JP2013081456A - System and method for purifying sugar chains - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a system and a method for purifying sugar chains, which can separate and purify a large quantity of sugar chains easily and with good accuracy.SOLUTION: A system 1 for purifying sugar chains is a system for purifying sugar chains from a sugar chain-containing liquid. It is equipped with a plate assembly 10, which is composed of a first upper plate 100, a second upper plate 100', a first lower plate 300, a second lower plate 200 and a support 700, which supports either upper plate between the first upper plate 100 and the second upper plate 100' and either lower plate between the first lower plate 300 and the second lower plate 200 in a state of being arranged and set up above and below, and a stage 3, on which the plate assembly 10 is placed. Then, in the plate assembly 10, the first upper plate 100 and the second upper plate 100' can be exchanged with each other and also the first lower plate 300 and the second lower plate 200 can be exchanged with each other on the stage 3.

Description

  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, and sialic acid, and derivatives thereof are linked in a chain by glycosidic bonds. .

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

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

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

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

  Furthermore, for separation and purification of sugar chains, for example, techniques such as ion exchange resin, reverse phase chromatography, activated carbon, gel filtration chromatography are used, but these separation means are not methods that specifically recognize sugars, Incorporation of impurities (peptides, proteins, etc.) is unavoidable, and the recovery rate often varies depending on the structure of the sugar chain.

  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.

  In general, gel filtration is performed using the difference in molecular weight between the labeled sugar chain and the contaminant to remove the contaminant. 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.

  Moreover, although a method of distilling off impurities by azeotropy has been tried as a simple method, it is difficult to sufficiently remove the impurities.

  Furthermore, in order to investigate the relationship between the sugar chain structure and various diseases, it is necessary to examine the sugar chain structures of a large number of specimens that can be statistically processed.

  In the conventional method as described above, it is necessary to go through complicated steps to separate sugar chains, and enormous cost and time are required. Therefore, a large amount of sugar chains can be easily separated and purified with excellent accuracy. There was a need for a means to do this.

  Furthermore, various methods for applying fluorescent labeling to sugar chains have been developed (for example, Patent Documents 1, 2, and 3). However, the labeling efficiency is not 100%. Unaccompanied sugar chains were mixed. This situation does not pose a major problem when fluorescence detection is performed using high performance liquid chromatography (HPLC) or capillary electrophoresis (CE method), but peaks are present when analyzing sugar chains by mass spectrometry. There was a problem of increasing complexity. In addition, when the labeling efficiency is poor, there is a possibility that the sensitivity is lowered in the analysis by HPLC or CE.

JP-A-7-20131 JP 2006-098367 A JP 2008-309501 A

Introduction to Glycobiology Chemistry Doujin November 1, 2005 First Edition

  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.

Such an object is achieved by the present inventions (1) to (20) below.
(1) A plate assembly including a plate having a plurality of wells used when purifying a sugar chain from a solution containing a sugar chain, a stage on which the plate assembly is placed, and the above-described plate on the stage A sugar chain purification apparatus comprising: dispensing means having a nozzle for sucking and discharging liquid with respect to a plate assembly; and moving means for relatively moving the plate assembly on the stage and the nozzle. ,
The plate assembly has a plate-like shape, and includes a plurality of first wells each formed with a recess opening on the upper surface thereof and having a through-hole penetrating the bottom of the recess, and the first well is penetrated. A first upper plate having a first filter installed to cover each of the holes;
A plurality of second wells each having a plate shape and formed of a recess opening on the upper surface thereof, each having a through hole penetrating the bottom of the recess, and the through holes of the second wells are respectively covered. A second upper plate installed and having a second filter different from the first filter;
A first lower plate having a plate-like shape and having a first storage portion configured by a recess opening on an upper surface thereof;
A second lower plate having a plurality of second reservoirs configured in a plate-like shape and having recesses opened on the upper surface thereof;
One upper plate of the first upper plate and the second upper plate and one lower plate of the first lower plate and the second lower plate are arranged vertically. And a support body that supports the assembled state,
In the plate assembly, the first upper plate and the second upper plate can be exchanged on the stage, and the first lower plate and the second lower plate can be exchanged. An apparatus for purifying a sugar chain, comprising:

(2) In the plate assembly, the 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 from the first state, and the second lower plate is supported by the support;
The first upper plate and the second upper plate are exchanged from the second state, 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;
The first lower plate and the second lower plate are exchanged from the third state, and the second lower plate can be in the fourth state supported by the support. The sugar chain purification apparatus according to (1).

(3) In the first state, each of the first wells communicates with the first storage part through the through hole,
In the second state, each of the first wells communicates with each of the second storage portions via the through holes,
In the third state, each second well communicates with the first reservoir through the through hole, and
In the fourth state, the second well is the sugar chain purifying apparatus according to (2), wherein each second well communicates with each second reservoir through a through hole.

(4) 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;
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 (3), wherein the second state, the third state, and the fourth state are set.

(5) The support includes an upper support member that supports the one upper plate, and a lower support member that is configured separately from the upper support member and supports the one lower plate,
The upper support member and the lower support member can be disassembled and assembled with the upper support member supporting the one upper plate and the lower support member supporting the one lower plate. The sugar chain purification apparatus according to any one of (1) to (4), which is configured.

  (6) 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 plate. The sugar chain purification apparatus according to (5), which is performed in a state where the lower support member is disassembled.

  (7) In the plate assembly, the lower surface of the one upper plate and the upper surface of the one lower plate are arranged apart from each other, and the separation distance is 0.5 mm or more and 10 mm or less. (6) The sugar chain purification apparatus according to any one of (6).

  (8) The sugar chain purification apparatus according to any one of (1) to (7), wherein the first filter is formed of a porous film or a nonwoven fabric.

  (9) The sugar chain purification apparatus according to (8), wherein the opening of the first filter is 0.1 to 50 μm.

  (10) The sugar chain purification apparatus according to any one of (1) to (9), wherein the second filter is made of silica gel.

  (11) The sugar chain purification apparatus according to any one of (1) to (10), wherein the dispensing unit includes a pump and a tube connecting the pump and the nozzle.

  (12) The moving unit according to any one of (1) to (11), wherein the moving unit includes a horizontal moving mechanism that moves the nozzle in the horizontal direction and a vertical moving mechanism that moves the nozzle in the vertical direction. Sugar chain purification equipment.

  (13) On the stage, a first upper plate mounting portion on which a plurality of the first upper plates are stacked and a second on which a plurality of the second upper plates are stacked. 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 any one of (1) to (12), wherein the second lower plate mounting portion is provided.

  (14) The sugar chain purification apparatus according to any one of (1) to (13), further including a washing tank for washing the nozzle.

(15) A method for purifying the sugar chain using the sugar chain purification apparatus according to (4) 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.

  (16) The sugar chain purification method according to (15), wherein in the fourth step, the sugar chain is labeled with a labeling reagent after the sugar chain is re-released from the capture carrier.

  (17) The sugar chain purification method according to (16), wherein the labeling reagent is a fluorescent substance composed of an aromatic amine.

  (18) The sugar chain purification method according to (17), wherein the concentration of the fluorescent substance in the solution containing the re-released sugar chain is 0.5 mol / L or more.

  (19) The fluorescent substance may be 2-aminobenzomide, 2-aminobenzoic acid, 8-aminopyrene-1,3,6-trisulfonate, 8-aminophathalene-1,3,6-trisulphonate, 2-Amino9 (10H) -acidone, 5-Aminofluorescein, Dansylethylenediamine, 7-Amino-4-methylcoumarin, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3- (Acetylamino) -6-aminoacidine (At least one kind selected from 17 or above) The method for purifying sugar chains as described in 18).

  (20) The sugar chain purification method according to any one of (15) to (19), wherein the capture carrier is polymer particles having a hydrazide group or an oxylamino group.

  According to the present invention, it is possible to separate and purify sugar chains easily and with excellent accuracy, and to perform fluorescent labeling 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.

1 is a perspective view showing a first embodiment of a sugar chain purification apparatus of the present invention. It is a perspective view which shows the plate assembly with which the sugar_chain | carbohydrate refinement | purification apparatus shown in FIG. 1 is provided. FIG. 3 is an exploded perspective view of the plate assembly shown in FIG. 2. FIG. 3 is a sectional view taken along line BB in FIG. 2 (a sectional view showing a first state of the plate assembly). FIG. 3 is a sectional view taken along line BB in FIG. 2 (a sectional view showing a second state of the plate assembly). FIG. 3 is a cross-sectional view taken along line BB in FIG. 2 (a cross-sectional view showing a third state of the plate assembly). FIG. 5 is a cross-sectional view taken along line BB in FIG. 2 (a cross-sectional view showing a fourth state of the plate assembly). It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is sectional drawing (sectional drawing seen from the arrow A direction in FIG. 1) which shows the process of refine | purifying a sugar chain in order by the sugar chain refinement | purification apparatus shown in FIG. It is a perspective view which shows 2nd Embodiment of the sugar chain refinement | purification apparatus of this invention. It is a perspective view which shows 3rd Embodiment of the sugar chain refinement | purification apparatus of this invention.

  Hereinafter, a sugar chain purification apparatus and a sugar chain purification method of the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings.

<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.

  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 in. 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.

  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, a second lower plate (multiwell plate) 200, an adjustment plate (spacer) 600, and a support 700 are provided, and 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.

  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.

  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.

  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.

  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.

  The stage 3 has a flat plate shape that is long in the y-axis direction, 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 and is located at substantially the center in the longitudinal direction of the stage 3. The sugar chains are purified from the sugar chain-containing liquid 23 on the plate assembly 10 placed on the plate assembly placement unit 31.

  The first upper plate mounting portion 32 is an area where one or more first upper plates 100 are stacked and placed in front of the stage 3 toward the purification apparatus 1. Each first upper plate 100 placed on the first upper plate placement portion 32 is unused and used for replacement.

  The second upper plate placement part 33 is located further to the right side than the first upper plate placement part 32 toward the purification apparatus 1, and one or more second upper plates 100 ′ are placed on top of each other. Area. 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 refining device 1, and one or more first lower plates 300 are stacked and mounted. It is an area to be placed. Each first lower plate 300 placed on the first lower plate placing portion 34 is unused and used for replacement.

  The second lower plate placing portion 35 is located further back than the first upper plate placing portion 32 toward the refining device 1, and one or more second lower plates 200 are placed on top of each other. It is an area to be placed. Each second lower plate 200 placed on the second lower plate placing portion 35 is unused and used for replacement.

In the purification apparatus 1, each plate is exchanged manually by an operator who operates the purification apparatus 1.
The detailed configuration of the plate assembly 10 will be described later.

  The cleaning tank mounting part 36 is an area where the cleaning tank 7 is mounted, located on the left side of the stage 3 toward the purification apparatus 1. The cleaning tank 7 mounted on the cleaning tank mounting part 36 is fixed by a fixing mechanism (not shown).

  The processing liquid mounting part 37 is located further to the left of the cleaning tank mounting part 36 toward the purification apparatus 1, and a multiwell plate 81 and tanks 82 to 87 each storing various liquids (processing liquids) are mounted thereon. It is an area to be placed.

  The constituent material of the stage 3 is not particularly limited, and various metal materials such as stainless steel, aluminum, and an 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 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 a gear pump or a vane pump, for example, 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. 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).

  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.

  Further, 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.

  With the moving means 5 having such a configuration, the nozzle 41 can 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 heater 61 includes the plate assembly 10 in a plan view, that is, the plate assembly 10 is disposed inside the heater 61 in a plan view.

  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). The support member 400) is transmitted in order, and the liquid on the first upper plate 100 can be heated and evaporated (see FIGS. 9 (5) and 12 (14)).

  Further, since the heater 61 is embedded in the stage 3, it is possible to reliably prevent an 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 is configured to detect the temperature on the plate assembly mounting portion 31 of the stage 3 and to control the voltage applied to the heater 61 according to the detection result. 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 cleaning tank 7 is composed of a member having a bottomed cylindrical shape, and a cleaning liquid 71 for cleaning the nozzle 41 can be stored inside the cleaning 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.

  Moreover, in the refiner | purifier 1, the washing | cleaning liquid 71 is comprised so that it may replace | exchange for an unused thing whenever it wash | cleans the nozzle 41, ie, whenever 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 and perform continuous cleaning with two or more cleaning liquids 71.

  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.

  As described above, one set of plate assemblies 10 is placed on the plate assembly placement 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.

  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), that is, constituted by a recess opening in the upper surface 108. A plurality of (in this embodiment, 96) wells (first 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), and are used when purifying sugar chains from the sugar chain-containing liquid 23.

  As shown in FIGS. 3 and 4, each of these wells 101 has a structure having a bottom portion 103 on the lower side thereof, and further, a through-hole penetrating the bottom portion 103 substantially at the center of the bottom portion 103. 104 is provided. Thereby, the outflow of the liquid supplied (stored) to the well 101 through the through-hole 104 to the outside is allowed from the bottom 103 side.

  The 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.

  Further, a film-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 through the through hole 104.

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

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

  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.

  The second upper plate 100 ′ has a flat plate shape as a whole and is provided in the thickness direction (vertical direction), that is, a plurality of second upper plates 100 ′ (this embodiment) configured by recesses opened in the upper surface 108 ′. 96) wells (second wells) 101 ′. 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.

  As shown in FIG. 6 and FIG. 7, each of these wells 101 ′ has a structure having a bottom portion 103 ′ on the lower side thereof, and further, the bottom portion 103 ′ is provided at a substantially central portion of the bottom portion 103 ′. A through hole 104 ′ is provided therethrough. Accordingly, the liquid supplied (accommodated) to the well 101 ′ through the through hole 104 ′ is allowed to flow out from the bottom 103 ′ side.

  Further, 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 attached to 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 from the bottom 103 'side to the outside through the through hole 104'.

  The filter 106 is made of a material different from that of the filter 105 and has a different capture function, 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.

  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.

  Then, a recess 405 surrounded by the upper outer wall 403 is formed. By inserting the first upper plate 100 or the second upper plate 100 ′ into the recess 405, the inserted upper plate becomes the first It is supported by the support base 400.

  Further, a groove is provided along the edge of the opening 402 of the bottom 401, and a packing (seal member) 407 is disposed so as to correspond to the groove. Accordingly, when the 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 a structure having an opening 408 in a direction orthogonal to the thickness direction (vertical direction) at the center of the two opposing long sides, thereby being inserted into the recess 405 ( When taking out the first upper plate 100 or the second upper plate 100 ′ placed), a finger can be put into the opening 408 to lift the upper plate inserted into the recess 405, The take-out operation can be easily performed.

  Further, a recess 406 surrounded by the lower outer wall 404 is formed, and a second support 502 is inserted by inserting a protrusion 502 included in a second support base 500 described later along the inner peripheral surface of the recess 406. A first support base 400 is fixed on the base 500.

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

  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 (first reservoir) 301 that opens on the upper surface 302.

As shown in FIG. 3, the first lower plate 300 has a configuration including a recess forming portion 303 in which a recess 301 is formed, and a frame portion 304 arranged so as to surround the periphery of the recess forming portion 303. In this case, the thickness of the recess forming portion 303 is larger than the thickness of the frame portion 304. As a result, the upper surface 302 of the recess forming part 303 protrudes from the upper surface of the frame part 205. 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.
The upper surface 302 of the well forming portion 202 protrudes from the upper surface of the frame portion 205 as described above, but is not limited thereto, and may be the same height as the upper surface of the frame portion 205. Also in this case, the sugar chain can be reliably separated and purified.

  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 other words, a plurality (96 in the present embodiment) of wells (second reservoirs) 201 each including a concave portion 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 includes a bottom 203 on the lower side thereof. Thereby, the liquid supplied (stored) to the well 201 is stored therein.

  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. The well formation portion 202 is thicker than 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 shape of the well forming portion 202 in plan view is set to a size that can be inserted into the opening 402 of the first support base 400. Accordingly, 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.

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

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

  Then, by selecting the adjustment plate 600 having an appropriate thickness according to the types of the first lower plate 300 and the second lower plate 200 to be inserted into the recess 505, the first state can be obtained in the first state. The upper plate 100 and the first lower plate 300 can be made as close as possible, and the first upper plate 100 and the second lower plate 200 can be made as close as possible in the second state. In the third state, the second upper plate 100 ′ and the first lower plate 300 can be brought as close as possible, and in the fourth state, the second upper plate 100 ′ and the second lower plate 300 200 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 portion 501 and the outer edge of the bottom portion 501. The outer wall 503 is provided.

  By being surrounded by the outer wall 503, a concave portion 505 is formed inside thereof, and the first lower plate 300 or the second lower plate 200 is inserted into the concave portion 505, so that the first lower plate 505 is inserted. The side plate 300 or the second lower plate 200 is supported by the second support base 500.

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

  A groove is provided on the upper surface of the outer wall 503 so as to surround the protruding portion 502, and a packing (seal member) 507 is disposed so as to correspond to the groove. Thereby, when the 1st support stand 400 is mounted on the 2nd support stand 500, the communication with the exterior between the 1st support stand 400 and the 2nd support stand 500 is interrupted | blocked.

  Furthermore, as shown in FIG. 3, the outer wall 503 has a through hole 506 that penetrates the recess 505 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.

  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.

  In addition, as the constituent material of the first support base 400 and the second support base 500, the resin material can be used. For example, Fe-based alloy such as stainless steel, Cu or Cu-based alloy, Al or Al-based Examples thereof include metal materials such as alloys, ceramic materials such as alumina, apatite, and aluminum nitride. One or more of these materials 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)).

  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.

  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.

  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. The lower plate 300 is inserted, and the first support base 400 is placed on the second support base 500 in this state. 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.

  As a result of such assembly, each well 101 of the first upper plate 100 is in communication with the recess 301 of the first lower plate 300 through the through-hole 104, and in this state, The first support base 400 is fixed on the second support base 500.

  In such a state, the packing 507 blocks communication between the first support base 400 and the second support base 500 between the first support base 400 and the first support base 400. Communication with the plate 100 is blocked. 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.

  And by operating the suction pump 13 connected to the through hole 506 and depressurizing the internal space 9a (recessed portion 505), the internal space 9a can be set to a negative pressure 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.

  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. It is also possible to apply centrifugal force or gravity to the liquid located above the filter 105. 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.

  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 support plate 200 is inserted, and the first support base 400 is placed on the second support base 500 in this state. 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.

  As a result of such 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, and in this state, the second well 101 The first support base 400 is fixed on the support base 500.

  Even 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.

  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. The lower plate 300 is inserted, and the first support base 400 is placed on the second support base 500 in this state. 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.

  As a result of such assembly, each well 101 ′ of the second upper plate 100 ′ communicates with the concave portion 301 of the first lower plate 300 through the through hole 104 ′ in a collective manner. Thus, the first support base 400 is fixed on the second support base 500.

  In such a state, the packing 507 blocks communication between the first support base 400 and the second support base 500 between the first support base 400 and the second support base 500. Communication with the plate 100 'is blocked. 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.

  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 set to a negative pressure 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. 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.

  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, and the first support base 400 is placed on the second support base 500 in this state. 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.

  By such 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 ′, and in this state, The first support base 400 is fixed on the second support base 500.

  Even 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.

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. Even in the fourth state, the lower surface 109 ′ of the second upper plate 100 ′ and the upper surface 204 of the second lower plate 200 can be made as close as possible, and the separation distance d ₄ can be set as small as possible. it can.

  In particular, even if the liquid that has passed through the through hole 104 is scattered in the second state, the liquid is supplied into the well 201 of the second lower plate 200 corresponding to each well 101 with high efficiency. (Recover). 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.

Note that 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-described effect can be exhibited more remarkably.

  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.

  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, the “sugar chain” and the “capture carrier that specifically binds to the sugar chain” are described. Will be described.

  Here, a case where the sugar chain is obtained from a glycoprotein provided with the sugar chain is taken 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.

  Then, this sample is subjected to a predetermined treatment such as SDS-PAGE (SDS-denaturing polyacrylamide gel electrophoresis) or two-dimensional electrophoresis combining isoelectric focusing and SDS-PAGE in the gel. Glycoprotein contained in the sample is separated. Thereafter, the sugar chain is released from the glycoprotein held in the gel using a sugar chain releasing means. The means for releasing the sugar chain is not particularly limited, and for example, methods such as glycosidase treatment using N-glycosidase or O-glycosidase, hydrazine decomposition, and β elimination by alkali treatment can be used. The gel after the sugar chain releasing treatment is taken out and rinsed with a washing solution such as water, so that the released sugar chain is eluted from the gel into the washing solution. And after precipitating a gel in a washing | cleaning liquid, the sugar_chain | carbohydrate containing liquid 23 is obtained by collect | recovering this supernatant. 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.

  In this embodiment, the release of sugar chains uses an electrophoretic treatment, but 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.

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

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

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

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

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

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

  If the polymer particles are solid particles or gel particles, the particles can be easily recovered using the well 101 provided in the 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).

  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.

  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. 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.

  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.

  The capture carrier supplying step is a step of supplying a capture carrier (polymer particle 20) that specifically binds to a sugar chain to each well 101 of the first upper plate 100. In this capture carrier supplying step, the plate assembly 10 is used as the first state.

  In the sugar chain capturing step, the sugar chain-containing liquid 23 is dispensed from the nozzle 41 to each well 101 of the first upper plate 100 to bring the sugar chain-containing liquid 23 and the polymer particles 20 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.

  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.

  The re-releasing step is a step of re-releasing the sugar chain bonded to the polymer particle 20. In this re-releasing process, 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. In this sugar chain purification step, the plate assembly 10 is used in the second state, the third state, and the fourth state in order. In the second state, the first lower plate 300 and the second lower plate 200 are exchanged on the stage 3 from the plate assembly 10 in the first state, and the second lower plate 200 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 from the plate assembly 10 in the second state, and the second upper plate 100 ′ is the first support. The base 400 (support 700) is supported, 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). In the fourth state, the first lower plate 300 and the second lower plate 200 are exchanged from the plate assembly 10 in the third state, and the second lower plate 200 is changed to the first state. The second support base 500 (support 700) supports the second support base 500.

[1] Capture carrier supply process [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 (Particle Dispersion Liquid Suction)] Further, the nozzle 41 is inserted into the tank 82 in which the particle dispersion liquid 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] Then, the moving means 5 is operated to move the nozzle 41 that has 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] 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, whereby the particle dispersion liquid in each well 101. Each 22 is aspirated.

  Thereby, in each well 101, the pure water 21 in the particle dispersion liquid 22 permeate | transmits the filter 105, respectively. 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.

  As a result, as shown in FIG. 8 (3), the polymer particles 20 are individually filled in the wells 101 of the first upper plate 100.

[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 through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.

[4] Sugar Chain Capture Step [4-1 (Sugar Chain-Containing Liquid Suction)] Next, the moving means 5 is operated to move the nozzle 41 to the well of the multiwell 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 (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 (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 through the stage 3, the second support base 500, the first support base 400, and the first upper plate 100, and the sugar chain-containing liquid 23 on the first upper plate 100 is heated. Is done. This heating is maintained in a certain temperature range (heating temperature) until the sugar chain-containing liquid 23 is dried.

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

  The pH of the reaction system at this time is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6. In addition, pH adjustment can be performed by adding various buffer solution or organic solvent to the sugar_chain | carbohydrate containing liquid 23 of each well 101 of the 1st upper plate 100, for example.

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

  Further, 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.

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

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

  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.

  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] 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.

[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 (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 (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 (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.

  In addition, when performing this process [5], the operation | movement 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] 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, impurities (proteins, peptides, lipids, etc.) other than sugar chains adsorbed nonspecifically on the surface of the polymer particles 20 are included. Can be mentioned.

  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 (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 (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 (Cleaning 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. Each of the cleaning liquids 24 is aspirated.

  As a result, the cleaning liquid 24 can be transmitted through the filter 105 and selected from the first upper plate 100 with the polymer particles 20 remaining on the first upper plate 100 as shown in FIG. Thus, the cleaning liquid 24 can be removed into the first lower plate 300 and the cleaning substance dissolved in the cleaning liquid 24 can be removed.

  Since the operation shown in FIGS. 11 (10) to (12) is repeated a plurality of times, the cleaning substance can be separated into the first lower plate 300 in a state dissolved in the cleaning liquid 24. The cleaning substance can be reliably removed from the polymer particles 20 in which the sugar chains are captured.

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

[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 through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.

[9] Re-releasing step Here, the sugar chain bonded to the polymer particle 20 is re-released, and the sugar chain is replaced with another compound (hereinafter also referred to as “compound A”). 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 (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 (Supply of Compound-Containing Liquid)] Then, as shown in FIG. 12 (13), the moving means 5 is operated to move the nozzle 41 that has 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.

  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, the substitution rate of the compound A to the sugar chain when the compound-containing liquid 25 is heated next can be improved.

  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 (Compound-containing liquid heating)] Next, as shown in FIG. 12 (14), the heater 61 is operated. As a result, the compound-containing liquid 25 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.

  As a result, the captured sugar chain is separated from the polymer particle 20, and compound A is added to the sugar chain almost simultaneously with it, so that the sugar chain is labeled with compound A. Hereinafter, the sugar chain labeled with the compound A may be referred to as “labeled sugar chain”.

  Moreover, the pH of the reaction system in this case becomes like this. Preferably it is 2-9, More preferably, it is 2-7, More preferably, it is 2-6. In addition, pH adjustment can be performed by adding various buffer solutions, after supplying the compound containing liquid 25 to each well 101 of the 1st upper plate 100, respectively.

  The temperature at the time of labeling is preferably 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.

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

By performing labeling under such conditions, the sugar chain is surely labeled with Compound A.
In addition, by setting it as the structure heated until the compound containing liquid 25 dries like this embodiment, the improvement of the reaction rate of a sugar_chain | carbohydrate and the compound A can be aimed at reliably.

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

[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] First / Second Lower Plate Replacement Step [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 (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 (Second Lower Plate Loading)] Next, as shown in FIG. 13 (17), the unused second lower plate 200 is loaded onto the empty second support base 500.

  [11-4 (Plate Assembly Assembly)] Next, as shown in FIG. 13 (18), the first support plate 500 loaded with the unused second lower plate 200 is loaded with 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.

  In addition, when performing this process [11], the operation | movement 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] 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 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”). .).

  Therefore, in this step [12], the labeled sugar chain is purified by removing the polymer particles 20 and the unused compound A.

  [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 (Dissolution Solution Supply)] Then, as shown in FIG. 14 (19), the moving means 5 is operated to move the nozzle 41 that has sucked the dissolution solution 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 (Solution Solution Suction)] Next, as shown in FIG. 14 (20), the suction pump 13 is operated to set the internal space 9 a 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.

  Then, as shown in FIG. 14 (21), the polymer particles 20 remain in each well 101 of the first upper plate 100, and the second lower plate corresponding to the well 101. In the well 201 of 200, 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 (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 (Disassembly of Plate Assembly)] As shown in FIG. 15 (22), the operator of the purification 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 (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 (Second Upper Plate Loading)] Next, as shown in FIG. 15 (24), the empty first support base 400 is loaded with an unused second upper plate 100 '.

  [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 (Second Lower Plate Removal)] Next, as shown in FIG. 17 (27), the second lower plate 200 is removed from the second support base 500.

  [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 (Plate Assembly 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 (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.

  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 excludes the labeled sugar chain and a part of the unused compound A that has flowed into the first lower plate 300 out of the unused compound A contained in the solution 26. The remaining unused compound A is adsorbed.

  [12-13 (Nonaqueous Solvent Suction)] Next, the moving means 5 is operated, and the nozzle 41 is inserted into the tank 86 in which the nonaqueous 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 (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 (Nonaqueous 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 in the state adsorbed on the filter 106 is washed away from the filter 106, that is, removed, together with the non-aqueous solvent 27. 1 flows into the lower plate 300. Even if the unused compound A is slightly adsorbed on the filter 106, the amount is substantially insignificant when measured by HPLC or a mass spectrometer described later. Further, the labeled sugar chain is still adsorbed on the filter 106.

  [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 (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 (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 (Second Lower Plate Loading)] Next, as shown in FIG. 20 (37), the unused second lower plate 200 is loaded onto the empty second support base 500.

  [12-20 (Assembly of Plate Assembly)] Next, as shown in FIG. 20 (38), the second support base 500 loaded with the unused second lower plate 200 is loaded with 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 (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 (Water Supply)] Then, as shown in FIG. 21 (39), the moving means 5 is operated to move the nozzle 41 that 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 (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, whereby each well 101 ′. The water 28 inside is aspirated. 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 chains adsorbed on the filter 106 are peeled off from the filter 106, and in the well 201 of the second lower plate 200 together with the water 28. In other words, it is purified (separated).

  [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 through the tip opening 411 of the nozzle 41, and the nozzle 41 is cleaned.

  Through the above steps, the sugar chain labeled with the compound A is purified using the plate assembly 10.

  In addition, the sugar chain labeled with Compound A can be analyzed by a technique such as mass spectrometry represented by MALDI-TOF MS, and high performance liquid chromatography (HPLC). In particular, when the sugar chain is labeled with N-aminooxyacetyl-tryptophanyl (arginine methyl ester) represented by the chemical formula (3) described above, high-sensitivity analysis can be performed using MALDI-TOF MS. In addition, when the sugar chain is labeled with, for example, 2-aminobenzamide, high-sensitivity analysis can be performed using HPLC.

  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.

  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 supports the first lower plate 300 or the second lower plate 200 so that it can be disassembled and assembled. 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 Replacement with the second lower plate 200 can be performed, and subsequent assembly can be easily performed.

  As described above, the purification apparatus 1 is configured such that plate exchange can be performed easily and quickly on the stage 3.

  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. A large amount of sugar chains can be separated and purified easily and with excellent accuracy.

  In addition, it is preferable that the step of deactivating the functional group of 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.

  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 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.

  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.

  Further, 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 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 prepared in an acetonitrile solution. In addition, since the hydrophobicity of the solution is increased by adding acetonitrile, 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, it is prepared so that the acetonitrile content is 90% or more (preferably 95%).

Second Embodiment
FIG. 22 is a perspective view showing a second embodiment of the sugar chain purification apparatus of the present invention.

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.
This embodiment is the same as the first embodiment except that the configuration of the heating means is different.

  In the purification apparatus 1 of this embodiment shown in FIG. 22, the heater 61 (heating means 6) is omitted from the stage 3, and instead, the heating means 6A includes a chamber 62 arranged at a position different from the stage 3. The thermostat is provided with a heater 63 for heating the inside of the chamber 62.

  The chamber 62 has a box shape, and includes a chamber body 64 having a mouth portion 641 through which the first upper plate 100 is inserted and removed, 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.

  When it is desired to heat the liquid in the well 101 of the first upper plate 100, the operator of the purification apparatus 1 moves the first upper plate 100 into the chamber 62, operates the heater 63, and heats the liquid. Can be done.

<Third Embodiment>
FIG. 23 is a perspective view showing a third embodiment of the sugar chain purification apparatus of the present invention.

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.
This embodiment is the same as the first embodiment except that the configuration of the heating means is different.

  In the refining device 1 of this embodiment shown in FIG. 23, the heater 61 (heating means 6) is omitted from the stage 3, and instead, the heating means 6B is adjacent to the plate assembly mounting portion 31 of the stage 3. And 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. In addition, it does not specifically limit as 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 stored in the filled state.

  The heater 67 is disposed outside 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.

  When the liquid in the well 101 of the first upper plate 100 is to be heated, the operator of the purification apparatus 1 moves the first upper plate 100 to the recess 661 filled with the heat medium and operates the heater 67. And heating can be performed.

<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.
The present embodiment is the same as the first embodiment except that in the re-releasing step, the timing between re-releasing and labeling is shifted, that is, the labeling is performed after re-releasing.

In this step, a fluorescent material composed of an aromatic amine is used as compound A.
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. .

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

  [9-1 ′] First, using the nozzle 41, the sugar chain release liquid for re-releasing the sugar chains is placed in each well 101 of the first upper plate 100 filled with the polymer particles 20 in which the sugar chains are captured. (Supplied).

  [9-2 '] Next, the sugar chain free liquid is maintained in a certain temperature range by operating the heater 61 to heat the sugar chain free liquid until the added sugar chain free liquid is dried. As a result, the captured sugar chain is separated from the polymer particle 20, whereby the sugar chain is liberated again.

  The pH of the reaction system at this time is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6. This pH adjustment is performed by adding the sugar chain free solution in the above step [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 at a temperature range of about 4 to 100 ° C, more preferably about 25 to 90 ° C, more preferably about 30 to 80 ° C, and most preferably about 60 to 80 ° C. Set to.

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

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

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

  [9-3 ′] Next, the compound-containing liquid 25 containing an aromatic amine as the compound A is used in the well 101 of the multiwell filter plate 100 in which the sugar chain is released from the polymer particle 20 using the nozzle 41. Add.

  The aromatic amine (compound A) is not particularly limited. 2-Amino9 (10H) -acidone, 5-Aminofluorescein, Dansylethylenediamine, 7-Amino-4-methylcoumarin, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3- (Acetinominor) -6-aminominorido-aminoidoid Among these, 2-aminobenzoamide or 2-aminobenzoic acid is preferable. These compounds are preferably used because of their availability as reagents and the convenience of the reaction.

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

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

  More specifically, when 2-aminobenzamide is used as an 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 resulting solution is added as compound-containing solution 25.

  [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.

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

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

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

  More specifically, when 2-aminobenzamide is used as the aromatic amine, the compound-containing liquid 25 is heated in a temperature range of 30 to 70 ° C. and reacted for about 1 to 10 hours.

By performing labeling under such conditions, the sugar chain is surely labeled with Compound A.
The sugar chain is labeled with the compound A also by the steps [9-1 ′] to [9-4 ′] as described above.

  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.

  Moreover, although the nozzle of the dispensing means is fixed to the nozzle head in each of the embodiments described above, the present invention is not limited to this, and the nozzle may be detachably attached to the nozzle head.

  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, but is not limited thereto, and the plate assembly on the stage with respect to the nozzle is not limited thereto. It may be configured to move a solid.

Moreover, the heating means may be abbreviate | omitted in the sugar chain refinement | purification apparatus.
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. However, the present invention is not limited to this, and a configuration in which the liquid is allowed to pass through the filter by setting the upper side of the filter to a positive pressure from the lower side thereof may be used.

Further, the movement of the plate and the disassembly / removal of the plate assembly are performed manually by the operator (operator) of the refining apparatus in each of the above embodiments. However, the present invention is not limited to this. You may automate using.
Further, the entire sugar chain purification apparatus may be stored in a chamber capable of heating the inside.

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 (20)

A plate assembly comprising a plate having a plurality of wells used when purifying a sugar chain from a solution containing a sugar chain, a stage on which the plate assembly is placed, and the plate assembly on the stage On the other hand, a glycan purification apparatus comprising dispensing means having a nozzle for sucking and discharging liquid, and moving means for relatively moving the plate assembly on the stage and the nozzle,
The plate assembly has a plate-like shape, and includes a plurality of first wells each formed with a recess opening on the upper surface thereof and having a through-hole penetrating the bottom of the recess, and the first well is penetrated. A first upper plate having a first filter installed to cover each of the holes;
A plurality of second wells each having a plate shape and formed of a recess opening on the upper surface thereof, each having a through hole penetrating the bottom of the recess, and the through holes of the second wells are respectively covered. A second upper plate installed and having a second filter different from the first filter;
A first lower plate having a plate-like shape and having a first storage portion configured by a recess opening on an upper surface thereof;
A second lower plate having a plurality of second reservoirs configured in a plate-like shape and having recesses opened on the upper surface thereof;
One upper plate of the first upper plate and the second upper plate and one lower plate of the first lower plate and the second lower plate are arranged vertically. And a support body that supports the assembled state,
In the plate assembly, the first upper plate and the second upper plate can be exchanged on the stage, and the first lower plate and the second lower plate can be exchanged. An apparatus for purifying a sugar chain, comprising: 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 from the first state, and the second lower plate is supported by the support;
The first upper plate and the second upper plate are exchanged from the second state, 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;
The first lower plate and the second lower plate may be exchanged from the third state, and the second lower plate may be in a fourth state supported by the support. Item 2. The sugar chain purification apparatus according to Item 1. In the first state, each of the first wells communicates with the first reservoir through the through hole,
In the second state, each of the first wells communicates with each of the second storage portions via the through holes,
In the third state, each second well communicates with the first reservoir through the through hole, and
3. The sugar chain purification apparatus according to claim 2, wherein in each of the fourth states, each of the second wells communicates with each of the second reservoirs through a through hole thereof. 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;
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 3, wherein the second state, the third state, and the fourth state are set. The support includes an upper support member that supports the one upper plate, and a lower support member that is configured separately from the upper support member and supports the one lower plate,
The upper support member and the lower support member can be disassembled and assembled with the upper support member supporting the one upper plate and the lower support member supporting the one lower plate. The sugar chain purification apparatus according to any one of claims 1 to 4, which is configured.   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 5, which is performed in a state where the member is decomposed.   In the said plate assembly, the lower surface of said one upper plate and the upper surface of said one lower plate are spaced apart, and the separation distance is 0.5 mm or more and 10 mm or less. The sugar chain purification apparatus according to 1.   The sugar chain purification apparatus according to any one of claims 1 to 7, wherein the first filter is formed of a porous film or a nonwoven fabric.   The sugar chain purification apparatus according to claim 8, wherein the opening of the first filter is 0.1 to 50 μm.   The sugar chain purification apparatus according to any one of claims 1 to 9, wherein the second filter is made of silica gel.   The sugar chain purification apparatus according to any one of claims 1 to 10, wherein the dispensing unit includes a pump and a tube connecting the pump and the nozzle.   The sugar chain purification apparatus according to any one of claims 1 to 11, 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.   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 any one of claims 1 to 12, further comprising a lower plate mounting portion.   The sugar chain purification apparatus according to any one of claims 1 to 13, further comprising a washing tank for washing the nozzle. A method for purifying the sugar chain using the sugar chain purification apparatus according to claim 4,
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.   16. The method for purifying a sugar chain according to claim 15, wherein in the fourth step, the sugar chain is labeled with a labeling reagent after re-release of the sugar chain from the capture carrier.   The sugar chain purification method according to claim 16, wherein the labeling reagent is a fluorescent substance composed of an aromatic amine.   The method for purifying a sugar chain according to claim 17, wherein the concentration of the fluorescent substance in the solution containing the re-released sugar chain is 0.5 mol / L or more.   The fluorescent materials include 2-aminobenzoamide, 2-aminobenzoic acid, 8-aminopyrene-1,3,6-trisulfonate, 8-aminophathalene-1,3,6-trisulfonate, 2-amino9 (10H) -acidone, 5-aminominocele. 19. The sugar according to claim 17, which 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. Strand purification method.   The sugar chain purification method according to any one of claims 15 to 19, wherein the capture carrier is polymer particles having a hydrazide group or an oxylamino group.

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