as to surround the well forming portion 202. Have The thickness of the well forming portion 202 is thicker than the thickness of the frame portion 205. As a result, the upper surface of the well forming portion 202 protrudes from the upper surface of the frame portion 205. The well forming portion 202 is set to a size that can be inserted into the opening 402 of the first support base 400 in a plan view. Accordingly, the first support base 400 is mounted on the second support base 500 in a state where the second lower plate 200 is disposed in the recess 505 of the second support base 500 (see FIGS. 5 and 7). Then, the well forming part 202 can be inserted into the opening 402 of the first support base 400.
　調整板６００は、第１の下側プレート３００または第２の下側プレート２００に先立って、第２の支持台５００が備える凹部５０５内に挿入して用いられるものである。 Prior to the first lower plate 300 or the second lower plate 200, the adjustment plate 600 is used by being inserted into the recess 505 provided in the second support base 500.
　この調整板６００は、平板状をなしており、その厚さの異なるものが複数枚用意されている。 The adjusting plate 600 has a flat plate shape, and a plurality of plates having different thicknesses are prepared.
　そして、凹部５０５内に挿入される第１の下側プレート３００、第２の下側プレート２００の種類に応じて、適切な厚さの調整板６００を選択することで、以下のような効果を得ることができる。第１の状態で第１の上側プレート１００と第１の下側プレート３００とをできる限り接近させることができる。第２の状態で第１の上側プレート１００と第２の下側プレート２００とをできる限り接近させることができる。第３の状態で第２の上側プレート１００’と第１の下側プレート３００とをできる限り接近させることができる。第４の状態で第２の上側プレート１００’と第２の下側プレート２００とをできる限り接近させることができる。特に、第２の状態で第１の上側プレート１００と第２の下側プレート２００とをできる限り接近させることができ、第４の状態で第２の上側プレート１００’と第２の下側プレート２００とをできる限り接近させることができるのは、後述する工程で有効である。 Then, by selecting the adjustment plate 600 having an appropriate thickness according to the type of the first lower plate 300 and the second lower plate 200 inserted into the recess 505, the following effects can be obtained. Obtainable. In the first state, the first upper plate 100 and the first lower plate 300 can be brought as close as possible. In the second state, the first upper plate 100 and the second lower plate 200 can be brought as close as possible. In the third state, the second upper plate 100 ′ and the first lower plate 300 can be as close as possible. In the fourth state, the second upper plate 100 ′ and the second lower plate 200 can be brought as close as possible. In particular, the first upper plate 100 and the second lower plate 200 can be brought as close as possible in the second state, and the second upper plate 100 ′ and the second lower plate in the fourth state. It is effective in the process described later that 200 can be brought as close as possible.
　第２の支持台５００は、第１の下側プレート３００または第２の下側プレート２００を支持するための部材である。図４～図７に示すように、第２の支持台５００は、第１の支持台４００と別体で構成され、平板状をなす底部５０１と、底部５０１の外縁に沿って上方に突出するように設けられた外壁５０３とを有している。 The second support base 500 is a member for supporting the first lower plate 300 or the second lower plate 200. As shown in FIGS. 4 to 7, the second support base 500 is configured separately from the first support base 400, and protrudes upward along the flat bottom 501 and the outer edge of the bottom 501. The outer wall 503 is provided.
　このような外壁５０３で取り囲まれることにより、その内側に凹部５０５が形成される。この凹部５０５内に第１の下側プレート３００または第２の下側プレート２００を挿入することで、第１の下側プレート３００または第２の下側プレート２００が第２の支持台５００により支持される。 By being surrounded by such an outer wall 503, a recess 505 is formed inside thereof. The first lower plate 300 or the second lower plate 200 is supported by the second support base 500 by inserting the first lower plate 300 or the second lower plate 200 into the recess 505. Is done.
　また、外壁５０３は、外壁５０３の内縁に沿って上方に突出する突出部５０２を有している。この突出部５０２は、第１の支持台４００が備える凹部４０６の内周面に沿って、挿入されるように設定されている。これにより、第２の支持台５００上に第１の支持台４００を載置した際に、突出部５０２が第１の支持台４００の凹部４０６に挿入されることで、第２の支持台５００上に第１の支持台４００が固定される。 The outer wall 503 has a protruding portion 502 that protrudes upward along the inner edge of the outer wall 503. The protrusion 502 is set to be inserted along the inner peripheral surface of the recess 406 provided in the first support base 400. As a result, when the first support base 400 is placed on the second support base 500, the protrusion 502 is inserted into the concave portion 406 of the first support base 400, whereby the second support base 500. The first support base 400 is fixed on the top.
　外壁５０３の上面には、突出部５０２を取り囲むように溝が設けられている。この溝に対応するようにパッキン（シール部材）５０７が配置されている。これにより、第２の支持台５００上に第１の支持台４００を載置した際に、第１の支持台４００、第２の支持台５００同士間で、プレート組立体１０の内部と外部との連通が遮断される。 A groove is provided on the upper surface of the outer wall 503 so as to surround the protruding portion 502. A packing (seal member) 507 is disposed so as to correspond to the groove. As a result, when the first support base 400 is placed on the second support base 500, between the first support base 400 and the second support base 500, the inside and the outside of the plate assembly 10 Is disconnected.
　さらに、図３に示すように、外壁５０３は、外壁５０３の側面に、外壁５０３を貫通する貫通孔５０６を有している。図１に示すように、貫通孔５０６には、ステージ３から立設する壁部１２の裏側にポンプ４２に隣接して配置され、真空ポンプで構成された吸引ポンプ１３が、チューブ１４を介して、連結されている。これにより、吸引ポンプ１３を作動させることで、凹部５０５内を減圧することができる。 Further, as shown in FIG. 3, the outer wall 503 has a through hole 506 penetrating the outer wall 503 on the side surface of the outer wall 503. As shown in FIG. 1, the through-hole 506 is disposed adjacent to the pump 42 on the back side of the wall portion 12 erected from the stage 3, and a suction pump 13 constituted by a vacuum pump is connected via the tube 14. Are connected. Thereby, the inside of the recessed part 505 can be pressure-reduced by operating the suction pump 13.
　なお、第１の上側プレート１００、第２の上側プレート１００’、第１の下側プレート３００、第２の下側プレート２００の構成材料としては、特に限定されないが、例えば、ポリプロピレン、ポリエチレン、ポリスチレン、ポリ塩化ビニル、ポリテトラフルオロエチレン等の樹脂材料が挙げられ、これらのうちの１種または２種以上を組み合わせて用いることができる。 The constituent materials of the first upper plate 100, the second upper plate 100 ′, the first lower plate 300, and the second lower plate 200 are not particularly limited. For example, polypropylene, polyethylene, polystyrene And resin materials such as polyvinyl chloride and polytetrafluoroethylene can be used, and one or more of them can be used in combination.
　また、第１の支持台４００、第２の支持台５００の構成材料としては、前記樹脂材料を用いることができる。その構成材料は、例えばステンレス鋼等のＦｅ系合金、ＣｕまたはＣｕ系合金、ＡｌまたはＡｌ系合金のような金属系材料、アルミナ、アパタイト、窒化アルミのようなセラミックス系材料等が挙げられ、これらのうちの１種または２種以上を組み合わせて用いることができる。第１の支持台４００、第２の支持台５００の構成材料として金属材料を用いることにより、ヒータ６１からの熱がステージ３、第２の支持台５００、第１の支持台４００を順に伝わり、第１の上側プレート１００上の液体を確実に加熱して蒸発させることができる（図９（５）、図１２（１４）参照）。 Further, as the constituent material of the first support base 400 and the second support base 500, the resin material can be used. Examples of the constituent materials include Fe-based alloys such as stainless steel, Cu- or Cu-based alloys, metal-based materials such as Al or Al-based alloys, and ceramic-based materials such as alumina, apatite, and aluminum nitride. 1 type or 2 types or more can be used in combination. By using a metal material as the constituent material of the first support base 400 and the second support base 500, heat from the heater 61 is transmitted in order through the stage 3, the second support base 500, and the first support base 400, The liquid on the first upper plate 100 can be reliably heated and evaporated (see FIGS. 9 (5) and 12 (14)).
　次に、プレート組立体１０での第１の状態、第２の状態、第３の状態、第４の状態について説明する。プレート組立体１０を第１の状態、第２の状態、第３の状態、第４の状態のいずれの状態とするのかは、当該プレート組立体１０が、後述する精製装置１で糖鎖を精製する各工程のうちのどの工程を経ているのかで選択される。 Next, the first state, the second state, the third state, and the fourth state in the plate assembly 10 will be described. Whether the plate assembly 10 is in the first state, the second state, the third state, or the fourth state depends on whether the plate assembly 10 purifies sugar chains with the purification device 1 described later. It is selected according to which process among the processes to be performed.
　なお、プレート組立体１０では、第１の上側プレート１００のウェル１０１同士のピッチと、第２の上側プレート１００’のウェル１０１’同士のピッチと、第２の下側プレート２００のウェル２０１同士のピッチとは、互いに同じ大きさに設定されている。 In the plate assembly 10, the pitch between the wells 101 of the first upper plate 100, the pitch between the wells 101 ′ of the second upper plate 100 ′, and the wells 201 of the second lower plate 200. The pitch is set to the same size.
　図４に示すように、第１の状態では、第１の支持台４００の凹部４０５に第１の上側プレート１００が挿入され、さらに第２の支持台５００の凹部５０５に調整板６００および第１の下側プレート３００が挿入されている。この状態で、第２の支持台５００上に、第１の支持台４００が載置される。このとき、第１の下側プレート３００の凹部形成部３０３が第１の支持台４００の開口部４０２に挿入され、さらに、第２の支持台５００の突出部５０２が第１の支持台４００の凹部４０６に挿入される。 As shown in FIG. 4, in the first state, the first upper plate 100 is inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the first plate are further inserted into the recess 505 of the second support base 500. A lower plate 300 is inserted. In this state, the first support base 400 is placed on the second support base 500. At this time, the recessed portion forming portion 303 of the first lower plate 300 is inserted into the opening 402 of the first support base 400, and the protruding portion 502 of the second support base 500 is further inserted into the first support base 400. It is inserted into the recess 406.
　このような組立により、第１の上側プレート１００の各ウェル１０１は、その貫通孔１０４を介して、第１の下側プレート３００の凹部３０１と一括して連通している。この状態で、第２の支持台５００上に第１の支持台４００が固定される。 By such an assembly, each well 101 of the first upper plate 100 communicates with the recess 301 of the first lower plate 300 through the through-hole 104 in a lump. In this state, the first support base 400 is fixed on the second support base 500.
　また、かかる状態では、パッキン５０７により、第１の支持台４００、第２の支持台５００同士間で、プレート組立体１０の内部と外部との連通が遮断される。また、パッキン４０７により、第１の支持台４００と第１の上側プレート１００との間での連通が遮断される。これにより、第１の上側プレート１００と、第１の支持台４００と、第２の支持台５００とで形成される（画成される）内部空間９ａを、外部に対して閉鎖された閉鎖空間とすることができる。 In this state, the packing 507 blocks communication between the inside and the outside of the plate assembly 10 between the first support base 400 and the second support base 500. Further, the packing 407 blocks communication between the first support base 400 and the first upper plate 100. Accordingly, the internal space 9a formed (defined) by the first upper plate 100, the first support base 400, and the second support base 500 is closed with respect to the outside. It can be.
　そして、貫通孔５０６に連結された吸引ポンプ１３を作動させて、内部空間９ａ（凹部５０５）を減圧することで、当該内部空間９ａをその外部に対して負圧とすることができる。そのため、第１の上側プレート１００の各ウェル１０１内では、それぞれ、フィルタ１０５を介して、その上下で圧力差が生じることから、この圧力差によって、ウェル１０１内の液体がフィルタ１０５を透過する。従って、各ウェル１０１から貫通孔１０４を介して液体を流出させて、第１の下側プレート３００の凹部３０１内に一括して液体を貯留することができる。 And by operating the suction pump 13 connected to the through-hole 506 to depressurize the internal space 9a (recessed portion 505), the internal space 9a can be made negative with respect to the outside. Therefore, in each well 101 of the first upper plate 100, a pressure difference is generated between the upper and lower sides of the well 105 through the filter 105. Therefore, the liquid in the well 101 passes through the filter 105 due to the pressure difference. Therefore, the liquid can flow out from each well 101 through the through-hole 104 and can be stored in the recess 301 of the first lower plate 300 in a lump.
　なお、ウェル１０１内の液体がフィルタ１０５を透過するには、本実施形態ではフィルタ１０５の下側をその上側より負圧とする圧力差を利用しているが、これに限定されず、例えば、フィルタ１０５の上側に位置する液体に付与する遠心力や、重力を利用することも可能である。例えば、ウェル１０１内の液体の粘度等が比較的低く、当該液体がフィルタ１０５を容易に透過する場合には、重力すなわち自由落下により、液体がフィルタ１０５を透過するようにすることができる。 In order to allow the liquid in the well 101 to pass through the filter 105, in the present embodiment, a pressure difference in which the lower side of the filter 105 is negative from the upper side thereof is used. Centrifugal force applied to the liquid located above the filter 105 or gravity can be used. For example, when the viscosity of the liquid in the well 101 is relatively low and the liquid easily passes through the filter 105, the liquid can pass through the filter 105 by gravity, that is, free fall.
　図５に示すように、第２の状態では、第１の支持台４００の凹部４０５に第１の上側プレート１００が挿入され、さらに第２の支持台５００の凹部５０５に調整板６００および第２の下側プレート２００が挿入されている。この状態で、第２の支持台５００上に、第１の支持台４００が載置されている。このとき、第２の下側プレート２００のウェル形成部２０２が第１の支持台４００の開口部４０２に挿入され、さらに、第２の支持台５００の突出部５０２が第１の支持台４００の凹部４０６に挿入される。 As shown in FIG. 5, in the second state, the first upper plate 100 is inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the second plate are further inserted into the recess 505 of the second support base 500. The lower plate 200 is inserted. In this state, the first support base 400 is placed on the second support base 500. At this time, the well forming portion 202 of the second lower plate 200 is inserted into the opening 402 of the first support base 400, and the protrusion 502 of the second support base 500 is further inserted into the first support base 400. It is inserted into the recess 406.
　このような組立により、第１の上側プレート１００の各ウェル１０１は、その貫通孔１０４を介して、第２の下側プレート２００のウェル２０１とそれぞれ連通している。この状態で、第２の支持台５００上に第１の支持台４００が固定される。 With this assembly, each well 101 of the first upper plate 100 communicates with the well 201 of the second lower plate 200 through the through hole 104. In this state, the first support base 400 is fixed on the second support base 500.
　また、第２の状態でも、内部空間９ａを、外部に対して閉鎖された閉鎖空間とすることができる。そして、貫通孔５０６に連結された吸引ポンプ１３を作動させて、内部空間９ａ（凹部５０５）を減圧することで、各ウェル１０１から貫通孔１０４を介して液体を流出させることができる。その結果、各ウェル１０１から流出した液体を、第２の下側プレート２００のウェル２０１にそれぞれ独立して貯留することができる。 Also in the second state, the internal space 9a can be a closed space closed to the outside. Then, by operating the suction pump 13 connected to the through-hole 506 and depressurizing the internal space 9 a (recess 505), 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 independently in each well 201 of the second lower plate 200.
　図６に示すように、第３の状態では、第１の支持台４００の凹部４０５に第２の上側プレート１００’が挿入され、さらに第２の支持台５００の凹部５０５に調整板６００および第１の下側プレート３００が挿入されている。この状態で、第２の支持台５００上に、第１の支持台４００が載置される。このとき、第１の状態と同様に、第１の下側プレート３００の凹部形成部３０３が第１の支持台４００の開口部４０２に挿入され、さらに、第２の支持台５００の突出部５０２が第１の支持台４００の凹部４０６に挿入される。 As shown in FIG. 6, in the third state, the second upper plate 100 ′ is inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the first plate are further inserted into the recess 505 of the second support base 500. 1 lower plate 300 is inserted. In this state, the first support base 400 is placed on the second support base 500. At this time, as in the first state, the recess forming portion 303 of the first lower plate 300 is inserted into the opening 402 of the first support base 400, and the protrusion 502 of the second support base 500 is further inserted. Is inserted into the recess 406 of the first support base 400.
　このような組立により、第２の上側プレート１００’の各ウェル１０１’は、その貫通孔１０４’を介して、第１の下側プレート３００の凹部３０１と一括して連通している。この状態で、第２の支持台５００上に第１の支持台４００が固定される。 By such assembly, each well 101 ′ of the second upper plate 100 ′ is in communication with the recess 301 of the first lower plate 300 through the through hole 104 ′. In this state, the first support base 400 is fixed on the second support base 500.
　また、かかる状態では、パッキン５０７により、第１の支持台４００、第２の支持台５００同士間で、プレート組立体１０の内部と外部との連通が遮断される。また、パッキン４０７により、第１の支持台４００と第２の上側プレート１００’との間での連通が遮断される。これにより、第２の上側プレート１００’と、第１の支持台４００と、第２の支持台５００とで形成される（画成される）内部空間９ｂを、外部に対して閉鎖された閉鎖空間とすることができる。 In this state, the packing 507 blocks communication between the inside and the outside of the plate assembly 10 between the first support base 400 and the second support base 500. Further, the packing 407 blocks communication between the first support base 400 and the second upper plate 100 ′. Accordingly, the internal space 9b formed (defined) by the second upper plate 100 ′, the first support base 400, and the second support base 500 is closed with respect to the outside. It can be a space.
　そして、貫通孔５０６に連結された吸引ポンプ１３を作動させて、内部空間９ｂ（凹部５０５）を減圧することで、当該内部空間９ｂをその外部に対して負圧とすることができる。そのため、第２の上側プレート１００’の各ウェル１０１’内では、それぞれ、フィルタ１０６を介して、その上下で圧力差が生じることから、この圧力差によって、ウェル１０１’内の液体がフィルタ１０６を透過する。従って、各ウェル１０１’から貫通孔１０４’を介して液体を流出させて、第１の下側プレート３００の凹部３０１内に一括して液体を貯留することができる。 Then, by operating the suction pump 13 connected to the through hole 506 to depressurize the internal space 9b (recessed portion 505), the internal space 9b can be made negative with respect to the outside. Therefore, in each well 101 ′ of the second upper plate 100 ′, a pressure difference is generated between the upper and lower sides through the filter 106, so that the liquid in the well 101 ′ causes the filter 106 to pass through the pressure difference. To Penetrate. Accordingly, the liquid can flow out from each well 101 ′ through the through hole 104 ′ and can be stored in the recess 301 of the first lower plate 300 in a lump.
　図７に示すように、第４の状態では、第１の支持台４００の凹部４０５に第２の上側プレート１００’が挿入され、さらに第２の支持台５００の凹部５０５に調整板６００および第２の下側プレート２００が挿入されている。この状態で、第２の支持台５００上に、第１の支持台４００が載置されている。このとき、第２の下側プレート２００のウェル形成部２０２が第１の支持台４００の開口部４０２に挿入され、さらに、第２の支持台５００の突出部５０２が第１の支持台４００の凹部４０６に挿入される。 As shown in FIG. 7, in the fourth state, the second upper plate 100 ′ is inserted into the recess 405 of the first support base 400, and the adjustment plate 600 and the first plate are further inserted into the recess 505 of the second support base 500. Two lower plates 200 are inserted. In this state, the first support base 400 is placed on the second support base 500. At this time, the well forming portion 202 of the second lower plate 200 is inserted into the opening 402 of the first support base 400, and the protrusion 502 of the second support base 500 is further inserted into the first support base 400. It is inserted into the recess 406.
　このような組立により、第２の上側プレート１００’の各ウェル１０１’は、その貫通孔１０４’を介して、第２の下側プレート２００のウェル２０１とそれぞれ連通している。この状態で、第２の支持台５００上に第１の支持台４００が固定される。 With this assembly, each well 101 'of the second upper plate 100' communicates with the well 201 of the second lower plate 200 through the through hole 104 '. In this state, the first support base 400 is fixed on the second support base 500.
　また、第４の状態でも、内部空間９ｂを、外部に対して閉鎖された閉鎖空間とすることができる。そして、貫通孔５０６に連結された吸引ポンプ１３を作動させて、内部空間９ｂ（凹部５０５）を減圧することで、各ウェル１０１’から貫通孔１０４’を介して液体を流出させることができる。その結果、各ウェル１０１’から流出した液体を、第２の下側プレート２００のウェル２０１にそれぞれ独立して貯留することができる。 Also in the fourth state, the internal space 9b can be a closed space closed to the outside. Then, by operating the suction pump 13 connected to the through hole 506 and depressurizing the internal space 9b (recessed portion 505), 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 independently in each well 201 of the second lower plate 200.
　前述したように、プレート組立体１０では、調整板６００により、第１の状態で第１の上側プレート１００の下面１０９と第１の下側プレート３００の上面３０２とをできる限り接近させて、これらの離間距離ｄ１をできる限り小さく設定することができる。第２の状態でも、第１の上側プレート１００の下面１０９と第２の下側プレート２００の上面２０４とをできる限り接近させて、これらの離間距離ｄ２をできる限り小さく設定することができる。第３の状態でも、第２の上側プレート１００’の下面１０９’と第１の下側プレート３００の上面３０２とをできる限り接近させて、これらの離間距離ｄ３をできる限り小さく設定することができる。第４の状態でも、第２の上側プレート１００’の下面１０９’と第２の下側プレート２００の上面２０４とをできる限り接近させて、これらの離間距離ｄ４をできる限り小さく設定することができる。 As described above, in the plate assembly 10, the adjustment plate 600 brings the lower surface 109 of the first upper plate 100 and the upper surface 302 of the first lower plate 300 as close as possible to each other in the first state. it can be set as small as possible a distance d 1. In the second state, it can be a first lower surface 109 of the upper plate 100 is brought closer as possible and the upper surface 204 of the second lower plate 200, set as small as possible these the distance d 2. Even in the third state, the lower surface 109 ′ of the second upper plate 100 ′ and the upper surface 302 of the first lower plate 300 can be as close as possible, and the separation distance d 3 can be set as small as possible. it can. In the fourth state, it is brought closer as possible to the 'lower surface 109' of the second upper plate 100 and the upper surface 204 of the second lower plate 200, smaller is as much as possible these separation distance d 4 it can.
　そして、特に第２の状態で、貫通孔１０４’を通過した液体がたとえ飛散したとしても、各ウェル１０１に対応する、第２の下側プレート２００のウェル２０１内に当該液体を高効率に供給する（回収する）ことができる。これと同様に、第４の状態でも、貫通孔１０４’を通過した液体がたとえ飛散したとしても、各ウェル１０１’に対応する、第２の下側プレート２００のウェル２０１内に当該液体を高効率に供給することができる。 In particular, even in the second state, even if the liquid that has passed through the through hole 104 ′ is scattered, the liquid is supplied into the well 201 of the second lower plate 200 corresponding to each well 101 with high efficiency. (Can be recovered). Similarly, even in the fourth state, even if the liquid that has passed through the through hole 104 ′ is scattered, the liquid is placed in the well 201 of the second lower plate 200 corresponding to each well 101 ′. Can be supplied with efficiency.
　なお、離間距離ｄ１～ｄ４としては、特に限定されず、例えば、好ましくは１０ｍｍ以下、より好ましくは０．５ｍｍ以上、１０ｍｍ以下、さらに好ましくは０．５ｍｍ以上、３ｍｍ以下に設定される。離間距離ｄ１～ｄ４をかかる範囲内に設定することにより、前記効果をより顕著に発揮させることができる。 The separation distances d 1 to d 4 are not particularly limited, and for example, are preferably set to 10 mm or less, more preferably 0.5 mm to 10 mm, and further preferably 0.5 mm to 3 mm. By setting the separation distances d 1 to d 4 within such a range, the above effects can be exhibited more remarkably.
　また、図７（図６も同様）に示すように、第２の上側プレート１００’の底面（下面１０９’）には、各貫通孔１０４’を囲むようにリング状の突起部１０７が突出形成されているのが好ましい。突起部１０７が形成されていることにより、貫通孔１０４’を通過した液体を第２の下側プレート２００のウェル２０１内に向かって導くことができ、よって、該液体をさらに高効率に回収することができる。 Further, as shown in FIG. 7 (similarly in FIG. 6), a ring-shaped protrusion 107 protrudes from the bottom surface (lower surface 109 ′) of the second upper plate 100 ′ so as to surround each through hole 104 ′. It is preferable. Since the protrusion 107 is formed, the liquid that has passed through the through hole 104 ′ can be guided into the well 201 of the second lower plate 200, and thus the liquid can be recovered more efficiently. be able to.
　次に、精製装置１で糖鎖を精製する工程について、図８～図２１を参照しつつ説明するが、その前に、「糖鎖」と「糖鎖と特異的に結合する捕捉担体」とについて説明する。 Next, the step of purifying the sugar chain with the purification apparatus 1 will be described with reference to FIGS. 8 to 21. Before that, “sugar chain” and “capture carrier that specifically binds to sugar chain” and Will be described.
　ここでは、糖鎖は、当該糖鎖を備える糖タンパク質から得られる場合を一例に挙げて説明する。糖タンパク質を含む試料としては、特に限定されないが、例えば、全血、血清、血漿、尿、唾液、細胞、組織等の生体試料が挙げられる。 Here, the case where the sugar chain is obtained from a glycoprotein provided with the sugar chain will be described as an example. Although it does not specifically limit as a sample containing a glycoprotein, For example, biological samples, such as whole blood, serum, plasma, urine, saliva, a cell, a structure | tissue, are mentioned.
　そして、この試料を、例えば、ＳＤＳ－ＰＡＧＥ（ＳＤＳ変性ポリアクリルアミドゲル電気泳動）や、等電点電気泳動とＳＤＳ－ＰＡＧＥを組み合わせた二次元電気泳動等の所定処理を施す。これにより、ゲル中において試料に含まれる糖タンパク質を分離する。その後、ゲル内に保持されている糖タンパク質から、糖鎖遊離方法を用いて糖鎖を遊離させる。糖鎖を遊離させる方法としては、特に限定されないが、例えば、Ｎ-グリコシダーゼまたはＯ-グリコシダーゼを用いたグリコシダーゼ処理、ヒドラジン分解、アルカリ処理によるβ脱離等の方法を用いることができる。この糖鎖遊離処理後のゲルを取り出し、このゲルを、例えば水等の洗浄液を用いてリンスする。そうすることで、遊離された糖鎖をゲルから洗浄液中に溶出させる。そして、洗浄液中においてゲルを沈殿させ、上清を得た後、この上清を回収することで、糖鎖含有液２３を得る。この糖鎖含有液２３は、複数（本実施形態では９６個）のウェル８１１を有するマルチウェルプレート８１内にそれぞれ予め貯留されている。 The sample is then 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. Thereby, the glycoprotein contained in the sample is separated in the gel. Thereafter, the sugar chain is released from the glycoprotein retained in the gel using a sugar chain releasing method. The method 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. The gel after the sugar chain release treatment is taken out, and the gel is rinsed using a cleaning solution such as water. By doing so, the released sugar chain is eluted from the gel into the washing solution. And after precipitating a gel in a washing | cleaning liquid and obtaining a supernatant liquid, the sugar_chain | carbohydrate containing liquid 23 is obtained by collect | recovering this supernatant liquid. The sugar chain-containing liquid 23 is stored in advance in a multi-well plate 81 having a plurality (96 in this embodiment) of wells 811.
　なお、糖鎖の遊離のために、本実施形態では電気泳動処理を用いているが、これに限定されず、例えば、アフィニティークロマトグラフィー処理またはイオン交換クロマトグラフィー処理を用いてもよい。また、前記試料からの糖タンパク質の精製を省略してもよい場合には、前記試料に含まれる糖タンパク質に対して直接糖鎖を遊離させる処理を施すようにしてもよい。 In this embodiment, electrophoresis treatment is used to release sugar chains. However, the present invention is not limited to this. For example, affinity chromatography treatment or ion exchange chromatography treatment may be used. In addition, when the purification of glycoprotein from the sample may be omitted, the glycoprotein contained in the sample may be subjected to a treatment for directly releasing the sugar chain.
　また、糖鎖は、生体内物質のなかで唯一、アルデヒド基をもつ物質である。すなわち、糖鎖は、水溶液等の状態で、環状のヘミアセタール型構造と、非環状型のアルデヒド型構造とが平衡で存在する物質である。 In addition, sugar chains are the only substances in the living body that have an aldehyde group. That is, a sugar chain is a substance in which a cyclic hemiacetal structure and an acyclic aldehyde structure exist in an equilibrium state in an aqueous solution or the like.
　これに対して、タンパク質、核酸および脂質等の糖鎖以外の生体内物質には、通常、アルデヒド基が含まれていない。 In contrast, in vivo substances other than sugar chains such as proteins, nucleic acids and lipids usually do not contain aldehyde groups.
　このことから、アルデヒド基と特異的に反応して、安定な結合を形成する官能基を有する捕捉担体を利用すれば、糖鎖のみを選択的に捕捉することが可能である。 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.
　アルデヒド基と特異的に反応する官能基としては、例えば、ヒドラジド基、オキシルアミノ基、アミノ基、セミチオカルバジド基およびそれらの誘導体が好ましく、ヒドラジド基またはオキシルアミノ基がより好ましく用いられる。オキシルアミノ基とアルデヒド基との反応によって生じるオキシム結合、およびヒドラジド基とアルデヒド基との反応によって生じるヒドラゾン結合は、酸処理等によって容易に切断されるため、糖鎖を担体に捕捉させたのち、糖鎖を担体から簡単に切り離すことができる。 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. The sugar chain can be easily detached from the carrier.
　なお、一般的に，生理活性物質の捕捉、担持にはアミノ基が多用されている。しかしながら、アミノ基とアルデヒド基の反応によって生じる結合（シッフ塩基）は結合力が弱いため、還元剤等を用いた二次処理が必要である。このことから、アミノ基は糖鎖の捕捉には好ましくない。 In general, amino groups are frequently used for capturing and supporting physiologically active substances. However, since the bond (Schiff base) generated by the reaction of the amino group and the aldehyde group has a weak binding force, a secondary treatment using a reducing agent or the like is necessary. For this reason, amino groups are not preferred for capturing sugar chains.
　糖鎖を捕捉するための担体としては、ポリマー粒子を用いることが好ましい。さらに、このポリマー粒子は、少なくとも表面の一部に糖鎖のアルデヒド基と特異的に反応する官能基を有した固体粒子またはゲル粒子であることが好ましい。 It is preferable to use polymer particles as a carrier for capturing sugar chains. Further, the polymer particles are preferably solid particles 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.
　ポリマー粒子が固体粒子またはゲル粒子であれば、ポリマー粒子に糖鎖を捕捉させた後に、第１の上側プレート１００が備えるウェル１０１を用いて、かかる粒子を容易に回収することができる。 If the polymer particles are solid particles or gel particles, the particles can be easily recovered using the well 101 provided in the first upper plate 100 after the sugar chains are captured by the polymer particles.
　このようなポリマー粒子としては、例えば、下記一般式（１）で表されるものが挙げられる。 Examples of such polymer particles include those represented by the following general formula (1).
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　精製装置１では、糖鎖と特異的に結合する捕捉担体として、ポリマー粒子２０を用い、ポリマー粒子２０上に糖鎖を捕捉する。 In the purification apparatus 1, polymer particles 20 are used as a capture carrier that specifically binds to sugar chains, and sugar chains are captured on the polymer particles 20.
　ポリマー粒子２０は、当該ポリマー粒子２０を純水２１中に分散させた粒子分散液２２として、タンク８２に予め貯留されている。 The polymer particles 20 are stored in advance in a tank 82 as a particle dispersion 22 in which the polymer particles 20 are dispersed in pure water 21.
　なお、ポリマー粒子２０の形状は、特に限定されないが、例えば、球状またはそれに類する形状が好ましい。ポリマー粒子２０が球状の場合、平均粒径は、好ましくは０．０５～１０００μｍ程度、より好ましくは０．０５～２００μｍ程度、さらに好ましくは０．１～２００μｍ程度、最も好ましくは０．１～１００μｍ程度に設定される。平均粒径が下限値未満では、ポリマー粒子２０を遠心分離やろ過で回収することが困難となるおそれがある。また、平均粒径が上限値を超えると、ポリマー粒子２０と、後述する試料溶液との接触面積が少なくなり、糖鎖捕捉の効率が低下するおそれがある。 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.
　さて、精製装置１では、以下の「捕捉担体供給工程（第１の工程）」、「糖鎖捕捉工程（第２の工程）」、「物質除去工程（第３の工程）」、「再遊離工程（第４の工程）」、「糖鎖精製工程（第５の工程）」が順に行なわれる。また、これらの工程の間に、必要に応じて、１以上の任意の目的の工程が行なわれる。 In the purification apparatus 1, the following "capture carrier supply step (first step)", "sugar chain capture step (second step)", "substance removal step (third step)", "re-release Step (fourth step) "and" sugar chain purification step (fifth step) "are sequentially performed. In addition, one or more arbitrary target steps are performed between these steps as necessary.
　捕捉担体供給工程は、第１の上側プレート１００の各ウェル１０１にそれぞれ糖鎖と特異的に結合する捕捉担体（ポリマー粒子２０）を供給する工程である。この捕捉担体供給工程では、プレート組立体１０を第１の状態として用いる。 The capture carrier supply step is a step of supplying capture carriers (polymer particles 20) that specifically bind to sugar chains to the wells 101 of the first upper plate 100, respectively. In this capture carrier supplying step, the plate assembly 10 is used as the first state.
　糖鎖捕捉工程は、第１の上側プレート１００の各ウェル１０１にそれぞれ糖鎖含有液２３をノズル４１で分注して、糖鎖含有液２３とポリマー粒子２０とを接触させ、ポリマー粒子２０上に糖鎖を捕捉する工程である。この糖鎖捕捉工程では、捕捉担体供給工程に引き続き、プレート組立体１０を第１の状態として用いる。 In the sugar chain capturing step, the sugar chain-containing liquid 23 is dispensed to each well 101 of the first upper plate 100 with the nozzle 41, and the sugar chain-containing liquid 23 and the polymer particles 20 are brought into contact with each other. This is a step of capturing a sugar chain. In this sugar chain capturing step, the plate assembly 10 is used as the first state following the capturing carrier supplying step.
　物質除去工程は、ポリマー粒子２０に結合した糖鎖以外の物質を除去する工程である。この物質除去工程では、プレート組立体１０を第１の状態として用いる。 The substance removing step is a step of removing substances other than the sugar chains bonded to the polymer particles 20. In this substance removing step, the plate assembly 10 is used as the first state.
　再遊離工程は、ポリマー粒子２０に結合した糖鎖を再遊離させる工程である。この再遊離工程では、物質除去工程に引き続き、プレート組立体１０を第１の状態として用いる。 The re-releasing step is a step for re-releasing the sugar chains bound to the polymer particles 20. In this re-releasing process, the plate assembly 10 is used as the first state following the substance removing process.
　糖鎖精製工程は、再遊離した糖鎖をポリマー粒子２０と分離して精製する工程である。この糖鎖精製工程では、プレート組立体１０を順に第２の状態、第３の状態、第４の状態に変えて用いる。第２の状態は、ステージ３上で、第１の状態のプレート組立体１０において第１の下側プレート３００と第２の下側プレート２００とを交換して、当該第２の下側プレート２００を第２の支持台５００（支持体７００）に支持することによりなる。第３の状態は、第２の状態のプレート組立体１０において第１の上側プレート１００と第２の上側プレート１００’とを交換して、当該第２の上側プレート１００’が第１の支持台４００（支持体７００）を支持し、第２の下側プレート２００と第１の下側プレート３００とを交換して、当該第１の下側プレート３００を第２の支持台５００（支持体７００）に支持することによりなる。また、第４の状態は、第３の状態のプレート組立体１０において第１の下側プレート３００と第２の下側プレート２００とを交換して、当該第２の下側プレート２００が第２の支持台５００（支持体７００）に支持することによりなる。 The sugar chain purification step is a step in which the re-released sugar chain is separated from the polymer particles 20 and purified. In this sugar chain purification step, the plate assembly 10 is used by sequentially changing to the second state, the third state, and the fourth state. In the second state, on the stage 3, the first lower plate 300 and the second lower plate 200 are exchanged in the plate assembly 10 in the first state, and the second lower plate 200 is replaced. Is supported by the second support base 500 (support 700). In the third state, the first upper plate 100 ′ and the second upper plate 100 ′ are exchanged in the plate assembly 10 in the second state, and the second upper plate 100 ′ becomes the first support base. 400 (support 700), the second lower plate 200 and the first lower plate 300 are exchanged, and the first lower plate 300 is replaced with the second support base 500 (support 700). ) To support. Further, in the fourth state, the first lower plate 300 and the second lower plate 200 are exchanged in the plate assembly 10 in the third state, and the second lower plate 200 is changed to the second state. This is achieved by supporting the support table 500 (support 700).
　［１］　捕捉担体供給工程
　［１－１（プレート組立体載置）］　図８（１）に示すように、プレート組立体１０を第１の状態とし、当該プレート組立体１０をステージ３（プレート組立体載置部３１）上に載置する。なお、ステージ３に埋設されているヒータ６１の作動は、未だ停止している。 [1] Capture Carrier Supply Step [1-1 (Plate Assembly Placement)] As shown in FIG. 8 (1), the plate assembly 10 is set to the first state, and the plate assembly 10 is moved to the stage 3 (plate It is mounted on the assembly mounting part 31). Note that the operation of the heater 61 embedded in the stage 3 is still stopped.
　［１－２（粒子分散液吸引）］　また、ノズル４１を、粒子分散液２２が貯留されているタンク８２内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して当該ノズル４１内に粒子分散液２２が吸引される。 [1-2 (Particle Dispersion Suction)] The nozzle 41 is inserted into the tank 82 in which the particle dispersion 22 is stored, and the pump 42 is operated in this state. As a result, the particle dispersion liquid 22 is sucked into the nozzle 41 through the tip opening 411 of the nozzle 41.
　［１－３］そして、移動手段５を作動させて、粒子分散液２２を吸引したノズル４１をプレート組立体１０上に移動させる。また、この移動に伴って、プレート組立体１０の第１の上側プレート１００の各ウェル１０１にそれぞれ粒子分散液２２（ポリマー粒子２０）を供給する（分注する）。 [1-3] Then, the moving means 5 is operated to move the nozzle 41 that sucked the particle dispersion 22 onto the plate assembly 10. Further, along with this movement, the particle dispersion liquid 22 (polymer particles 20) is supplied (dispensed) to each well 101 of the first upper plate 100 of the plate assembly 10 respectively.
　［２］　吸引工程
　次に、図８（２）に示すように、吸引ポンプ１３を作動させて、プレート組立体１０の内部空間９ａを負圧とする。これにより、各ウェル１０１内の粒子分散液２２をそれぞれ吸引する。 [2] Suction Step Next, as shown in FIG. 8 (2), the suction pump 13 is operated to set the internal space 9 a of the plate assembly 10 to a negative pressure. Thereby, the particle dispersion liquid 22 in each well 101 is sucked, respectively.
　これにより、各ウェル１０１では、粒子分散液２２中の純水２１はフィルタ１０５を透過する。これに対して、フィルタ１０５の細孔径と、ポリマー粒子２０の粒径との関係により、ポリマー粒子２０はフィルタ１０５を透過することができない。そのため、フィルタ１０５を透過した純水２１が、貫通孔１０４を介して、第１の下側プレート３００（凹部３０１）内に選択的に供給される。 Thereby, in each well 101, the pure water 21 in the particle dispersion liquid 22 passes through the filter 105. On the other hand, the polymer particles 20 cannot pass through the filter 105 due to the relationship between the pore diameter of the filter 105 and the particle diameter of the polymer particles 20. Therefore, the pure water 21 that has passed through the filter 105 is selectively supplied into the first lower plate 300 (recessed portion 301) through the through hole 104.
　その結果、図８（３）に示すように、ポリマー粒子２０が第１の上側プレート１００のウェル１０１内に単独で残存する。 As a result, as shown in FIG. 8 (3), the polymer particles 20 remain alone in the well 101 of the first upper plate 100.
　［３］　ノズル洗浄工程
　次に、移動手段５を作動させて、ノズル４１を、洗浄液７１が充填された洗浄槽７内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して洗浄液７１がノズル４１内に出入りを繰り返し、ノズル４１が洗浄される。 [3] Nozzle Cleaning Step Next, the moving means 5 is operated, the nozzle 41 is inserted into the cleaning tank 7 filled with the cleaning liquid 71, and the pump 42 is operated in that state. Accordingly, the cleaning liquid 71 repeatedly enters and exits the nozzle 41 through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.
　［４］　糖鎖捕捉工程
　［４－１（糖鎖含有液吸引）］　次に、移動手段５を作動させて、ノズル４１を、糖鎖含有液２３が貯留されているマルチウェルプレート８１のウェル８１１内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して当該ノズル４１内に糖鎖含有液２３が吸引される。糖鎖含有液２３は、アセトニトリルに代表される揮発性の有機溶剤およびｐＨ調整剤としての酸（酢酸等）を含んでいてもよい。 [4] Sugar Chain Capture Step [4-1 (Sugar Chain-Containing Liquid Suction)] Next, the moving means 5 is operated and the nozzle 41 is moved to the well of the multi-well plate 81 in which the sugar chain-containing liquid 23 is stored. It inserts in 811 and the pump 42 is operated in that state. As a result, the sugar chain-containing liquid 23 is sucked into the nozzle 41 through the tip opening 411 of the nozzle 41. The sugar chain-containing liquid 23 may contain a volatile organic solvent typified by acetonitrile and an acid (such as acetic acid) as a pH adjuster.
　［４－２（糖鎖含有液供給）］　そして、図９（４）に示すように、移動手段５を作動させて、糖鎖含有液２３を吸引したノズル４１をプレート組立体１０上に移動させる。また、この移動に伴って、プレート組立体１０の第１の上側プレート１００の各ウェル１０１にそれぞれ糖鎖含有液２３を供給する（分注する）。 [4-2 (Sugar chain-containing liquid supply)] Then, as shown in FIG. 9 (4), the moving means 5 is operated to move the nozzle 41 that sucked the sugar chain-containing liquid 23 onto the plate assembly 10. Let Further, along with this movement, the sugar chain-containing liquid 23 is supplied (dispensed) to each well 101 of the first upper plate 100 of the plate assembly 10.
　［４－３（糖鎖含有液加熱）］　次に、図９（５）に示すように、ヒータ６１を作動させる。これにより、ヒータ６１の熱がステージ３、第２の支持台５００、第１の支持台４００、第１の上側プレート１００を順に伝わる。これにより、第１の上側プレート１００上の糖鎖含有液２３が加熱される。この加熱は、糖鎖含有液２３が乾燥するまで、一定の温度範囲（加熱温度）に保たれる。 [4-3 (Sugar chain-containing liquid heating)] Next, as shown in FIG. 9 (5), the heater 61 is operated. Thereby, the heat of the heater 61 is sequentially transmitted to the stage 3, the second support base 500, the first support base 400, and the first upper plate 100. As a result, the sugar chain-containing liquid 23 on the first upper plate 100 is heated. This heating is maintained in a certain temperature range (heating temperature) until the sugar chain-containing liquid 23 is dried.
　これにより、ポリマー粒子２０と、糖鎖含有液２３中に含まれる糖鎖とが反応し、ポリマー粒子２０上に糖鎖が捕捉される。 Thereby, 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.
　この際の反応液（糖鎖含有液２３）のｐＨは、好ましくは２～９、より好ましくは２～７であり、さらに好ましくは２～６である。なお、ｐＨ調整は、例えば、第１の上側プレート１００の各ウェル１０１の糖鎖含有液２３に、各種緩衝液または有機溶媒を添加することにより行うことができる。 At this time, the pH of the reaction solution (sugar chain-containing solution 23) 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 organic solvent to the sugar_chain | carbohydrate containing liquid 23 of each well 101 of the 1st upper plate 100, for example.
　糖鎖捕捉時の反応液の温度は、好ましくは４～１００℃程度、より好ましくは２５～９０℃程度、さらに好ましくは３０～８０℃程度、最も好ましくは６０～８０℃程度の温度範囲に保たれるように設定する。 The temperature of the reaction solution at the time of sugar chain capture is preferably kept in the temperature range of about 4 to 100 ° C, more preferably about 25 to 90 ° C, still more preferably about 30 to 80 ° C, and most preferably about 60 to 80 ° C. Set to sag.
　また、反応時間、すなわち、糖鎖含有液２３が乾燥するまでの時間は、かかる温度範囲に設定した場合、通常、０．１～３時間程度、好ましくは、０．６～２時間程度に設定される。 In addition, the reaction time, that is, the time until the sugar chain-containing liquid 23 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. Is done.
　かかる条件で、ポリマー粒子２０と糖鎖とを反応させることで、ポリマー粒子２０上に糖鎖が確実に捕捉されることとなる。 By reacting the polymer particles 20 and sugar chains under such conditions, the sugar chains are surely captured on the polymer particles 20.
　なお、本実施形態のように、糖鎖含有液２３が乾燥するまで、糖鎖含有液２３を加熱することにより、ポリマー粒子２０と糖鎖との反応率の向上を確実に図ることができる。 In addition, the reaction rate of the polymer particle 20 and the sugar chain can be reliably improved by heating the sugar chain-containing liquid 23 until the sugar chain-containing liquid 23 is dried as in the present embodiment.
　また、ポリマー粒子２０がヒドラジド基を有するものである場合、ヒドラジド基と、糖鎖が有する還元末端との間で、下記式（２）で表される反応が進行することで、ポリマー粒子２０上に糖鎖が捕捉される。 Further, 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.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
　以上のように、第１の状態のプレート組立体１０を用いることで、第１の上側プレート１００の各ウェル１０１内では、供給された液体の反応を進行させることができる。さらに、プレート組立体１０の内部空間９ａの吸引により、ウェル１０１に供給した液体中に含まれる固定成分と、液状成分との分離を容易に行うことができる。 As described above, by using the plate assembly 10 in the first state, the reaction of the supplied liquid can proceed in each well 101 of the first upper plate 100. 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 internal space 9 a of the plate assembly 10.
　［５］　ノズル洗浄工程
　次に、移動手段５を作動させて、ノズル４１を、洗浄液７１が充填された洗浄槽７内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して洗浄液７１がノズル４１内に出入りを繰り返し、ノズル４１が洗浄される。 [5] Nozzle Cleaning Step Next, the moving means 5 is operated, the nozzle 41 is inserted into the cleaning tank 7 filled with the cleaning liquid 71, and the pump 42 is operated in that state. Accordingly, the cleaning liquid 71 repeatedly enters and exits the nozzle 41 through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.
　［６］　使用済み第１の下側プレート交換工程
　［６－１（プレート組立体分解）］　図１０（６）に示すように、精製装置１の操作者は、ステージ３上でプレート組立体１０の支持体７００を、第１の上側プレート１００を支持した第１の支持台４００と、使用済みの（純水２１が貯留された）第１の下側プレート３００を支持した第２の支持台５００とに分解する。この分解操作は、第１の支持台４００を第１の上側プレート１００ごと持ち上げることにより、行なわれる。 [6] Used Lower Lower Plate Replacement Process [6-1 (Plate Assembly Disassembly)] As shown in FIG. 10 (6), the operator of the refining apparatus 1 operates the plate assembly 10 on the stage 3. The first support 700 that supports the first upper plate 100 and the second support that supports the used first lower plate 300 (which stores the pure water 21). Breaks down to 500. This disassembly operation is performed by lifting the first support base 400 together with the first upper plate 100.
　［６－２（第１の下側プレート取り外し）］　次に、図１０（７）に示すように、第２の支持台５００から使用済みの第１の下側プレート３００を取り除く。 [6-2 (Removal of First Lower Plate)] Next, as shown in FIG. 10 (7), the used first lower plate 300 is removed from the second support base 500.
　［６－３（第１の下側プレート装填）］　次に、図１０（８）に示すように、空の第２の支持台５００に未使用の第１の下側プレート３００を装填する。 [6-3 (First Lower Plate Loading)] Next, as shown in FIG. 10 (8), the unused first lower plate 300 is loaded on the empty second support base 500.
　［６－４（プレート組立体組み立て）］　次に、図１０（９）に示すように、未使用の第１の下側プレート３００が装填された第２の支持台５００上に、第１の上側プレート１００を支持した第１の支持台４００を重ね、プレート組立体１０を再度第１の状態とする。 [6-4 (Assembly of plate assembly)] Next, as shown in FIG. 10 (9), the first support plate 500 loaded with the unused first lower plate 300 is loaded with the first The first support base 400 that supports the upper plate 100 is stacked, and the plate assembly 10 is set to the first state again.
　なお、本工程［６］を行なうときには、ヒータ６１の作動を停止する。第１の下側プレート３００の交換は、操作者の手作業によるので、当該操作者が火傷を負うのを確実に防止することができる。 In addition, when performing this process [6], the operation of the heater 61 is stopped. Since the replacement of the first lower plate 300 is performed manually by the operator, it is possible to reliably prevent the operator from being burned.
　［７］　物質除去工程
　ここで、上記のように捕捉担体としてポリマー粒子２０を用いた場合、ポリマー粒子２０に捕捉された糖鎖以外の物質（以下、この物質を「洗浄物質」ということもある）としては、例えば、ポリマー粒子２０に捕捉されなかった糖鎖の他、ポリマー粒子２０の表面に、非特異的に吸着している糖鎖以外の夾雑物（タンパク質、ペプチド、脂質等）が挙げられる。 [7] Substance Removal Step Here, when the polymer particle 20 is used as the capture carrier as described above, a substance other than the sugar chain captured by the polymer particle 20 (hereinafter, this substance may be referred to as “cleaning substance”). ), For example, in addition to sugar chains not captured by the polymer particles 20, contaminants other than sugar chains non-specifically adsorbed on the surface of the polymer particles 20 (proteins, peptides, lipids, etc.). It is done.
　そのため、本工程［７］では、これらの物質を洗浄液２４で洗浄することで除去する。洗浄液２４としては、特に限定されないが、水、各種緩衝液、各種有機溶媒等が挙げられ、これらを適宜組み合わせて用いることができる。 Therefore, in this step [7], these substances are removed by washing with the washing liquid 24. 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.
　［７－１（洗浄液吸引）］　まず、移動手段５を作動させて、ノズル４１を、洗浄液２４が貯留されているタンク８３内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して当該ノズル４１内に洗浄液２４が吸引される。 [7-1 (Cleaning liquid suction)] First, the moving means 5 is operated, the nozzle 41 is inserted into the tank 83 in which the cleaning liquid 24 is stored, and the pump 42 is operated in that state. As a result, the cleaning liquid 24 is sucked into the nozzle 41 through the tip opening 411 of the nozzle 41.
　［７－２（洗浄液供給）］　そして、図１１（１０）に示すように、移動手段５を作動させて、洗浄液２４を吸引したノズル４１をプレート組立体１０上に移動させる。また、この移動に伴って、プレート組立体１０の第１の上側プレート１００の各ウェル１０１にそれぞれ洗浄液２４を供給する（添加する）。 [7-2 (Supply of cleaning liquid)] Then, as shown in FIG. 11 (10), the moving means 5 is operated to move the nozzle 41 that has sucked the cleaning liquid 24 onto the plate assembly 10. Further, along with this movement, the cleaning liquid 24 is supplied (added) to each well 101 of the first upper plate 100 of the plate assembly 10.
　［７－３（洗浄液吸引）］　次に、図１１（１１）に示すように、吸引ポンプ１３を作動させて、プレート組立体１０の内部空間９ａを負圧とする。これにより、各ウェル１０１内の洗浄液２４をそれぞれ吸引する。 [7-3 (Washing liquid suction)] Next, as shown in FIG. 11 (11), the suction pump 13 is operated to set the internal space 9a of the plate assembly 10 to a negative pressure. As a result, the cleaning liquid 24 in each well 101 is aspirated.
　その結果、洗浄液２４がフィルタ１０５を透過することができる。図１１（１２）に示すように、第１の上側プレート１００にポリマー粒子２０を残存させた状態で、第１の上側プレート１００から選択的に洗浄液２４を、第１の下側プレート３００内に除去することができる。また、洗浄液２４中に溶解した洗浄物質を除去することができる。 As a result, the cleaning liquid 24 can pass through the filter 105. As shown in FIG. 11 (12), with the polymer particles 20 remaining on the first upper plate 100, the cleaning liquid 24 is selectively passed from the first upper plate 100 into the first lower plate 300. Can be removed. Moreover, the cleaning substance dissolved in the cleaning liquid 24 can be removed.
　図１１（１０）～（１２）に示す作動を、複数回繰り返して行うことで、洗浄物質を洗浄液２４中に溶解させた状態で、第１の下側プレート３００に洗浄物質を分離することができる。そのため、洗浄物質を糖鎖が捕捉されたポリマー粒子２０から確実に除去することができる。 The operations shown in FIGS. 11 (10) to (12) are repeated a plurality of times, so that the cleaning substance can be separated into the first lower plate 300 in a state where the cleaning substance is dissolved in the cleaning liquid 24. it can. Therefore, it is possible to reliably remove the cleaning substance from the polymer particles 20 in which the sugar chains are captured.
　なお、この場合、まず水または緩衝液を洗浄液２４として用いて十分にポリマー粒子２０を洗浄したのち、さらに有機溶媒の洗浄液２４でポリマー粒子２０を洗浄し、必要に応じてこれら洗浄液２４による洗浄を繰り返し行い、最後に有機溶媒の洗浄液２４でポリマー粒子２０を洗浄するのが好ましい。これにより、洗浄物質、特に、非特異的にポリマー粒子２０の表面に吸着する夾雑物をより確実に除去することができるようになる。 In this case, first, the polymer particles 20 are sufficiently washed with water or a buffer solution as the washing solution 24, and then the polymer particles 20 are washed with the washing solution 24 of an organic solvent, and the washing with the washing solution 24 is performed as necessary. It is preferable that the polymer particles 20 be washed repeatedly with an organic solvent washing solution 24 lastly. Thereby, it becomes possible to more reliably remove the cleaning substance, in particular, impurities adsorbed nonspecifically on the surface of the polymer particle 20.
　［８］　ノズル洗浄工程
　次に、移動手段５を作動させて、ノズル４１を、洗浄液７１が充填された洗浄槽７内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して洗浄液７１がノズル４１内に出入りを繰り返し、ノズル４１が洗浄される。 [8] Nozzle Cleaning Step Next, the moving means 5 is operated, the nozzle 41 is inserted into the cleaning tank 7 filled with the cleaning liquid 71, and the pump 42 is operated in that state. Accordingly, the cleaning liquid 71 repeatedly enters and exits the nozzle 41 through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.
　［９］　再遊離工程
　ここでは、ポリマー粒子２０に結合した糖鎖を再遊離させる。さらに、この糖鎖を別の化合物（以下、「化合物Ａ」と言うこともある。）で置換する、すなわち、化合物Ａで糖鎖をラベル化する。なお、この化合物Ａとしては、蛍光物質、吸光物質および放射性物質等を備えるラベル化試薬が好ましく用いられる。 [9] Re-release step Here, the sugar chain bonded to the polymer particle 20 is re-released. Further, this sugar chain is substituted with another compound (hereinafter sometimes referred to as “compound A”), that is, the sugar chain is labeled with compound A. As this compound A, a labeling reagent comprising a fluorescent substance, a light-absorbing substance, a radioactive substance and the like is preferably used.
　［９－１（化合物含有液吸引）］　まず、移動手段５を作動させて、ノズル４１を、化合物Ａを含有する化合物含有液２５が貯留されているタンク８４内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して当該ノズル４１内に化合物含有液２５が吸引される。 [9-1 (Compound-containing liquid suction)] First, the moving means 5 is operated, and the nozzle 41 is inserted into the tank 84 in which the compound-containing liquid 25 containing the compound A is stored. 42 is activated. As a result, the compound-containing liquid 25 is sucked into the nozzle 41 through the tip opening 411 of the nozzle 41.
　［９－２（化合物含有液供給）］そして、図１２（１３）に示すように、移動手段５を作動させて、化合物含有液２５を吸引したノズル４１をプレート組立体１０上に移動させる。また、この移動に伴って、プレート組立体１０の第１の上側プレート１００の各ウェル１０１にそれぞれ化合物含有液２５を供給する。 [9-2 (Compound-containing liquid supply)] Then, as shown in FIG. 12 (13), the moving means 5 is operated to move the nozzle 41 that sucked the compound-containing liquid 25 onto the plate assembly 10. In accordance with this movement, the compound-containing liquid 25 is supplied to each well 101 of the first upper plate 100 of the plate assembly 10.
　第１の上側プレート１００のウェル１０１内に添加する化合物Ａの添加量は、糖鎖が捕捉されたポリマー粒子２０に対して、過剰量となっているのが好ましい。これにより、次で化合物含有液２５を加熱した際に、糖鎖に対する化合物Ａの置換率の向上を図ることができる。 The amount of compound A added to the well 101 of the first upper plate 100 is preferably excessive with respect to the polymer particles 20 in which the sugar chains are captured. Thereby, when the compound containing liquid 25 is heated next, the substitution rate of the compound A with respect to a sugar chain can be improved.
　具体的には、化合物Ａの添加量は、ポリマー粒子２０が有する糖鎖と特異的に反応する官能基量に対して、好ましくは１．５倍量以上、より好ましくは３倍量以上、さらに好ましくは５倍量以上であり、最も好ましくは１０倍量以上に設定される。 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.
　［９－３（化合物含有液加熱）］　次に、図１２（１４）に示すように、ヒータ６１を作動させる。これにより、第１の上側プレート１００上のウェル１０１内の化合物含有液２５が加熱される。この加熱は、化合物含有液２５が乾燥するまで、一定の温度範囲（加熱温度）に保たれる。 [9-3 (Compound-containing liquid heating)] Next, as shown in FIG. 12 (14), the heater 61 is operated. As a result, the compound-containing liquid 25 in the well 101 on the first upper plate 100 is heated. This heating is maintained in a certain temperature range (heating temperature) until the compound-containing liquid 25 is dried.
　これにより、捕捉された糖鎖はポリマー粒子２０から切り離され、それとほぼ同時に糖鎖に化合物Ａが付加することとなる。そのため、糖鎖は化合物Ａでラベル化されることとなる。以下、化合物Ａでラベル化された糖鎖を「ラベル化糖鎖」と言うこともある。 Thereby, the captured sugar chain is separated from the polymer particle 20, and at the same time, the compound A is added to the sugar chain. Therefore, the sugar chain is labeled with compound A. Hereinafter, the sugar chain labeled with the compound A may be referred to as “labeled sugar chain”.
　また、この際の反応液（化合物含有液２５）のｐＨは、好ましくは２～９、より好ましくは２～７であり、さらに好ましくは２～６である。なお、ｐＨ調整は、例えば、第１の上側プレート１００の各ウェル１０１にそれぞれ化合物含有液２５を供給した後に、各種緩衝液をウェル１０１に添加することにより行うことができる。 In addition, the pH of the reaction solution (compound-containing solution 25) at this time is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6. The pH adjustment can be performed, for example, by adding various buffer solutions to the well 101 after supplying the compound-containing solution 25 to each well 101 of the first upper plate 100.
　ラベル化時の反応液の温度は、好ましくは４～１００℃程度、より好ましくは２５～９０℃程度、さらに好ましくは３０～８０℃程度、最も好ましくは６０～８０℃程度の温度範囲に保たれるように設定する。 The temperature of the reaction solution at the time of labeling is preferably kept in a temperature range of about 4 to 100 ° C., more preferably about 25 to 90 ° C., further preferably about 30 to 80 ° C., and most preferably about 60 to 80 ° C. Set to
　また、反応時間、すなわち溶液が乾燥するまでの時間は、かかる温度範囲に設定した場合、通常、０．１～３時間程度、好ましくは、０．６～２時間程度に設定される。 Also, the reaction time, that is, the time until the solution dries, is usually set to about 0.1 to 3 hours, preferably about 0.6 to 2 hours when set in such a temperature range.
　かかる条件で、ラベル化を行うことで、糖鎖が確実に化合物Ａによりラベル化される。 By performing labeling under such conditions, the sugar chain is surely labeled with compound A.
　なお、本実施形態のように、化合物含有液２５が乾燥するまで、化合物含有液２５を加熱することにより、糖鎖と化合物Ａとの反応率の向上を確実に図ることができる。 In addition, the reaction rate of a sugar chain and the compound A can be improved reliably by heating the compound-containing liquid 25 until the compound-containing liquid 25 is dried as in this embodiment.
　なお、化合物Ａとしては、アミノオキシ基またはヒドラジド基を有する化合物が好ましく用いられる。ポリマー粒子２０がヒドラジド基を有するものである場合、特に、下記化学式（３）で表されるN-aminooxyacetyl-tryptophanyl（arginine methyl ester）が好ましく用いられる。 In addition, as the compound A, a compound having an aminooxy group or a hydrazide group is preferably used. When the polymer particle 20 has a hydrazide group, N-aminooxyacetyl-tryptophanyl (arginineargmethyl ester) represented by the following chemical formula (3) is particularly preferably used.
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
　［１０］　ノズル洗浄工程
　次に、移動手段５を作動させて、ノズル４１を、洗浄液７１が充填された洗浄槽７内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して洗浄液７１が出入りを繰り返し、ノズル４１が洗浄される。 [10] Nozzle Cleaning Step Next, the moving means 5 is operated, the nozzle 41 is inserted into the cleaning tank 7 filled with the cleaning liquid 71, and the pump 42 is operated in that state. Accordingly, the cleaning liquid 71 repeatedly enters and exits through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.
　［１１］　第１・第２下側プレート交換工程
　［１１－１（プレート組立体分解）］　図１３（１５）に示すように、精製装置１の操作者は、ステージ３上でプレート組立体１０の支持体７００を、第１の上側プレート１００を支持した第１の支持台４００と、純水２１と洗浄液２４との混合液が貯留された第１の下側プレート３００を支持した第２の支持台５００とに分解する。この分解操作は、第１の支持台４００を第１の上側プレート１００ごと持ち上げることにより、行なわれる。 [11] First / Second Lower Plate Replacement Process [11-1 (Disassembly of Plate Assembly)] As shown in FIG. 13 (15), the operator of the refining apparatus 1 operates the plate assembly 10 on the stage 3. The second support 700 is supported by a first support 400 that supports the first upper plate 100 and a first lower plate 300 that stores a mixture of pure water 21 and cleaning liquid 24. Disassembled into a support 500. This disassembly operation is performed by lifting the first support base 400 together with the first upper plate 100.
　［１１－２（第１の下側プレート取り外し）］　次に、図１３（１６）に示すように、第２の支持台５００から第１の下側プレート３００を取り除く。 [11-2 (Removal of First Lower Plate)] Next, as shown in FIG. 13 (16), the first lower plate 300 is removed from the second support base 500.
　［１１－３（第２の下側プレート装填）］　次に、図１３（１７）に示すように、空の第２の支持台５００に未使用の第２の下側プレート２００を装填する。 [11-3 (Second Lower Plate Loading)] Next, as shown in FIG. 13 (17), the unused second lower plate 200 is loaded on the empty second support base 500.
　［１１－４（プレート組立体組み立て）］　次に、図１３（１８）に示すように、未使用の第２の下側プレート２００が装填された第２の支持台５００上に、第１の上側プレート１００を支持した第１の支持台４００を重ね、プレート組立体１０を第２の状態とする。 [11-4 (Assembly of plate assembly)] Next, as shown in FIG. 13 (18), on the second support base 500 loaded with the unused second lower plate 200, the first The first support base 400 that supports the upper plate 100 is overlapped to place the plate assembly 10 in the second state.
　なお、本工程［１１］を行なうときには、ヒータ６１の作動を停止する。第１の下側プレート３００と第２の下側プレート２００との交換は、操作者の手作業によるので、当該操作者が火傷を負うのを確実に防止することができる。 In addition, when performing this process [11], the operation of the heater 61 is stopped. Since the replacement of the first lower plate 300 and the second lower plate 200 is performed manually by the operator, it is possible to reliably prevent the operator from being burned.
　［１２］　糖鎖精製工程
　ここで、上記のようにポリマー粒子２０に捕捉された糖鎖を再遊離させることで、ラベル化糖鎖を得た場合、化合物Ａでラベル化された糖鎖以外に、第１の上側プレート１００のウェル１０１中に含まれる物質としては、糖鎖が遊離したポリマー粒子２０および糖鎖のラベル化に使用されなかった化合物Ａ（以下、「未使用化合物Ａ」と言うこともある。）が挙げられる。 [12] Sugar chain purification step Here, when the labeled sugar chain is obtained by re-releasing the sugar chain captured by the polymer particles 20 as described above, in addition to the sugar chain labeled with the compound A, The substances contained in the well 101 of the first upper plate 100 include polymer particles 20 from which sugar chains are released and compound A that has not been used for labeling sugar chains (hereinafter referred to as “unused compound A”). Sometimes).
　そのため、本工程［１２］では、これらポリマー粒子２０および未使用化合物Ａを除去することで、ラベル化糖鎖の精製を行う。 Therefore, in this step [12], the labeled sugar chain is purified by removing the polymer particles 20 and the unused compound A.
　［１２－１（溶解液吸引）］　まず、移動手段５を作動させて、ノズル４１を、乾燥したラベル化糖鎖を溶解し得る液剤である溶解液２６が貯留されているタンク８５内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して当該ノズル４１内に溶解液２６が吸引される。この溶解液２６としては、特に限定されるものではないが、水、各種緩衝液、各種有機溶剤等が挙げられる。 [12-1 (Solution Solution Suction)] First, the moving means 5 is operated, and the nozzle 41 is inserted into the tank 85 in which the solution 26 that is a solution capable of dissolving the dried labeled sugar chain is stored. In this state, the pump 42 is operated. As a result, the solution 26 is sucked into the nozzle 41 through the tip opening 411 of the nozzle 41. The solution 26 is not particularly limited, and examples thereof include water, various buffer solutions, and various organic solvents.
　［１２－２（溶解液供給）］　そして、図１４（１９）に示すように、移動手段５を作動させて、溶解液２６を吸引したノズル４１をプレート組立体１０上に移動させる。また、この移動に伴って、プレート組立体１０の第１の上側プレート１００の各ウェル１０１にそれぞれ溶解液２６を供給する。 [12-2 (Dissolved liquid supply)] Then, as shown in FIG. 14 (19), the moving means 5 is operated to move the nozzle 41 that has sucked the dissolved liquid 26 onto the plate assembly 10. Further, along with this movement, the solution 26 is supplied to each well 101 of the first upper plate 100 of the plate assembly 10.
　［１２－３（溶解液吸引）］　次に、図１４（２０）に示すように、吸引ポンプ１３を作動させて、プレート組立体１０の内部空間９ａを負圧とし、これにより、各ウェル１０１内の溶解液２６をそれぞれ吸引する。これにより、第１の上側プレート１００の各ウェル１０１では、それぞれ、溶解液２６がフィルタ１０５を透過する。 [12-3 (Solution Solution Suction)] Next, as shown in FIG. 14 (20), the suction pump 13 is operated to set the internal space 9a of the plate assembly 10 to a negative pressure. Each of the dissolved liquids 26 is aspirated. As a result, the solution 26 passes through the filter 105 in each well 101 of the first upper plate 100.
　そして、図１４（２１）に示すように、第１の上側プレート１００の各ウェル１０１では、ポリマー粒子２０が残存しており、このウェル１０１に対応する、第２の下側プレート２００のウェル２０１では、溶解液２６が貯留されている。また、その際には、溶解液２６中に溶解したラベル化糖鎖も第２の下側プレート２００のウェル２０１に移動させることができる。その結果、ラベル化糖鎖とポリマー粒子２０とが分離される。また、未使用化合物Ａも、通常、溶解液２６に対して溶解性を示すため、溶解液２６に溶解した状態で、第２の下側プレート２００のウェル２０１に移動する。 Then, as shown in FIG. 14 (21), the polymer particles 20 remain in each well 101 of the first upper plate 100, and the well 201 of the second lower plate 200 corresponding to the well 101. Then, the solution 26 is stored. At that time, the labeled sugar chain dissolved in the solution 26 can also be moved to the well 201 of the second lower plate 200. As a result, the labeled sugar chain and the polymer particle 20 are separated. In addition, the unused compound A also usually shows solubility in the solution 26, and thus moves to the well 201 of the second lower plate 200 in a state dissolved in the solution 26.
　［１２－４（ノズル洗浄）］　次に、移動手段５を作動させて、ノズル４１を、洗浄液７１が充填された洗浄槽７内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して洗浄液７１が出入りを繰り返し、ノズル４１が洗浄される。 [12-4 (Nozzle cleaning)] Next, the moving means 5 is operated, the nozzle 41 is inserted into the cleaning tank 7 filled with the cleaning liquid 71, and the pump 42 is operated in that state. Accordingly, the cleaning liquid 71 repeatedly enters and exits through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.
　［１２－５（プレート組立体分解）］　図１５（２２）に示すように、精製装置１の操作者は、ステージ３上でプレート組立体１０の支持体７００を、第１の上側プレート１００を支持した第１の支持台４００と、溶解液２６が貯留された第２の下側プレート２００を支持した第２の支持台５００とに分解する。この分解操作は、第１の支持台４００を第１の上側プレート１００ごと持ち上げることにより、行なわれる。 [12-5 (Disassembly of plate assembly)] As shown in FIG. 15 (22), the operator of the refining apparatus 1 moves the support 700 of the plate assembly 10 on the stage 3 and the first upper plate 100. The first support base 400 that is supported and the second support base 500 that supports the second lower plate 200 in which the solution 26 is stored are disassembled. This disassembly operation is performed by lifting the first support base 400 together with the first upper plate 100.
　［１２－６（第１の上側プレート取り外し）］　次に、図１５（２３）に示すように、第１の支持台４００を第２の支持台５００の隣に載置して、第１の支持台４００から、使用済みの（ポリマー粒子２０が残留した）第１の上側プレート１００を取り除く。 [12-6 (Removal of First Upper Plate)] Next, as shown in FIG. 15 (23), the first support base 400 is placed next to the second support base 500, and the first The used first upper plate 100 (with the polymer particles 20 remaining) is removed from the support base 400.
　［１２－７（第２の上側プレート装填）］　次に、図１５（２４）に示すように、空の第１の支持台４００に未使用の第２の上側プレート１００’を装填する。 [12-7 (Second Upper Plate Loading)] Next, as shown in FIG. 15 (24), the unused first upper plate 100 'is loaded on the empty first support base 400.
　［１２－８（洗浄液移し換え）］　次に、図１６（２５）および（２６）に示すように、移動手段５を作動させつつ、ノズル４１を用いて、第２の下側プレート２００のｍ行ｎ列のウェル２０１内の溶解液２６を、未使用の第２の上側プレート１００’のｍ行ｎ列（ウェル２０１と同じ行列）のウェル１０１’に移し換える（本実施形態では、ｍは１～１２までの整数であり、ｎは１～８までの整数である）。 [12-8 (Transfer of Cleaning Solution)] Next, as shown in FIGS. 16 (25) and (26), the nozzle 41 is used to move the second lower plate 200 m while operating the moving means 5. The lysate 26 in the well 201 in the row n column is transferred to the well 101 ′ in the m row n column (the same matrix as the well 201) of the unused second upper plate 100 ′ (in this embodiment, m is An integer from 1 to 12, and n is an integer from 1 to 8.
　［１２－９（第２の下側プレート取り外し）］　次に、図１７（２７）に示すように、第２の支持台５００から第２の下側プレート２００を取り除く。 [12-9 (Removal of Second Lower Plate)] Next, as shown in FIG. 17 (27), the second lower plate 200 is removed from the second support base 500.
　［１２－１０（第１の下側プレート装填）］　次に、図１７（２８）に示すように、空の第２の支持台５００に未使用の第１の下側プレート３００を装填する。 [12-10 (First Lower Plate Loading)] Next, as shown in FIG. 17 (28), the unused first lower plate 300 is loaded on the empty second support base 500.
　［１２－１１（プレート組立体組み立て）］　次に、図１７（２９）に示すように、未使用の第１の下側プレート３００が装填された第２の支持台５００上に、第２の上側プレート１００’を支持した第１の支持台４００を重ね、プレート組立体１０を第３の状態とする。 [12-11 (Assembly of plate assembly)] Next, as shown in FIG. 17 (29), on the second support base 500 loaded with the unused first lower plate 300, the second The first support base 400 that supports the upper plate 100 ′ is overlapped to place the plate assembly 10 in the third state.
　［１２－１２（溶解液吸引）］　次に、図１８（３０）に示すように、吸引ポンプ１３を作動させて、プレート組立体１０の内部空間９ｂを負圧とし、これにより、各ウェル１０１’内の溶解液２６をそれぞれ吸引する。 [12-12 (Solution Solution Suction)] Next, as shown in FIG. 18 (30), the suction pump 13 is operated to set the internal space 9b of the plate assembly 10 to a negative pressure. Aspirate the solution 26 in each.
　この際、フィルタ１０６をアセトニトリル等の非水溶媒で希釈した溶解液２６が透過するため、当該溶解液２６に含まれるラベル化糖鎖と未使用化合物Ａとは、シリカゲルで構成されたフィルタ１０６に吸着する。フィルタ１０６（シリカゲル）への吸着力は、未使用化合物Ａよりもラベル化糖鎖の方が高い。したがって、図１８（３１）に示すように、第１の下側プレート３００（凹部３０１）内には、未使用化合物Ａの一部が溶解液２６とともに流入する（貯留される）。なお、フィルタ１０６には、ラベル化糖鎖と、溶解液２６に含まれていた未使用化合物Ａのうちの、前記第１の下側プレート３００内に流入した未使用化合物Ａを除く残りの未使用化合物Ａとが吸着した状態となっている。 At this time, since the solution 26 diluted with a non-aqueous solvent such as acetonitrile passes through the filter 106, the labeled sugar chain and the unused compound A contained in the solution 26 are passed through the filter 106 made of silica gel. Adsorb. The adsorptive power to the filter 106 (silica gel) is higher in the labeled sugar chain than in the unused compound A. Accordingly, as shown in FIG. 18 (31), a part of the unused compound A flows (is stored) together with the solution 26 into the first lower plate 300 (recessed portion 301). The filter 106 has a labeling sugar chain and the remaining unused compounds A excluding the unused compounds A flowing into the first lower plate 300 out of the unused compounds A contained in the solution 26. The used compound A is in an adsorbed state.
　［１２－１３（非水溶媒吸引）］　次に、移動手段５を作動させて、ノズル４１を、アセトニトリル等の非水溶媒２７が貯留されているタンク８６内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して当該ノズル４１内に非水溶媒２７が吸引される。 [12-13 (Non-aqueous solvent suction)] Next, the moving means 5 is operated, and the nozzle 41 is inserted into the tank 86 in which the non-aqueous solvent 27 such as acetonitrile is stored. Is activated. As a result, the non-aqueous solvent 27 is sucked into the nozzle 41 through the tip opening 411 of the nozzle 41.
　［１２－１４（非水溶媒供給）］　そして、図１９（３２）に示すように、移動手段５を作動させて、非水溶媒２７を吸引したノズル４１をプレート組立体１０上に移動させる。また、この移動に伴って、プレート組立体１０の第２の上側プレート１００’の各ウェル１０１’にそれぞれ非水溶媒２７を供給する。 [12-14 (Nonaqueous solvent supply)] Then, as shown in FIG. 19 (32), the moving means 5 is operated to move the nozzle 41 that sucked the nonaqueous solvent 27 onto the plate assembly 10. Further, along with this movement, the non-aqueous solvent 27 is supplied to each well 101 ′ of the second upper plate 100 ′ of the plate assembly 10.
　［１２－１５（非水溶媒吸引）］　次に、図１９（３３）に示すように、吸引ポンプ１３を作動させて、プレート組立体１０の内部空間９ｂを負圧とし、これにより、各ウェル１０１’内の非水溶媒２７をそれぞれ吸引する。これにより、第２の上側プレート１００’の各ウェル１０１’では、それぞれ、非水溶媒２７がフィルタ１０６を透過する。この際、図１９（３４）に示すように、フィルタ１０６に吸着した未使用化合物Ａのほとんどが、フィルタ１０６から洗い流されて、すなわち、除去されて、非水溶媒２７ともに第１の下側プレート３００内に流入する。なお、未使用化合物Ａが若干フィルタ１０６に吸着していたとしても、その量は、後述するＨＰＬＣや質量分析装置を用いた未使用化合物Ａの量の測定時に、実質的に影響のない程度である。また、フィルタ１０６には、依然としてラベル化糖鎖が吸着した状態となっている。 [12-15 (Non-aqueous solvent suction)] Next, as shown in FIG. 19 (33), the suction pump 13 is operated to set the internal space 9b of the plate assembly 10 to a negative pressure. The nonaqueous solvent 27 in 101 ′ is sucked respectively. As a result, the nonaqueous solvent 27 passes through the filter 106 in each well 101 ′ of the second upper plate 100 ′. At this time, as shown in FIG. 19 (34), most of the unused compound A adsorbed on the filter 106 is washed away from the filter 106, that is, removed, and the first lower plate together with the non-aqueous solvent 27. Flows into 300. Even if the unused compound A is slightly adsorbed on the filter 106, the amount thereof is such that there is substantially no influence when measuring the amount of the unused compound A using an HPLC or mass spectrometer described later. is there. Further, the labeled sugar chain is still adsorbed on the filter 106.
　［１２－１６（ノズル洗浄）］　次に、移動手段５を作動させて、ノズル４１を、洗浄液７１が充填された洗浄槽７内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して洗浄液７１が出入りを繰り返し、ノズル４１が洗浄される。 [12-16 (Nozzle Cleaning)] Next, the moving means 5 is operated, the nozzle 41 is inserted into the cleaning tank 7 filled with the cleaning liquid 71, and the pump 42 is operated in that state. Accordingly, the cleaning liquid 71 repeatedly enters and exits through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.
　［１２－１７（プレート組立体分解）］　図２０（３５）に示すように、精製装置１の操作者は、ステージ３上でプレート組立体１０の支持体７００を、第２の上側プレート１００’を支持した第１の支持台４００と、溶解液２６と非水溶媒２７との混合液が貯留された第１の下側プレート３００を支持した第２の支持台５００とに分解する。この分解操作は、第１の支持台４００を第２の上側プレート１００’ごと持ち上げることにより、行なわれる。 [12-17 (Disassembly of plate assembly)] As shown in FIG. 20 (35), the operator of the refining apparatus 1 mounts the support 700 of the plate assembly 10 on the stage 3 and the second upper plate 100 ′. Is divided into a first support base 400 that supports the first lower plate 300 in which a mixed liquid of the solution 26 and the non-aqueous solvent 27 is stored. This disassembly operation is performed by lifting the first support base 400 together with the second upper plate 100 '.
　［１２－１８（第１の下側プレート取り外し）］　次に、図２０（３６）に示すように、第２の支持台５００から第１の下側プレート３００を取り除く。 [12-18 (Removal of First Lower Plate)] Next, as shown in FIG. 20 (36), the first lower plate 300 is removed from the second support base 500.
　［１２－１９（第２の下側プレート装填）］　次に、図２０（３７）に示すように、空の第２の支持台５００に未使用の第２の下側プレート２００を装填する。 [12-19 (Second Lower Plate Loading)] Next, as shown in FIG. 20 (37), the unused second lower plate 200 is loaded on the empty second support base 500.
　［１２－２０（プレート組立体組み立て）］　次に、図２０（３８）に示すように、未使用の第２の下側プレート２００が装填された第２の支持台５００上に、第２の上側プレート１００’を支持した第１の支持台４００を重ね、プレート組立体１０を第４の状態とする。 [12-20 (Assembly of plate assembly)] Next, as shown in FIG. 20 (38), on the second support base 500 loaded with the unused second lower plate 200, the second The first support base 400 supporting the upper plate 100 ′ is overlapped to set the plate assembly 10 to the fourth state.
　［１２－２１（水吸引）］　次に、移動手段５を作動させて、ノズル４１を、水２８が貯留されているタンク８７内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して当該ノズル４１内に水２８が吸引される。 [12-21 (Water Suction)] Next, the moving means 5 is operated, the nozzle 41 is inserted into the tank 87 in which the water 28 is stored, and the pump 42 is operated in that state. Accordingly, the water 28 is sucked into the nozzle 41 through the tip opening 411 of the nozzle 41.
　［１２－２２（水供給）］　そして、図２１（３９）に示すように、移動手段５を作動させて、水２８を吸引したノズル４１をプレート組立体１０上に移動させる。また、この移動に伴って、プレート組立体１０の第２の上側プレート１００’の各ウェル１０１’にそれぞれ水２８を供給する。 [12-22 (Water Supply)] Then, as shown in FIG. 21 (39), the moving means 5 is operated to move the nozzle 41 that has sucked the water 28 onto the plate assembly 10. Further, along with this movement, water 28 is supplied to each well 101 ′ of the second upper plate 100 ′ of the plate assembly 10.
　［１２－２３（水吸引）］　次に、図２１（４０）に示すように、吸引ポンプ１３を作動させて、プレート組立体１０の内部空間９ｂを負圧とする。これにより、各ウェル１０１’内の水２８をそれぞれ吸引する。これにより、第２の上側プレート１００’の各ウェル１０１’では、それぞれ、水２８がフィルタ１０６を透過する。この際、図２１（４１）に示すように、フィルタ１０６に吸着した状態となっているラベル化糖鎖が、フィルタ１０６から脱離して、水２８とともに第２の下側プレート２００のウェル２０１内に流入する。すなわち、ラベル化糖鎖が精製（分離）される。 [12-23 (Water Suction)] Next, as shown in FIG. 21 (40), the suction pump 13 is operated to set the internal space 9b of the plate assembly 10 to a negative pressure. Thereby, the water 28 in each well 101 'is sucked. As a result, the water 28 passes through the filter 106 in each well 101 ′ of the second upper plate 100 ′. At this time, as shown in FIG. 21 (41), the labeled sugar chain adsorbed on the filter 106 is detached from the filter 106, and is contained in the well 201 of the second lower plate 200 together with the water 28. Flow into. That is, the labeled sugar chain is purified (separated).
　［１２－２４（ノズル洗浄）］　次に、移動手段５を作動させて、ノズル４１を、洗浄液７１が充填された洗浄槽７内に挿入し、その状態でポンプ４２を作動させる。これにより、ノズル４１の先端開口部４１１を介して洗浄液７１がノズル４１内に出入りを繰り返し、ノズル４１が洗浄される。 [12-24 (nozzle cleaning)] Next, the moving means 5 is operated, the nozzle 41 is inserted into the cleaning tank 7 filled with the cleaning liquid 71, and the pump 42 is operated in that state. Accordingly, the cleaning liquid 71 repeatedly enters and exits the nozzle 41 through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.
　以上のような工程を経ることで、プレート組立体１０を用いて、化合物Ａでラベル化された糖鎖が精製される。 Through the above steps, the sugar chain labeled with the compound A is purified using the plate assembly 10.
　また、化合物Ａでラベル化された糖鎖は、MALDI-TOF MSに代表される質量分析、さらに高速液体クロマトグラフィー（ＨＰＬＣ）等の手法で分析することができる。特に、糖鎖が前述した化学式（３）で表されるN-aminooxyacetyl-tryptophanyl(arginine methyl ester)でラベル化されている場合、MALDI-TOF MSを用いて高感度分析を行うことができる。また、糖鎖が例えば２－アミノベンズアミドでラベル化されている場合、ＨＰＬＣを用いて高感度分析が可能である。 In addition, the sugar chain labeled with Compound A can be analyzed by mass spectrometry represented by MALDI-TOFTOMS, and further by a technique such as 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. In addition, when the sugar chain is labeled with, for example, 2-aminobenzamide, high-sensitivity analysis can be performed using HPLC.
　このような精製装置１によれば、ステージ３上のプレート組立体１０に対し、第１の上側プレート１００と第２の上側プレート１００’とを交換することができるとともに、第１の下側プレート３００と第２の下側プレート２００とを交換することができる。 According to the refining device 1, the first upper plate 100 and the second upper plate 100 ′ can be exchanged with respect to the plate assembly 10 on the stage 3, and the first lower plate 300 and the second lower plate 200 can be exchanged.
　特に、前記のように、第１の支持台４００と第２の支持台５００とは、第１の支持台４００が第１の上側プレート１００または第２の上側プレート１００’を支持し、第２の支持台５００が第１の下側プレート３００または第２の下側プレート２００を支持した状態で、分解可能、組立可能に構成されている。そして、第１の支持台４００と第２の支持台５００とが分解された状態で、第１の上側プレート１００と第２の上側プレート１００’との交換と、第１の下側プレート３００と第２の下側プレート２００との交換とをそれぞれ行なうことができる。その後、組立プレート１０の組立も容易に行なうことができる。 In particular, as described above, the first support base 400 and the second support base 500 are configured such that the first support base 400 supports the first upper plate 100 or the second upper plate 100 ′, The support base 500 is configured to be disassembleable and assembleable in a state where the first lower plate 300 or the second lower plate 200 is supported. Then, in a state in which the first support base 400 and the second support base 500 are disassembled, the first upper plate 100 and the second upper plate 100 ′ are exchanged, and the first lower plate 300 Exchange with the second lower plate 200 can be performed. Thereafter, the assembly plate 10 can be easily assembled.
　このように精製装置１では、プレート交換をステージ３上で容易かつ迅速に行なうことができるよう構成されている。 Thus, the purification apparatus 1 is configured so that the plate can be exchanged easily and quickly on the stage 3.
　これにより、各工程での使用に適するように、ステージ３上のプレート組立体１０を第１の状態～第４の状態のうちのいずれかの状態に容易かつ確実にすることができる。よって、簡単にかつ優れた精度で多量の糖鎖を分離精製することができる。 Thereby, the plate assembly 10 on the stage 3 can be easily and surely set in any one of the first state to the fourth state so as to be suitable for use in each process. Therefore, a large amount of sugar chains can be separated and purified easily and with excellent accuracy.
　なお、［７］物質除去工程の直後か、または［９］再遊離工程の直前に、捕捉担体（ポリマー粒子２０）が備える官能基を失活させる工程が行なわれるのが好ましい。この工程では、前記工程［４］において、糖鎖の捕捉に用いられなかった、捕捉担体が備える官能基（糖鎖のアルデヒド基と特異的に反応する官能基）を失活させる。これにより、捕捉担体に糖鎖以外の不純物が化学的に結合するのを確実に防止することができるため、糖鎖の精製率の向上を図ることができる。 In addition, it is preferable that the step of deactivating the functional group included in the capture carrier (polymer particle 20) is performed immediately after the [7] substance removing step or just before the [9] re-releasing step. In this step, the functional group (functional group that reacts specifically with the aldehyde group of the sugar chain) provided in the capture carrier that was not used for capturing the sugar chain in the step [4] is deactivated. As a result, it is possible to reliably prevent impurities other than sugar chains from chemically binding to the capture carrier, so that the purification rate of sugar chains can be improved.
　捕捉担体が備える官能基の失活は、例えば、前記官能基を失活させる機能を有する失活液を、捕捉担体に接触させ、それを静置することにより行うことができる。失活液としては、特に限定されないが、官能基がヒドラジド基である場合、例えば、無水酢酸、無水コハク酸等の酸無水物が挙げられる。これにより、官能基を容易に失活させることができる。 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 and allowing it to stand. Although it does not specifically limit as a deactivation liquid, When a functional group is a hydrazide group, acid anhydrides, such as an acetic anhydride and a succinic anhydride, are mentioned, for example. Thereby, a functional group can be deactivated easily.
　反応条件としては、失活液：１０％無水酢酸／メタノール、温度：常温（室温）、静置時間：３０分間が好ましいが、これに限定されることはない。また、プレート組立体１０を第１の状態として使用する。 The reaction conditions are preferably a quenching solution: 10% acetic anhydride / methanol, temperature: normal temperature (room temperature), and standing time: 30 minutes, but are not limited thereto. Further, the plate assembly 10 is used as the first state.
　また、［１２－４（ノズル洗浄）］と［１２－５（プレート組立体分解）］との間に、「非水溶媒供給」が行なわれるのが好ましい。この非水溶媒供給とは、アセトニトリルを第１の上側プレート１００の各ウェル１０１に供給し、アセトニトリルを吸引することをいう。この非水溶媒供給により、ウェル１０１内に残っているラベル化糖鎖を残らず第２の下側プレート２００に回収する（洗い流す）とともに、第２の下側プレート２００に溜まっているラベル化糖鎖の溶液をアセトニトリル溶液に変えることができる。なお、アセトニトリルを加えることにより、溶液の疎水性が上がる。そのため、ラベル化糖鎖（親水性）が、シリカゲルで構成されたフィルタ１０６（親水性）に保持され易くなる。ラベル化糖鎖が水溶液の状態では、フィルタ１０６に保持され難い。通常、アセトニトリル含量が９０％以上（好ましくは９５％）となるように、アセトニトリル溶液が調製される。 Also, it is preferable that “non-aqueous solvent supply” is performed between [12-4 (nozzle cleaning)] and [12-5 (plate assembly disassembly)]. This non-aqueous solvent supply means that acetonitrile is supplied to each well 101 of the first upper plate 100 and the acetonitrile is sucked. By supplying the non-aqueous solvent, all the labeled sugar chains remaining in the well 101 are recovered (washed out) to the second lower plate 200, and the labeled sugars accumulated in the second lower plate 200 are collected. The chain solution can be changed to an acetonitrile solution. In addition, the hydrophobicity of a solution increases by adding acetonitrile. Therefore, the labeled sugar chain (hydrophilic) is easily held by the filter 106 (hydrophilic) made of silica gel. When the labeled sugar chain is in an aqueous solution state, it is difficult to be held by the filter 106. Usually, an acetonitrile solution is prepared so that the acetonitrile content is 90% or more (preferably 95%).
　＜第２実施形態＞
　図２２は、本発明の糖鎖精製装置の第２実施形態を示す斜視図である。 <Second Embodiment>
FIG. 22 is a perspective view showing a second embodiment of the sugar chain purification apparatus of the present invention.
　以下、この図を参照して本発明の糖鎖精製装置および糖鎖精製方法の第２実施形態について説明するが、前述した実施形態との相違点を中心に説明し、同様の事項はその説明を省略する。 Hereinafter, the second embodiment of the sugar chain purification apparatus and the sugar chain purification method of the present invention will be described with reference to this figure. However, the difference from the above-described embodiment will be mainly described, and the same matters will be described. Is omitted.
　本実施形態は、加熱手段の構成が異なること以外は前記第１実施形態と同様である。 This embodiment is the same as the first embodiment except that the configuration of the heating means is different.
　図２２に示す本実施形態の精製装置１では、ステージ３からヒータ６１（加熱手段６）が省略されている。それに代えて、加熱手段６Ａは、ステージ３と異なる位置に配置されたチャンバ６２と、チャンバ６２内を加熱するヒータ６３とを備えた恒温槽である。 In the refining device 1 of this embodiment shown in FIG. 22, the heater 61 (heating means 6) is omitted from the stage 3. Instead, the heating means 6 </ b> A is a thermostatic chamber including a chamber 62 disposed at a position different from the stage 3 and a heater 63 that heats the inside of the chamber 62.
　チャンバ６２は、箱状をなし、第１の上側プレート１００が出し入れされる口部６４１を有するチャンバ本体６４と、口部６４１を開閉する扉６５とを有している。 The chamber 62 has a box shape, and includes a chamber main body 64 having a mouth portion 641 through which the first upper plate 100 is taken in and out, and a door 65 for opening and closing the mouth portion 641.
　また、ヒータ６３は、チャンバ６２内に配置され、通電により発熱するニクロム線等のような電熱線で構成されている。 Further, the heater 63 is disposed in the chamber 62 and is configured by a heating wire such as a nichrome wire that generates heat when energized.
　そして、第１の上側プレート１００のウェル１０１内の液体を加熱するときには、精製装置１の操作者は、第１の上側プレート１００をチャンバ６２内に移す。その後、ヒータ６３を作動させ、液体の加熱を行なうことができる。 Then, when the liquid in the well 101 of the first upper plate 100 is heated, the operator of the purification apparatus 1 moves the first upper plate 100 into the chamber 62. Thereafter, the heater 63 is operated to heat the liquid.
　＜第３実施形態＞
　図２３は、本発明の糖鎖精製装置の第３実施形態を示す斜視図である。 <Third Embodiment>
FIG. 23 is a perspective view showing a third embodiment of the sugar chain purification apparatus of the present invention.
　以下、この図を参照して本発明の糖鎖精製装置および糖鎖精製方法の第３実施形態について説明するが、前述した実施形態との相違点を中心に説明し、同様の事項はその説明を省略する。 Hereinafter, the third embodiment of the sugar chain purification apparatus and the sugar chain purification method of the present invention will be described with reference to this figure. However, the difference from the above-described embodiment will be mainly described, and the same matters will be described. Is omitted.
　本実施形態は、加熱手段の構成が異なること以外は前記第１実施形態と同様である。 This embodiment is the same as the first embodiment except that the configuration of the heating means is different.
　図２３に示す本実施形態の精製装置１では、ステージ３からヒータ６１（加熱手段６）が省略されている。それに代えて、加熱手段６Ｂは、ステージ３のプレート組立体載置部３１に隣接して配置され、熱媒体が充填された凹部６６１を有する加熱装置本体６６と、凹部６６１内を加熱するヒータ６７とを備えた恒温槽（ヒートブロック）である。なお、熱媒体としては、特に限定されず、例えば、水等のような液体を用いることができる。 23, the heater 61 (heating means 6) is omitted from the stage 3 in the purification apparatus 1 of the present embodiment shown in FIG. Instead, the heating means 6B is disposed adjacent to the plate assembly mounting portion 31 of the stage 3 and has a heating device body 66 having a recess 661 filled with a heat medium, and a heater 67 for heating the inside of the recess 661. And a thermostat (heat block). In addition, it does not specifically limit as a heat medium, For example, liquids, such as water, can be used.
　加熱装置本体６６は、図中の上方に向かって開口した凹部６６１を有している。この凹部６６１に熱媒体を充填して、その充填状態のまま第１の上側プレート１００を加熱装置本体６内に収納することができる。 The heating device main body 66 has a recess 661 that opens upward in the drawing. The recess 661 can be filled with a heat medium, and the first upper plate 100 can be accommodated in the heating apparatus main body 6 in the filled state.
　また、ヒータ６７は、凹部６６１の外側に配置され、通電により発熱するニクロム線等のような電熱線で構成されている。このヒータ６７の熱は、熱媒体を介して第１の上側プレート１００に伝達される。 Further, the heater 67 is arranged on the outside of the recess 661, and is configured by a heating wire such as a nichrome wire that generates heat when energized. The heat of the heater 67 is transmitted to the first upper plate 100 through the heat medium.
　そして、第１の上側プレート１００のウェル１０１内の液体を加熱するときには、精製装置１の操作者は、熱媒体が充填された凹部６６１に第１の上側プレート１００を移す。その後、ヒータ６７を作動させ、液体の加熱を行なうことができる。 Then, when the liquid in the well 101 of the first upper plate 100 is heated, the operator of the purification apparatus 1 moves the first upper plate 100 to the recess 661 filled with the heat medium. Thereafter, the heater 67 is operated to heat the liquid.
　＜第４実施形態＞
　以下、本発明の糖鎖精製装置および糖鎖精製方法の第４実施形態について説明するが、前述した実施形態との相違点を中心に説明し、同様の事項はその説明を省略する。 <Fourth embodiment>
Hereinafter, the fourth embodiment of the sugar chain purification apparatus and the sugar chain purification method of the present invention will be described. The description will focus on differences from the above-described embodiment, and the description of the same matters will be omitted.
　本実施形態は、再遊離工程では、糖鎖の再遊離と糖鎖のラベル化とのタイミングがズレている、すなわち、糖鎖の再遊離の後に糖鎖をラベル化すること以外は前記第１実施形態と同様である。 In this embodiment, in the re-releasing step, the timing of the re-releasing of the sugar chain and the labeling of the sugar chain are shifted, that is, the first except that the sugar chain is labeled after the re-releasing of the sugar chain. This is the same as the embodiment.
　本工程では、化合物Ａとして、芳香族アミンで構成される蛍光物質が用いられる。 In this step, a fluorescent material composed of an aromatic amine is used as compound A.
　化合物Ａとして、このような芳香族アミンを用いた場合、ポリマー粒子２０上に捕捉された糖鎖は、まずポリマー粒子２０から切り離されて再遊離した後に、化合物Ａによりラベル化されることとなる。 When such an aromatic amine is used as the compound A, the sugar chain captured on the polymer particle 20 is first separated from the polymer particle 20 and re-released, and then labeled with the compound A. .
　以下、化合物Ａとして芳香族アミンを用いて、糖鎖を化合物Ａでラベル化する場合について説明する。 Hereinafter, the case where an aromatic amine is used as compound A and the sugar chain is labeled with compound A will be described.
　［９－１’］まず、ノズル４１を用いて、糖鎖を再遊離させる糖鎖遊離液を、糖鎖が捕捉されたポリマー粒子２０を含む第１の上側プレート１００の各ウェル１０１内に添加する（供給する）。 [9-1 ′] First, using the nozzle 41, a sugar chain release solution for re-releasing sugar chains is added to each well 101 of the first upper plate 100 including the polymer particles 20 in which the sugar chains are captured. Do (supply).
　［９－２’］次に、ヒータ６１を作動させて糖鎖遊離液を加熱する。そうすることで、添加した糖鎖遊離液が乾燥するまで、糖鎖遊離液を一定の温度範囲に保つ。これにより、捕捉された糖鎖がポリマー粒子２０から切り離されることで、糖鎖は再遊離する。 [9-2 '] Next, the sugar chain free solution is heated by operating the heater 61. By doing so, 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.
　この際の反応液（糖鎖遊離液）のｐＨは、好ましくは２～９、より好ましくは２～７であり、さらに好ましくは２～６である。このｐＨ調整が、前記工程［９－１’］における糖鎖遊離液のウェル１０１内への添加により行われ、各種緩衝液が糖鎖遊離液として用いられる。 In this case, the pH of the reaction solution (sugar chain free solution) 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 into the well 101 in the step [9-1 '], and various buffer solutions are used as the sugar chain free solution.
　糖鎖遊離時の温度は、好ましくは４～１００℃程度、より好ましくは２５～９０℃程度、さらに好ましくは３０～８０℃程度、最も好ましくは６０～８０℃程度の温度範囲に保たれるように設定する。 The temperature at the time of sugar chain release is preferably kept in a temperature range of about 4 to 100 ° C., more preferably about 25 to 90 ° C., further preferably about 30 to 80 ° C., and most preferably about 60 to 80 ° C. Set to.
　また、反応時間、すなわち溶液（糖鎖遊離液）が乾燥するまでの時間は、かかる温度範囲に設定した場合、通常、０．１～３時間程度、好ましくは、０．６～２時間程度に設定される。 In addition, the reaction time, that is, the time until the solution (sugar chain free solution) 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.
　かかる条件で、糖鎖の再遊離を行うことで、糖鎖がポリマー粒子２０から確実に切り離される。 Under such conditions, the sugar chain is reliably separated from the polymer particle 20 by re-releasing the sugar chain.
　なお、本実施形態のように、糖鎖遊離液が乾燥するまで、糖鎖遊離液を加熱することにより、糖鎖の遊離率の向上を確実に図ることができる。 It should be noted that, as in this embodiment, by heating the sugar chain free solution until the sugar chain free solution is dried, the release rate of the sugar chains can be reliably improved.
　［９－３’］次に、化合物Ａとして芳香族アミンを含有する化合物含有液２５を、ポリマー粒子２０から糖鎖が遊離している、マルチウェルフィルタプレート１００のウェル１０１内にノズル４１を用いて添加する。 [9-3 ′] Next, the compound 41 containing the aromatic amine as the compound A is used in the well 101 of the multiwell filter plate 100 where the sugar chain is released from the polymer particle 20 using the nozzle 41. Add.
　芳香族アミン（化合物Ａ）としては、特に限定されないが、例えば、２－Ａｍｉｎｏｂｅｎｚａｍｉｄｅ、２－Ａｍｉｎｏｂｅｎｚｏｉｃ ａｃｉｄ、８－Ａｍｉｎｏｐｙｒｅｎｅ－１，３，６－ｔｒｉｓｕｌｆｏｎａｔｅ、８－Ａｍｉｎｏｎａｐｈｔｈａｌｅｎｅ－１，３，６－ｔｒｉｓｕｌｐｈｏｎａｔｅ、２－Ａｍｉｎｏ９（１０Ｈ）－ａｃｒｉｄｏｎｅ、５－Ａｍｉｎｏｆｌｕｏｒｅｓｃｅｉｎ、Ｄａｎｓｙｌｅｔｈｙｌｅｎｅｄｉａｍｉｎｅ、７－Ａｍｉｎｏ－４－ｍｅｔｈｙｌｃｏｕｍａｒｉｎｅ、３－Ａｍｉｎｏｂｅｎｚｏｉｃ ａｃｉｄ、７－Ａｍｉｎｏ－１－ｎａｐｈｔｈｏｌおよび３－（Ａｃｅｔｙｌａｍｉｎｏ）－６－ａｍｉｎｏａｃｒｉｄｉｎｅ等が挙げられる。中でも２－ａｍｉｎｏｂｅｎｚａｍｉｄｅまたは２－ａｍｉｎｏｂｅｎｚｏｉｃ　ａｃｉｄであるのが好ましい。これらの化合物は、試薬としての入手、反応の簡便性から好適に用いられる。 The aromatic amine (compound A) is not particularly limited, and examples thereof include 2-aminobenzoamide, 2-aminobenzoic acid, 8-aminopyrene-1,3,6-trisulfonate, 8-aminophenylene-1,3,6-trisulfonate, 2-Amino9 (10H) -acridone, 5-Aminofluorescein, Dansylethylenediamine, 7-Amino-4-methylcoumarin, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3- (Acetylamino) -6-acidolamino-6-acidolamino-6-acidolamino Of 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.
　また、芳香族アミンとして２－ａｍｉｎｏｂｅｎｚａｍｉｄｅまたは２－ａｍｉｎｏｂｅｎｚｏｉｃ　ａｃｉｄを用いる場合、一般的な条件では、それらは０．３５ｍｏｌ／Ｌで使用される。しかしながら、本工程では、化合物含有液２５添加後のウェル１０１内の溶液、すなわち、再遊離した糖鎖を含む溶液における芳香族アミンの濃度は、好ましくは０．５ｍｏｌ／Ｌ以上、より好ましくは１．４ｍｏｌ／Ｌ以上に設定される。これにより、化合物Ａによる糖鎖の標識効率を向上させることが可能となる。ただし、前記芳香族アミンの濃度が３ｍｏｌ／Ｌ以上になると、後の糖鎖精製工程において、糖鎖のラベル化反応に使用されなかった芳香族アミン（化合物Ａ）を除去するのが困難となるおそれがある。そのため、最も好ましい芳香族アミンの濃度は１．４ｍｏｌ／Ｌ以上３ｍｏｌ／Ｌ以下に設定される。 Also, when 2-aminobenzamide or 2-aminobenzoic acid is used as the aromatic amine, they are used at 0.35 mol / L under general conditions. However, in this step, the concentration of the aromatic amine in the solution in the well 101 after the addition of the compound-containing solution 25, that is, the solution containing the re-released sugar chain is preferably 0.5 mol / L or more, more preferably 1 It is set to 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 of the aromatic amine is 3 mol / L or more, it becomes difficult to remove the aromatic amine (compound A) that has not been used for the sugar chain labeling reaction in the subsequent sugar chain purification step. There is a fear. Therefore, the most preferable aromatic amine concentration is set to 1.4 mol / L or more and 3 mol / L or less.
　また、化合物含有液２５の液量に関して、ウェル１０１内に含まれるポリマー粒子２０で化合物含有液２５の液量を規定する場合、その液量は、通常はポリマー粒子２０が浸る程度の液量、例えば、５ｍｇのポリマー粒子２０に対して５０μＬ程度に設定される。しかしながら、本実施形態では、化合物含有液２５の液量（容量）を通常の液量の倍量の１００μＬ程度に設定するのが好ましい。これにより、化合物Ａによる糖鎖の標識効率を向上させることが可能となる。ただし、液量は１００μＬを超えても良いが、一定量を超えると、後の糖鎖精製工程において、糖鎖のラベル化反応に使用されなかった芳香族アミン（化合物Ａ）を除去するのが困難となるおそれがある。そのため、最も好まし液量は１００μＬ～２００μＬの間に設定される。 In addition, regarding the amount of the compound-containing solution 25, when the amount of the compound-containing solution 25 is defined by the polymer particles 20 contained in the well 101, the amount of the solution is usually such that the polymer particles 20 are immersed, For example, it is set to about 50 μL with respect to 5 mg of polymer particles 20. However, in the present embodiment, it is preferable to set the liquid volume (volume) of the compound-containing liquid 25 to about 100 μL that is twice the normal liquid 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 amount, the aromatic amine (compound A) that has not been used for the sugar chain labeling reaction is removed in the subsequent sugar chain purification step. May be difficult. Therefore, the most preferable amount of liquid is set between 100 μL and 200 μL.
　より具体的には、芳香族アミンとして２－ａｍｉｎｏｂｅｎｚａｍｉｄｅを用いる場合、ウェル１０１内に、1.4 M 2-Aminobenzamid, 1 M sodium cyanoborohydrideの濃度になるように、３０％酢酸／ジメチルスルホキシド（ＤＭＳＯ）に溶解させた溶液１００μＬを化合物含有液２５として添加する。 More specifically, when 2-aminobenzamide is used as the aromatic amine, the well 101 is dissolved in 30% acetic acid / dimethyl sulfoxide (DMSO) so as to have a concentration of 1.4 M 2-Aminobenzamid, 1 M sodium cyanoborohydride. 100 μL of the prepared solution is added as the compound-containing solution 25.
　［９－４’］次に、ヒータ６１を作動させて化合物含有液２５を加熱することで、化合物含有液２５を一定の温度範囲に保つ。これにより、ポリマー粒子２０から再遊離している糖鎖は化合物Ａと反応し、その結果、糖鎖は化合物Ａでラベル化されることとなる。 [9-4 '] Next, the compound-containing liquid 25 is kept in a certain temperature range by operating the heater 61 to heat the compound-containing liquid 25. 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.
　この際の反応液（化合物含有液２５）のｐＨは、好ましくは２～９、より好ましくは２～７であり、さらに好ましくは２～６である。 In this case, the pH of the reaction solution (compound-containing solution 25) is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6.
　ラベル化時の化合物含有液２５の温度は、好ましくは０～１００℃程度、より好ましくは４～９５℃程度、さらに好ましくは３０～９０℃程度の温度範囲に保たれるように設定する。 The temperature of the compound-containing liquid 25 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.
　また、反応時間は、かかる温度範囲に設定した場合、通常、０．１～２０時間程度、好ましくは、０．６～１２時間程度に設定される。 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.
　より具体的には、芳香族アミンとして２－ａｍｉｎｏｂｅｎｚａｍｉｄｅを用いた場合、化合物含有液２５を３０～７０℃の温度範囲で加熱して、１～１０時間程度、反応する。 More specifically, when 2-aminobenzamide is used as the aromatic amine, the compound-containing liquid 25 is heated in the temperature range of 30 to 70 ° C. and reacted for about 1 to 10 hours.
　かかる条件で、ラベル化を行うことで、糖鎖が確実に化合物Ａによりラベル化される。 By performing labeling under such conditions, the sugar chain is surely labeled with compound A.
　以上のような工程［９－１’］～［９－４’］によっても、糖鎖が化合物Ａでラベル化される。 The sugar chain is labeled with the compound A also by the steps [9-1 ′] to [9-4 ′] as described above.
　以上、本発明の糖鎖精製装置および糖鎖精製方法を図示の実施形態について説明したが、本発明は、これに限定されるものではなく、糖鎖精製装置を構成する各部は、同様の機能を発揮し得る任意の構成のものと置換することができる。また、任意の構成物が付加されていてもよい。 As described above, the sugar chain purification apparatus and the sugar chain purification method of the present invention have been described with reference to the illustrated embodiment. However, the present invention is not limited to this, and each part constituting the sugar chain purification apparatus has the same function. Can be substituted with any structure capable of exhibiting Moreover, arbitrary components may be added.
　また、分注手段のノズルは、前記各実施形態ではノズルヘッドに固定されているが、これに限定されず、ノズルヘッドに着脱自在に装着されていてもよい。 In addition, the nozzle of the dispensing means is fixed to the nozzle head in each of the above embodiments, but is not limited to this, and may be detachably attached to the nozzle head.
　また、移動手段は、前記各実施形態ではステージ上のプレート組立体に対して分注手段のノズルを移動するよう構成されていたが、これに限定されず、ノズルに対してステージ上のプレート組立体を移動するよう構成されていてもよい。 In addition, the moving means is configured to move the nozzle of the dispensing means with respect to the plate assembly on the stage in each of the above embodiments. It may be configured to move a solid.
　また、糖鎖精製装置は、加熱手段が省略されていてもよい。 Further, the sugar chain purification apparatus may omit the heating means.
　また、糖鎖精製装置は、前記各実施形態ではプレート組立体において、上側プレート（第１の上側プレート、第２の上側プレート）の各ウェルが有するフィルタの下側を、その上側より負圧とすることでフィルタに液体を透過させたが、これに限定されず、フィルタの上側を、その下側より正圧とすることでフィルタに液体を透過させてもよい。 In the above-described embodiments, the sugar chain purification apparatus is configured such that, in the plate assembly, the lower side of the filter of each well of the upper plate (first upper plate, second upper plate) has a negative pressure from the upper side. Thus, the liquid is allowed to pass through the filter, but the present invention is not limited to this, and the liquid may pass through the filter by setting the upper side of the filter to a positive pressure from the lower side.
　また、プレート組立体の、第１の上側プレートと第２の上側プレートとの交換、第１の下側プレートと第２の下側プレートとの交換は、ステージ上で実行されなくてもよい。 Further, the exchange of the first upper plate and the second upper plate and the exchange of the first lower plate and the second lower plate of the plate assembly may not be performed on the stage.
　また、プレートの移動およびプレート組立体の分解、部材の着脱を、前記各実施形態では精製装置の操作者（作業者）の手作業で行ったが、これに限定されず、例えば、ロボットアーム等の手段を用いて自動化してもよい。 In addition, the movement of the plate, the disassembly of the plate assembly, and the attachment / detachment of the members are performed manually by the operator (operator) of the refining apparatus in each of the above embodiments. You may automate using the means of.
　また、糖鎖精製装置全体が、内部が加熱可能なチャンバ内に格納されていてもよい。 Further, the entire sugar chain purification apparatus may be stored in a chamber in which the inside can be heated.
　本発明は、糖鎖精製装置を提供する。その糖鎖精製装置は、糖鎖を含有する溶液中から前記糖鎖を精製する際に用いられるプレート組立体と、該プレート組立体が載置されるステージと、該ステージ上の前記プレート組立体に対し、液体を吸引し、吐出するノズルを有する分注手段と、前記ステージ上の前記プレート組立体と前記ノズルとを相対的に移動させる移動手段とを備えている。前記プレート組立体は、板状をなし、その上面に開口する第１の凹部で構成され、該第１の凹部の底部を貫通する第１の貫通孔が形成された複数の第１のウェルと、該各第１のウェルの前記第１の貫通孔をそれぞれ覆うように前記第１の凹部の前記底部に設置された第１のフィルタとを有する第１の上側プレートと、板状をなし、その上面に開口する第２の凹部で構成され、該第２の凹部の底部を貫通する第２の貫通孔が形成された複数の第２のウェルと、該各第２のウェルの前記第２の貫通孔をそれぞれ覆うように前記第２の凹部の前記底部に設置され、前記第１のフィルタと異なる第２のフィルタとを有する第２の上側プレートと、板状をなし、その上面に開口する第３の凹部で構成された１つの第１の貯留部を有する第１の下側プレートと、板状をなし、その上面に開口する第４の凹部で構成された複数の第２の貯留部を有する第２の下側プレートと、　前記第１の上側プレートおよび前記第２の上側プレートのうちの一方と、前記第１の下側プレートおよび前記第２の下側プレートのうちの一方とを上下に配置して組み立てた状態で支持する１つの支持体とを備え、前記プレート組立体は、前記ステージ上で、前記１つの支持体に対して、前記第１の上側プレートと前記第２の上側プレートとが交換されるとともに、前記第１の下側プレートと前記第２の下側プレートとが交換されるよう構成されている。これにより、糖鎖を簡便にかつ優れた精度で分離精製し、収率よく糖鎖を蛍光標識することが可能となる。したがって、本発明は産業上の利用可能性を有する。 The present invention provides a sugar chain purification apparatus. The sugar chain purification apparatus includes a plate assembly used when purifying the sugar chain from a solution containing sugar chains, a stage on which the plate assembly is mounted, and the plate assembly on the stage. On the other hand, a dispensing means having a nozzle for sucking and discharging a liquid and a moving means for relatively moving the plate assembly and the nozzle on the stage are provided. The plate assembly includes a plurality of first wells that are plate-shaped and configured by a first recess opening on an upper surface of the plate assembly, and a first through hole penetrating the bottom of the first recess. A first upper plate having a first filter installed at the bottom of the first recess so as to cover the first through holes of the first wells, and a plate shape; A plurality of second wells each having a second through hole formed with a second recess opening on the upper surface thereof and penetrating the bottom of the second recess, and the second well of each second well. A second upper plate that is installed at the bottom of the second recess so as to cover each of the through-holes, has a second filter different from the first filter, has a plate shape, and has an opening on the upper surface thereof A first lower play having one first storage portion constituted by a third recess And a second lower plate having a plurality of second storage portions each having a plate shape and having a fourth recess opening on an upper surface thereof, the first upper plate and the second upper plate One of the first lower plate and one of the second lower plate and one support member that supports the plate assembly in an assembled state by being arranged one above the other. On the stage, the first upper plate and the second upper plate are exchanged with respect to the one support body, and the first lower plate and the second lower plate are exchanged. The plate is configured to be exchanged. This makes it possible to separate and purify sugar chains easily and with excellent accuracy, and to fluorescently label sugar chains with high yield. Therefore, the present invention has industrial applicability.
　１　　　　　　糖鎖精製装置（精製装置）
　３　　　　　　ステージ
　３１　　　　　プレート組立体載置部
　３２　　　　　第１の上側プレート載置部
　３３　　　　　第２の上側プレート載置部
　３４　　　　　第１の下側プレート載置部
　３５　　　　　第２の下側プレート載置部
　３６　　　　　洗浄槽載置部
　３７　　　　　処理液載置部
　４　　　　　　分注手段
　４１　　　　　ノズル（ピペッタ）
　４１１　　　　先端開口部
　４２　　　　　ポンプ
　４３　　　　　チューブ
　４４　　　　　ノズルヘッド
　５　　　　　　移動手段
　５１　　　　　ｘ軸方向移動機構（水平方向移動機構）
　５２　　　　　ｙ軸方向移動機構（水平方向移動機構）
　５３　　　　　ｚ軸方向移動機構（鉛直方向移動機構）
　６、６Ａ、６Ｂ　加熱手段
　６１　　　　　ヒータ
　６２　　　　　チャンバ
　６３　　　　　ヒータ
　６４　　　　　チャンバ本体
　６４１　　　　口部
　６５　　　　　扉
　６６　　　　　加熱装置本体
　６６１　　　　凹部
　６７　　　　　ヒータ
　７　　　　　　洗浄槽
　７１　　　　　洗浄液
　８１　　　　　マルチウェルプレート
　８１１　　　　ウェル
　８２、８３、８４、８５、８６、８７　タンク
　９ａ、９ｂ　　内部空間
　１０　　　　　プレート組立体
　１１　　　　　コントロールパネル
　１１１　　　　液晶画面
　１２　　　　　壁部
　１３　　　　　吸引ポンプ
　１４　　　　　チューブ
　２０　　　　　ポリマー粒子
　２１　　　　　純水
　２２　　　　　粒子分散液
　２３　　　　　糖鎖含有液（溶液）
　２４　　　　　洗浄液
　２５　　　　　化合物含有液
　２６　　　　　溶解液
　２７　　　　　非水溶媒
　２８　　　　　水
　１００　　　　第１の上側プレート（マルチウェルフィルタプレート）
　１００’　　　第２の上側プレート（クリーンアッププレート）
　１０１　　　　ウェル（第１のウェル）
　１０１’　　　ウェル（第２のウェル）
　１０３、１０３’　底部
　１０４、１０４’　貫通孔
　１０５　　　　フィルタ（第１のフィルタ）
　１０６　　　　フィルタ（第２のフィルタ）
　１０７　　　　突起部
　１０８、１０８’　上面
　１０９、１０９’　下面
　２００　　　　第２の下側プレート（マルチウェルプレート）
　２０１　　　　ウェル（第２の貯留部）
　２０２　　　　ウェル形成部
　２０３　　　　底部
　２０４　　　　上面
　２０５　　　　フレーム部
　３００　　　　第１の下側プレート（廃液トレイ）
　３０１　　　　凹部（第１の貯留部）
　３０２　　　　上面
　３０３　　　　凹部形成部
　３０４　　　　フレーム部
　４００　　　　第１の支持台（上側支持部材）
　４０１　　　　底部
　４０２　　　　開口部
　４０３　　　　上側外壁
　４０４　　　　下側外壁
　４０５、４０６　凹部
　４０７　　　　パッキン（シール部材）
　４０８　　　　開口部
　５００　　　　第２の支持台（下側支持部材）
　５０１　　　　底部
　５０２　　　　突出部
　５０３　　　　外壁
　５０５　　　　凹部
　５０６　　　　貫通孔
　５０７　　　　パッキン（シール部材）
　６００　　　　調整板（スペーサ）
　７００　　　　支持体
　ｄ１、ｄ２、ｄ３、ｄ４　離間距離 1 Sugar chain purification equipment (purification equipment)
3 Stage 31 Plate Assembly Placement Part 32 First Upper Plate Placement Part 33 Second Upper Plate Placement Part 34 First Lower Plate Placement Part 35 Second Lower Plate Placement Part 36 Cleaning Tank Placement part 37 Treatment liquid placement part 4 Dispensing means 41 Nozzle (pipetter)
411 End opening 42 Pump 43 Tube 44 Nozzle head 5 Moving means 51 X-axis direction moving mechanism (horizontal direction moving mechanism)
52 y-axis direction moving mechanism (horizontal direction moving mechanism)
53 z-axis direction moving mechanism (vertical direction moving mechanism)
6, 6A, 6B Heating means 61 Heater 62 Chamber 63 Heater 64 Chamber main body 641 Mouth 65 Door 66 Heating device main body 661 Recessed 67 Heater 7 Cleaning tank 71 Cleaning liquid 81 Multiwell plate 811 Well 82, 83, 84, 85, 86, 87 Tank 9a, 9b Internal space 10 Plate assembly 11 Control panel 111 Liquid crystal screen 12 Wall part 13 Suction pump 14 Tube 20 Polymer particle 21 Pure water 22 Particle dispersion liquid 23 Sugar chain containing liquid (solution)
24 Washing solution 25 Compound-containing solution 26 Dissolving solution 27 Non-aqueous solvent 28 Water 100 First upper plate (multiwell filter plate)
100 'second upper plate (cleanup plate)
101 well (first well)
101 'well (second well)
103, 103 ′ bottom 104, 104 ′ through-hole 105 filter (first filter)
106 Filter (second filter)
107 Protrusion 108, 108 'Upper surface 109, 109' Lower surface 200 Second lower plate (multiwell plate)
201 well (second reservoir)
202 Well forming portion 203 Bottom portion 204 Upper surface 205 Frame portion 300 First lower plate (waste liquid tray)
301 Concave portion (first storage portion)
302 Upper surface 303 Concave forming portion 304 Frame portion 400 First support base (upper support member)
401 Bottom 402 Opening 403 Upper outer wall 404 Lower outer wall 405, 406 Recess 407 Packing (seal member)
408 Opening 500 Second support base (lower support member)
501 Bottom 502 Projection 503 Outer wall 505 Recess 506 Through-hole 507 Packing (seal member)
600 Adjustment plate (spacer)
700 Supports d 1 , d 2 , d 3 , d 4 separation distance

Claims (25)

  1. 　糖鎖を含有する溶液中から前記糖鎖を精製する際に用いられるプレート組立体と、該プレート組立体が載置されるステージと、該ステージ上の前記プレート組立体に対し、液体を吸引し、吐出するノズルを有する分注手段と、前記ステージ上の前記プレート組立体と前記ノズルとを相対的に移動させる移動手段とを備える糖鎖精製装置であって、
    　前記プレート組立体は、板状をなし、その上面に開口する第１の凹部で構成され、該第１の凹部の底部を貫通する第１の貫通孔が形成された複数の第１のウェルと、該各第１のウェルの前記第１の貫通孔をそれぞれ覆うように前記第１の凹部の前記底部に設置された第１のフィルタとを有する第１の上側プレートと、
    　板状をなし、その上面に開口する第２の凹部で構成され、該第２の凹部の底部を貫通する第２の貫通孔が形成された複数の第２のウェルと、該各第２のウェルの前記第２の貫通孔をそれぞれ覆うように前記第２の凹部の前記底部に設置され、前記第１のフィルタと異なる第２のフィルタとを有する第２の上側プレートと、
    　板状をなし、その上面に開口する第３の凹部で構成された１つの第１の貯留部を有する第１の下側プレートと、
    　板状をなし、その上面に開口する第４の凹部で構成された複数の第２の貯留部を有する第２の下側プレートと、
    　前記第１の上側プレートおよび前記第２の上側プレートのうちの一方と、前記第１の下側プレートおよび前記第２の下側プレートのうちの一方とを上下に配置して組み立てた状態で支持する１つの支持体とを備え、
    　前記プレート組立体は、前記ステージ上で、前記１つの支持体に対して、前記第１の上側プレートと前記第２の上側プレートとが交換されるとともに、前記第１の下側プレートと前記第２の下側プレートとが交換されるよう構成されていることを特徴とする糖鎖精製装置。     A plate assembly used for purifying the sugar chain from a solution containing a sugar chain, a stage on which the plate assembly is mounted, and a liquid is sucked into the plate assembly on the stage. A sugar chain purification apparatus comprising dispensing means having a nozzle for discharging, and moving means for relatively moving the plate assembly on the stage and the nozzle,
    The plate assembly includes a plurality of first wells that are plate-shaped and configured by a first recess opening on an upper surface of the plate assembly, and a first through hole penetrating the bottom of the first recess. A first upper plate having a first filter installed at the bottom of the first recess so as to cover the first through hole of each first well,
    A plurality of second wells each having a plate-like shape and configured by a second recess opening on an upper surface thereof and having a second through-hole penetrating the bottom of the second recess; A second upper plate installed at the bottom of the second recess so as to cover the second through-holes of the wells and having a second filter different from the first filter;
    A first lower plate having a first storage portion formed of a third recess that is plate-shaped and opens on an upper surface thereof;
    A second lower plate having a plurality of second storage portions each having a plate shape and configured by a fourth recess opening on an upper surface thereof;
    One of the first upper plate and the second upper plate and one of the first lower plate and the second lower plate are vertically arranged and supported in an assembled state And a single support that
    In the plate assembly, the first upper plate and the second upper plate are exchanged with respect to the one support on the stage, and the first lower plate and the first plate are exchanged. 2. A sugar chain purification apparatus, wherein the lower plate is exchanged.
  2. 　前記プレート組立体を加熱する加熱手段さらに備える請求項１に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 1, further comprising heating means for heating the plate assembly.
  3. 　前記第１の下側プレートと前記第２の下側プレートとの交換が行われるときには、前記加熱手段の作動が停止する請求項２に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 2, wherein when the first lower plate and the second lower plate are exchanged, the operation of the heating means is stopped.
  4. 　前記プレート組立体は、前記支持体に前記第１の上側プレートが支持され、前記第１の下側プレートが支持された第１の状態と、
    　前記第１の状態において前記第１の下側プレートと前記第２の下側プレートとが交換され、該第２の下側プレートが前記支持体に支持された第２の状態と、
    　前記第２の状態において前記第１の上側プレートと前記第２の上側プレートとが交換され、該第２の上側プレートが前記支持体に支持され、前記第２の下側プレートと前記第１の下側プレートとが交換され、該第１の下側プレートが前記支持体に支持された第３の状態と、
    　前記第３の状態において前記第１の下側プレートと前記第２の下側プレートとが交換され、該第２の下側プレートが前記支持体に支持された第４の状態とを取り得る請求項２に記載の糖鎖精製装置。     The plate assembly includes a first state in which the first upper plate is supported by the support and the first lower plate is supported;
    A second state in which the first lower plate and the second lower plate are exchanged in the first state, and the second lower plate is supported by the support;
    In the second state, the first upper plate and the second upper plate are exchanged, the second upper plate is supported by the support, the second lower plate and the first plate A third state in which the lower plate is replaced and the first lower plate is supported by the support;
    In the third state, the first lower plate and the second lower plate are exchanged, and the second lower plate can be in a fourth state supported by the support. Item 3. The sugar chain purification apparatus according to Item 2.
  5. 　前記第１の状態では、前記各第１のウェルは、その前記第１の貫通孔を介して、前記第１の貯留部と一括して連通しており、
    　前記第２の状態では、前記各第１のウェルは、その前記第１の貫通孔を介して、前記各第２の貯留部とそれぞれ連通しており、
    　前記第３の状態では、前記各第２のウェルは、その前記第２の貫通孔を介して、前記第１の貯留部と一括して連通しており、
    　前記第４の状態では、前記各第２のウェルは、その前記第２の貫通孔を介して、前記各第２の貯留部とそれぞれ連通している請求項４に記載の糖鎖精製装置。     In the first state, each of the first wells communicates with the first reservoir through the first through hole.
    In the second state, each of the first wells communicates with each of the second storage portions via the first through hole,
    In the third state, each of the second wells communicates with the first reservoir through the second through hole, and
    5. The sugar chain purification apparatus according to claim 4, wherein, in the fourth state, each of the second wells communicates with each of the second storage portions via the second through hole.
  6. 　当該糖鎖精製装置は、前記各第１のウェルにそれぞれ前記糖鎖と特異的に結合する捕捉担体を供給する第１の工程と、
    　前記各第１のウェルにそれぞれ前記溶液を前記ノズルから分注して、該溶液と前記捕捉担体とを接触させ、該捕捉担体上に前記糖鎖を捕捉する第２の工程と、
    　前記捕捉担体に結合した糖鎖以外の物質を前記ウェルから除去する第３の工程と、
    　前記捕捉担体に結合した糖鎖を再遊離させる第４の工程と、
    　再遊離した前記糖鎖を前記捕捉担体と分離して精製する第５の工程とを順に行なうよう構成され、
    　前記第１の工程、前記第２の工程、前記第３の工程および前記第４の工程では、前記プレート組立体を前記第１の状態とし、前記第５の工程では、前記プレート組立体を順に前記第２の状態、前記第３の状態、前記第４の状態とする請求項５に記載の糖鎖精製装置。     The sugar chain purification apparatus includes a first step of supplying a capture carrier that specifically binds to the sugar chain to each of the first wells;
    A second step of dispensing the solution into the first wells from the nozzle, bringing the solution into contact with the capture carrier, and capturing the sugar chain on the capture carrier;
    A third step of removing substances other than sugar chains bound to the capture carrier from the well;
    A fourth step of re-releasing the sugar chain bound to the capture carrier;
    A fifth step of separating the purified sugar chain from the capture carrier and purifying the sugar chain in sequence,
    In the first step, the second step, the third step, and the fourth step, the plate assembly is set to the first state, and in the fifth step, the plate assembly is sequentially changed. The sugar chain purification apparatus according to claim 5, wherein the second state, the third state, and the fourth state are set.
  7. 　前記加熱手段は、前記第１の工程、前記第２の工程、前記第３の工程、前記第４の工程および前記第５の工程の各工程で、前記プレート組立体または前記第１の上側プレートに対する加熱温度を変更可能に構成されている請求項６に記載の糖鎖精製装置。 The heating means includes the plate assembly or the first upper plate in each of the first step, the second step, the third step, the fourth step, and the fifth step. The sugar chain refining device according to claim 6, which is configured to be able to change a heating temperature with respect to.
  8. 　前記加熱手段は、前記ステージに埋設されたヒータを有する請求項２に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 2, wherein the heating means includes a heater embedded in the stage.
  9. 　前記加熱手段は、ヒータを有する恒温槽である請求項２に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 2, wherein the heating means is a thermostatic bath having a heater.
  10. 　前記支持体は、前記第１の上側プレートおよび前記第２の上側プレートのうちの前記一方を支持する上側支持部材と、該上側支持部材と別体で構成され、前記第１の下側プレートおよび前記第２の下側プレートのうちの前記一方を支持する下側支持部材とを有し、
    　前記上側支持部材と前記下側支持部材とは、前記上側支持部材が前記第１の上側プレートおよび前記第２の上側プレートのうちの前記一方を支持し、前記下側支持部材が前記第１の下側プレートおよび前記第２の下側プレートのうちの前記一方を支持した状態で、分解可能、組立可能に構成されている請求項１に記載の糖鎖精製装置。     The support is configured by an upper support member that supports the one of the first upper plate and the second upper plate, and a separate body from the upper support member, and the first lower plate and A lower support member that supports the one of the second lower plates;
    The upper support member and the lower support member are such that the upper support member supports the one of the first upper plate and the second upper plate, and the lower support member is the first support member. 2. The sugar chain purification apparatus according to claim 1, wherein the sugar chain purification apparatus is configured to be disassembleable and assembleable in a state where the one of the lower plate and the second lower plate is supported.
  11. 　前記第１の上側プレートと前記第２の上側プレートとの交換と、前記第１の下側プレートと前記第２の下側プレートとの交換とは、それぞれ、前記上側支持部材と前記下側支持部材とが分解された状態で行なわれる請求項１０に記載の糖鎖精製装置。 The replacement of the first upper plate and the second upper plate and the replacement of the first lower plate and the second lower plate are respectively the upper support member and the lower support. The sugar chain purification apparatus according to claim 10, which is performed in a state in which the member is decomposed.
  12. 　前記プレート組立体では、前記第１の上側プレートおよび前記第２の上側プレートのうちの前記一方の下面と、前記第１の下側プレートおよび前記第２の下側プレートのうちの前記一方の上面とが離間して配置され、その離間距離が０．５ｍｍ以上１０ｍｍ以下である請求項１に記載の糖鎖精製装置。 In the plate assembly, the lower surface of the one of the first upper plate and the second upper plate, and the upper surface of the one of the first lower plate and the second lower plate The sugar chain purification apparatus according to claim 1, wherein the separation distance is 0.5 mm or more and 10 mm or less.
  13. 　前記第１のフィルタは、多孔性フィルムまたは不織布で構成されている請求項１に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 1, wherein the first filter is composed of a porous film or a nonwoven fabric.
  14. 　前記第１のフィルタの目開きは、０．１～５０μｍである請求項１３に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 13, wherein the opening of the first filter is 0.1 to 50 µm.
  15. 　前記第２のフィルタは、シリカゲルで構成されている請求項１に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 1, wherein the second filter is made of silica gel.
  16. 　前記分注手段は、ポンプと、該ポンプと前記ノズルとを連結するチューブとを有する請求項１に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 1, wherein the dispensing means has a pump and a tube connecting the pump and the nozzle.
  17. 　前記移動手段は、前記ノズルを水平方向に移動させる水平方向移動機構と、前記ノズルを鉛直方向に移動させる鉛直方向移動機構とを有する請求項１に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 1, wherein the moving means includes a horizontal movement mechanism that moves the nozzle in the horizontal direction and a vertical movement mechanism that moves the nozzle in the vertical direction.
  18. 　前記ステージには、前記第１の上側プレートが複数枚重ねて載置される第１の上側プレート載置部と、前記第２の上側プレートが複数枚重ねて載置される第２の上側プレート載置部と、前記第１の下側プレートが複数枚重ねて載置される第１の下側プレート載置部と、前記第２の下側プレートが複数枚重ねて載置される第２の下側プレート載置部とが設けられている請求項１に記載の糖鎖精製装置。 On the stage, a first upper plate placing portion on which a plurality of the first upper plates are placed and a second upper plate on which a plurality of the second upper plates are placed. A mounting portion, a first lower plate mounting portion on which a plurality of the first lower plates are stacked, and a second on which a plurality of the second lower plates are stacked. The sugar chain purification apparatus according to claim 1, further comprising a lower plate mounting portion.
  19. 　前記ノズルを洗浄する洗浄槽をさらに備える請求項１に記載の糖鎖精製装置。 The sugar chain purification apparatus according to claim 1, further comprising a washing tank for washing the nozzle.
  20. 　請求項６に記載の糖鎖精製装置を用いて、前記糖鎖を精製する方法であって、
    　前記第４の工程において、前記捕捉担体に結合した糖鎖を再遊離させるとともに、該糖鎖をラベル化試薬でラベル化することを特徴とする糖鎖精製方法。     A method for purifying the sugar chain using the sugar chain purification apparatus according to claim 6,
    In the fourth step, a sugar chain purification method, wherein the sugar chain bound to the capture carrier is re-released and the sugar chain is labeled with a labeling reagent.
  21. 　前記第４の工程で、前記捕捉担体からの前記糖鎖の再遊離の後に、前記糖鎖を前記ラベル化試薬でラベル化する請求項２０に記載の糖鎖精製方法。 21. The method for purifying a sugar chain according to claim 20, wherein in the fourth step, the sugar chain is labeled with the labeling reagent after the sugar chain is re-released from the capture carrier.
  22. 　前記ラベル化試薬は、芳香族アミンで構成される蛍光物質である請求項２１に記載の糖鎖精製方法。 The sugar chain purification method according to claim 21, wherein the labeling reagent is a fluorescent substance composed of an aromatic amine.
  23. 　再遊離した前記糖鎖を含む前記溶液における前記蛍光物質の濃度は、０．５ｍｏｌ／Ｌ以上である請求項２２に記載の糖鎖精製方法。 The method for purifying a sugar chain according to claim 22, wherein the concentration of the fluorescent substance in the solution containing the re-released sugar chain is 0.5 mol / L or more.
  24. 　前記蛍光物質は、２－Ａｍｉｎｏｂｅｎｚａｍｉｄｅ、２－Ａｍｉｎｏｂｅｎｚｏｉｃ  ａｃｉｄ、８－Ａｍｉｎｏｐｙｒｅｎｅ－１，３，６－ｔｒｉｓｕｌｆｏｎａｔｅ、８－Ａｍｉｎｏｎａｐｈｔｈａｌｅｎｅ－１，３，６－ｔｒｉｓｕｌｐｈｏｎａｔｅ、２－Ａｍｉｎｏ９（１０Ｈ）－ａｃｒｉｄｏｎｅ、５－Ａｍｉｎｏｆｌｕｏｒｅｓｃｅｉｎ、Ｄａｎｓｙｌｅｔｈｙｌｅｎｅｄｉａｍｉｎｅ、７－Ａｍｉｎｏ－４－ｍｅｔｈｙｌｃｏｕｍａｒｉｎｅ、３－Ａｍｉｎｏｂｅｎｚｏｉｃ  ａｃｉｄ、７－Ａｍｉｎｏ－１－ｎａｐｈｔｈｏｌ、３－（Ａｃｅｔｙｌａｍｉｎｏ）－６－ａｍｉｎｏａｃｒｉｄｉｎｅから選ばれる少なくとも１種である請求項２２に記載の糖鎖精製方法。 The fluorescent substances include 2-Aminobenzoamide, 2-Aminobenzoic acid, 8-Aminopyrene-1,3,6-trisulfonate, 8-Aminophathalene-1,3,6-trisulphonate, 2-Amino9 (10H) -acidone, 5-Aminofluor, 23. The sugar chain purification according to claim 22, wherein the sugar chain is at least one selected from the group consisting of: Dynetyrenediamine, 7-Amino-4-methylcoumarin, 3-Aminobenzoic acid, 7-Amino-1-naphthol, and 3- (Acetylamino) -6-aminoacidine. Method.
  25. 　前記捕捉担体は、ヒドラジド基またはオキシルアミノ基を有するポリマー粒子である請求項２０に記載の糖鎖精製方法。 The sugar chain purification method according to claim 20, wherein the capture carrier is a polymer particle having a hydrazide group or an oxylamino group.
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