WO2013047707A1 - Dispositif et procédé de raffinage de chaîne saccharide - Google Patents

Dispositif et procédé de raffinage de chaîne saccharide 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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English (en)
Japanese (ja)
Inventor
秀行 島岡
碧 阪口
Original Assignee
住友ベークライト株式会社
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Filing date
Publication date
Priority claimed from JP2012213078A external-priority patent/JP6123206B2/ja
Priority claimed from JP2012213077A external-priority patent/JP2013081456A/ja
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/fr

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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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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un dispositif de raffinage de chaîne saccharide et un procédé de raffinage de chaîne saccharide apte à séparer et de raffiner de grands volumes de chaînes saccharides simplement et avec une précision remarquable. Un dispositif (1) de raffinage de chaîne saccharide, qui raffine des chaînes saccharides provenant d'un liquide contenant de telles chaînes, est pourvu : d'un ensemble (10) de plaques formé d'une première plaque (100) supérieure, d'une seconde plaque (100') supérieure, d'une première plaque (300) inférieure, d'une seconde plaque (200) inférieure, et d'un corps (700) de support qui porte soit la première plaque (100) supérieure, soit la seconde plaque (100') supérieure et soit la première plaque (300) inférieure, soit la seconde plaque (200) inférieure alors qu'elles sont assemblées en un agencement vertical ; d'un plateau (3) sur lequel l'ensemble (10) de plaques est monté. Dans l'ensemble (10) de plaques, sur le plateau (3), la première plaque (100) supérieure et la seconde plaque (100') supérieure sont permutées et la première plaque (300) inférieure et la seconde plaque (200) inférieure sont permutées.
PCT/JP2012/074983 2011-09-29 2012-09-27 Dispositif et procédé de raffinage de chaîne saccharide WO2013047707A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
JP2011215141 2011-09-29
JP2011215142 2011-09-29
JP2011-215141 2011-09-29
JP2011-215142 2011-09-29
JP2012213078A JP6123206B2 (ja) 2011-09-29 2012-09-26 糖鎖精製装置および糖鎖精製方法
JP2012-213078 2012-09-26
JP2012-213077 2012-09-26
JP2012213077A JP2013081456A (ja) 2011-09-29 2012-09-26 糖鎖精製装置および糖鎖精製方法

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CN104236979A (zh) * 2014-10-09 2014-12-24 南京农业大学 一种可同时提取糖链的装置
CN107827996A (zh) * 2017-12-29 2018-03-23 福建海兴保健食品有限公司 一种从新鲜绿藻中提取绿藻蛋白多糖的生产线及其工艺

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JP2006231166A (ja) * 2005-02-23 2006-09-07 Fukae Kasei Kk 液体試料の処理用器具及びその使用方法
WO2008018170A1 (fr) * 2006-08-09 2008-02-14 Sumitomo Bakelite Co., Ltd. Substance capable de capturer les chaînes glucidiques et procédé l'utilisant

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JP2006231166A (ja) * 2005-02-23 2006-09-07 Fukae Kasei Kk 液体試料の処理用器具及びその使用方法
WO2008018170A1 (fr) * 2006-08-09 2008-02-14 Sumitomo Bakelite Co., Ltd. Substance capable de capturer les chaînes glucidiques et procédé l'utilisant

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104236979A (zh) * 2014-10-09 2014-12-24 南京农业大学 一种可同时提取糖链的装置
CN107827996A (zh) * 2017-12-29 2018-03-23 福建海兴保健食品有限公司 一种从新鲜绿藻中提取绿藻蛋白多糖的生产线及其工艺
CN107827996B (zh) * 2017-12-29 2023-04-14 福建海兴保健食品有限公司 一种从新鲜绿藻中提取绿藻蛋白多糖的生产线及其工艺

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