JP2013063064A - Processing tool - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a processing tool storable of a liquid to be stored in the processing tool with excellent accuracy without waste.SOLUTION: The processing tool includes a bottomed and cylindrical body part 1 storable of the liquid and a reaction vessel (processing tool body) 10 having an opening part 2 provided on an opening side of the body part 1, wherein the opening part 2 is provided with a transfer promoting means. Having such transfer promoting means, the transfer of a liquid droplet of the liquid adhering on an inner surface of the opening 2 into the body part 1 can be promoted when the processing tool is mounted to a centrifuge to centrifuge in a state of storing the liquid in the body part 1 of the reaction vessel 10. The transfer promoting means is preferably composed of an inclined surface 6.

Description

  The present invention relates to a treatment tool, and particularly to a treatment tool used when purifying a sugar chain of 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.

  As a method for analyzing sugar chains on the cell surface, for example, there is a method of fractionating cell surface proteins using a large apparatus such as ultracentrifugation and then collecting and analyzing the bound sugar chains.

  However, when this method is used, it cannot be said that all the glycoproteins present on the surface can be recovered, and an ultracentrifuge, which is an expensive device, is required. Therefore, there is a need for a method that does not require a special device and that can easily and comprehensively recover surface sugar chains.

  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. 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 costs and time are required. A means for separation and purification has been demanded. Furthermore, development of the processing tool used in order to implement | achieve this isolation | separation refinement means was calculated | required.

Introduction to Glycobiology Chemistry Doujin November 1, 2005 First Edition

  The objective of this invention is providing the processing tool which can store the liquid which should be stored in a processing tool with the outstanding precision without waste.

Such an object is achieved by the present invention described in the following (1) to (9).
(1) A processing tool to be mounted on a centrifuge and used.
A processing tool main body comprising a bottomed cylindrical main body capable of storing a liquid and a cylindrical mouth provided on the opening side of the main body;
Movement of the liquid adhering to the inner surface of the mouth into the main body when the processing tool is mounted on the centrifuge and centrifuged while the liquid is stored in the main body of the processing tool main body. A processing tool characterized in that a movement promoting means for promoting the movement is provided in the mouth.

  (2) The processing tool according to (1), wherein the movement promoting unit includes an area gradually decreasing portion formed by gradually decreasing the opening area of the mouth portion toward the main body portion.

  (3) The processing tool according to (2), wherein the area gradually decreasing portion is provided over the entire axial direction of the mouth portion.

  (4) The processing tool according to (1), wherein the movement promoting unit has a function of regulating movement of the attached liquid along a circumferential direction of the mouth.

  (5) The processing tool according to (4), wherein the movement promoting unit is configured by a ridge or a groove along the axial direction of the mouth.

(6) A through hole is formed in the bottom of the main body,
The processing tool according to any one of (1) to (5), wherein the processing tool main body further includes a filter provided to close the through hole.

  (7) Further, the apparatus further includes an outer container that is attached to the processing tool main body, and stores the liquid in the main body portion that has passed through the through-hole when the processing tool is attached to the centrifuge and centrifuged. The processing tool as described in 6).

(8) The outer container is composed of a cylindrical member whose one end is closed,
The processing tool according to (7), wherein the processing tool main body includes an engaging portion that engages with an opening edge when the outer container is mounted near a boundary between the main body portion and the mouth portion. .

  (9) The processing tool according to any one of (1) to (8), further including a lid portion that is attached to the mouth portion of the processing tool body and sealed.

  According to the present invention, since the liquid to be stored in the processing tool can be moved to the main body of the reaction vessel provided in the processing tool with no waste, the liquid waste can be prevented or suppressed accurately. it can.

  For this reason, when this treatment tool is used for purification of sugar chains, it is possible to purify sugar chains with excellent accuracy with simple operations and without waste.

It is the longitudinal cross-sectional view (a) and top view (b) which show 1st Embodiment of the reaction container with which the processing tool of this invention is provided. It is a top view which shows 2nd Embodiment of the reaction container with which the processing tool of this invention is provided. It is a top view which shows 3rd Embodiment of the reaction container with which the processing tool of this invention is provided. It is a top view which shows 4th Embodiment of the reaction container with which the processing tool of this invention is provided. It is a longitudinal cross-sectional view which shows an example of the processing tool used when refine | purifying a sugar chain from glycoprotein. It is a longitudinal cross-sectional view for demonstrating the method of refine | purifying a sugar chain from glycoprotein using the processing tool shown in FIG. It is a longitudinal cross-sectional view for demonstrating the method of refine | purifying a sugar chain from glycoprotein using the processing tool shown in FIG. It is a longitudinal cross-sectional view for demonstrating the method of refine | purifying a sugar chain from glycoprotein using the processing tool shown in FIG. It is a fragmentary longitudinal cross-sectional view which shows typically an example of the centrifuge used when refine | purifying a sugar chain from glycoprotein using the processing tool shown in FIG.

Hereinafter, the processing tool of the present invention will be described in detail based on a preferred embodiment.
In the following, an example of a method for purifying a sugar chain from a glycoprotein using a processing tool (sugar chain purification) will be described prior to the description of the processing tool of the present invention.

<Glycan purification>
FIG. 5 is a longitudinal sectional view showing an example of a treatment tool used when purifying a sugar chain from glycoprotein, and FIGS. 6 to 8 are methods for purifying a sugar chain from glycoprotein using the treatment tool shown in FIG. FIG. 9 is a partial longitudinal sectional view schematically showing an example of a centrifuge used for purifying sugar chains from glycoproteins using the processing tool shown in FIG. In the following description, the upper side in FIGS. 5 to 8 is referred to as “upper” and the lower side is referred to as “lower”. In the glycoprotein purification method described below, a case where a sugar chain is purified from a glycoprotein in a gel separated by electrophoresis will be described.

  In the sugar chain purification method shown below, [1] a step of obtaining a gel retaining glycoprotein, [2] a step of releasing the sugar chain by treating the gel with a sugar chain releasing means, [3] A step of eluting the released sugar chain from the gel to obtain a solution containing the sugar chain; and [4] contacting the solution containing the sugar chain with a capture carrier that specifically binds to the sugar chain, A step of capturing a sugar chain, [5] a step of removing a substance other than the sugar chain that was not bound to the capture carrier, and [6] re-releasing the sugar chain bound to the capture carrier, and the sugar chain. And [7] a step of purifying the labeled sugar chain.

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

  The sample containing the prepared glycoprotein is not particularly limited, and examples thereof include biological samples such as whole blood, serum, plasma, urine, saliva, cells, and tissues. Moreover, it is possible to use a glycoprotein that has been purified in advance using a predetermined method or is not purified.

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

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

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

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

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

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

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

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

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

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

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

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

  In contrast, in vivo substances other than sugar chains such as proteins, nucleic acids and lipids 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 collected by means such as centrifugation or filtration after sugar chains are captured by the polymer particles. It is also possible to use polymer particles packed in a column.

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

  Hereinafter, a case where a sugar chain is captured on the polymer particle 20 by using the polymer particle 20 as a capture carrier and further using various means such as centrifugation and filtration will be described as an example.

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

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

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

  [4-2] Next, a reaction container (processing tool main body) 100 as shown in FIG.

  The reaction container 100 includes a main body 101 that can store a liquid, and a mouth portion 102 provided on the opening side (upper side) of the main body 101. And normally, it mounts | wears with the outer container 200 which is a cylindrical member with which one end obstruct | occluded like an Eppendorf tube, and is used as one member with which the processing tool 300 is provided (refer FIG.5 (b)).

  The main body 101 has a bottomed cylindrical shape as a whole, both an inner diameter and an outer diameter are substantially constant in the height direction, and includes a bottom surface 103 on the lower side.

  Further, a through hole 104 is provided substantially corresponding to the center of the bottom surface 103, whereby the liquid supplied (stored) to the main body 101 through the through hole 104 is discharged to the outside. That is, the liquid can be supplied to the outer container 200.

  Further, a filter 105 is disposed below the main body 101 so as to close the through hole 104. Thereby, when the reaction container 100 is left still, the liquid supplied to the main body 101 is inhibited from flowing out to the outside (outer container 200) through the through hole 104. On the other hand, when the processing tool 300 is attached to the centrifuge and the reaction vessel 100 is centrifuged, the liquid is allowed to pass through the filter 105, so that the liquid passes through the through-hole 104. It is leaked outside. That is, the liquid is supplied to the outer container 200.

  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 pore diameter of the pores provided in the filter 105 is preferably set to about 0.1 to 50 μm, more preferably about 0.5 to 20 μm, and still more preferably 1 to 10 μm. Thereby, the permeation of the liquid component contained in the liquid is allowed, and the permeation of the polymer particle 20 is not allowed.

  The mouth portion 102 is provided on the upper side of the main body portion 101, and the entire shape thereof is cylindrical, and the inner diameter and the outer diameter are both larger than the inner diameter and the outer diameter of the main body portion 101.

  By increasing the inner diameter in this way, the liquid can be easily and reliably injected into the main body 101.

  Further, by increasing the outer diameter, the mouth portion 102 protrudes in a circumferential shape on the outer side as compared with the main body portion 101. Therefore, when the reaction container 100 is attached to the outer container 200, the vicinity of the boundary between the mouth part 102 and the main body part 101 functions as an engaging part that engages with the opening edge part 201 of the outer container 200.

  With the reaction container 100 having such a configuration, the reaction of the supplied liquid can be advanced in the main body 101 when the reaction container 100 is left standing. Further, the liquid is selectively supplied to the outer container 200 through the through-hole 104 in a state in which the solid component (polymer particles 20) contained in the liquid remains in the main body 101 by centrifugation of the reaction container 100. (Storage). That is, the stationary component contained in the liquid supplied to the main body 101 and the liquid can be easily separated by centrifuging the reaction vessel 100.

  [4-3] Next, as shown in FIG. 6A, after supplying the particle dispersion 22 prepared in advance in the step [3] into the reaction vessel 100, the reaction vessel 100 is attached to the outer vessel 200. The treated tool 300 is centrifuged using a centrifuge (see FIG. 6B).

  As a result, as shown in FIG. 6 (c), the pure water 21 contained in the particle dispersion liquid 22 is selectively supplied into the outer container 200, so that the polymer particles 20 are singly contained in the reaction container 100. It will be in the state filled with.

  Although it does not specifically limit as a centrifuge used here, For example, the centrifuge 500 as shown in FIG. 9 is used.

  The centrifuge 500 includes a casing 501, a rotating body 530 that fixes the processing tool 300, a driving unit 520 that rotates the rotating body 530, and a support plate 511 that supports the driving unit 520.

  The rotating body 530 includes a plurality of holes 531 into which the processing tool 300 can be inserted (mounted). In this embodiment, the entire shape is a disk shape, and the vertical cross-sectional shape passing through the center thereof is the upper bottom. The bottom is a trapezoid with a longer bottom.

  In this rotator 530, each hole 531 is provided at a line-symmetrical position with the central axis of the rotator 530 as the symmetric axis so that the inclination of the center axis 532 becomes an angle θ1 with respect to the support plate 511. It has been.

  The angle θ1 is set to about 30 to 80 °, preferably about 40 to 60 °, and more preferably about 45 °. By setting the angle θ1 within such a range, even when the rotating body 530 is stationary, when the processing tool 300 is inserted into the hole 531, the particle dispersion liquid 22 leaks from the reaction vessel 100 due to this inclination. Can be reliably prevented. Further, when the rotating body 530 rotates, the pure water 21 in the particle dispersion 22 can be reliably transmitted through the filter 105 by the centrifugal force generated thereby.

  The driving means 520 has a rotating shaft 522 extending downward from the central axis of the rotating body 530, and a motor 521 connected to the lower end of the rotating shaft 522 and rotating the rotating shaft 522 around the axis. By rotating the motor 521, the rotating body 530 rotates around the rotation shaft 522.

  The support plate 511 constitutes a partition that vertically separates the inside of the casing 501, and a through-hole penetrating in the thickness direction is formed substantially at the center of the support plate 511, and a rotating body is provided above the support plate 511. The motor 521 is arranged on the lower side of the 530, and these are connected via a rotating shaft 522 arranged in the through hole. With this configuration, the driving unit 520 is supported in the casing 501 by the support plate 511, and as a result, each part constituting the apparatus is supported in the casing 501.

  In the centrifuge 500 having such a configuration, when the rotating body 530 is rotated by driving the driving unit 520 in a state where the processing tool 300 is inserted into the rotating body 530, the centrifugal force generated thereby causes the inside of the particle dispersion 22 The pure water 21 passes through the filter 105. On the other hand, due to the relationship between the pore size of the filter 105 and the particle size of the polymer particle 20, the polymer particle 20 cannot pass through the filter 105. Therefore, the pure water 21 that has passed through the filter 105 is selectively supplied into the outer container 200 through the through-hole 104, and as a result, the polymer particles 20 are filled alone in the reaction container 100.

  [4-4] Next, as shown in FIG. 6D, the solution containing the sugar chain obtained in the step [3] (hereinafter referred to as “sugar chain-containing liquid 23”) is polymer particles. 20 is supplied into the reaction vessel 100 filled with 20.

  [4-5] Next, by heating the sugar chain-containing liquid 23, the sugar chain-containing liquid 23 is kept in a certain temperature range until the supplied sugar chain-containing liquid 23 is dried (see FIG. 6E). .)

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

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

  The temperature at the time of sugar chain capture is preferably kept in a temperature range of about 4 to 90 ° C, more preferably about 4 to 70 ° C, more preferably about 30 to 80 ° C, and most preferably about 40 to 80 ° C. Set to.

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

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

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

  Through the steps [4-1] to [4-5] as described above, sugar chains are captured on the polymer particles 20.

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

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

  Hereinafter, in the same manner as in the above step [4], a substance other than the sugar chain captured by the polymer particles 20 by means such as centrifugation or filtration using the reaction vessel 100 (hereinafter, this substance is referred to as “cleaning substance”). The case of cleaning and removing will be described.

  [5-1] First, as shown in FIG. 7A, the cleaning liquid 24 is added to the polymer particles 20 dried in the reaction vessel 100 obtained in the step [4-5].

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

  [5-2] Next, the processing tool 300 in which the reaction vessel 100 is mounted on the outer vessel 200 is centrifuged using a centrifuge (see FIG. 7B).

  As a result, as shown in FIG. 7C, the cleaning liquid 24 is selectively removed from the reaction container 100 while the polymer particles 20 remain in the reaction container 100 by allowing the cleaning liquid 24 to pass through the filter 105. In addition, the cleaning substance dissolved in the cleaning liquid 24 can be removed.

  By performing the cleaning composed of these steps [5-1] and [5-2] a plurality of times (repeatedly), the cleaning substance is separated into the outer container 200 in a state dissolved in the cleaning liquid 24. Therefore, the cleaning substance can be surely removed from the polymer particles 20 in which the sugar chains are captured.

  In this case, the polymer particles 20 are sufficiently washed with water or a buffer solution containing a surfactant as the washing liquid 24, then further washed with the organic solvent washing liquid 24, and finally with water as the washing liquid 24. Is preferred. Thereby, it becomes possible to more reliably remove the cleaning substance, in particular, impurities adsorbed nonspecifically on the surface of the polymer particle 20.

  [6] Next, the sugar chain bound to the capture carrier is released again, and this sugar chain is replaced with another compound (hereinafter sometimes referred to as “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.

  Hereinafter, similarly to the steps [4] and [5], the case where the sugar chain is labeled with the compound A using the reaction vessel 100 will be described.

  [6-1] First, as shown in FIG. 7D, the compound-containing liquid 25 containing the compound A is added into the reaction vessel 100 filled with the polymer particles 20 in which the sugar chains have been captured.

  The amount of compound A added to the reaction vessel 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 in the next step [6-2] 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.

  [6-2] Next, by heating the compound-containing liquid 25, the compound-containing liquid 25 is kept in a certain temperature range until the added compound-containing liquid 25 is dried (see FIG. 7E).

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

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

  The temperature at the time of labeling is preferably kept in a temperature range of about 4 to 90 ° C, more preferably about 4 to 70 ° C, more preferably about 30 to 80 ° C, and most preferably about 40 to 80 ° C. Set.

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

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

  The sugar chain is labeled with the compound A through the steps [6-1] and [6-2] as described above.

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

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

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

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

  [6-1 '] First, a sugar chain releasing solution for re-releasing sugar chains is added to the reaction vessel 100 filled with the polymer particles 20 in which the sugar chains are captured.

  [6-2 '] Next, by heating the sugar chain free solution, the sugar chain free solution is kept in a certain temperature range until the added sugar chain free solution is dried.

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

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

  [6-3 '] Next, the compound-containing liquid 25 containing an aromatic amine as the compound A is added into the reaction vessel 100 in which the sugar chains are released from the polymer particles 20.

  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, in the reaction vessel 100 after the addition of the compound-containing solution 25, The concentration of the aromatic amine in the solution, that is, 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 step [7]. It is set to 4 mol / L or more and 3 mol / L or less.

  Further, when the amount of the liquid is defined by the polymer particles 20 filled in the reaction vessel 100, the amount of the liquid 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. However, it is preferable to set the liquid volume (volume) 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 amount may exceed 100 μL, but if it exceeds a certain amount, it may be difficult to remove the aromatic amine (compound A) that was not used in the reaction in the post-process [7]. Therefore, the most preferred liquid amount is set between 100 μL and 200 μL.

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

  [6-4 '] Next, the compound-containing liquid 25 is heated to keep the compound-containing liquid 25 in a certain temperature range.

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

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

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

  In addition, when an aromatic amine is used as the compound A, drying of the compound-containing liquid 25 as described in the above step [6-2] can be omitted.

  The sugar chain is labeled with the compound A also by the steps [6-1 ′] to [6-4 ′] as described above.

  Furthermore, in this step, when it is not necessary to label the sugar chain, substitution of the sugar chain with Compound A can be omitted.

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

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

  Therefore, in this step [7], the labeled sugar chain is purified by removing the polymer particles 20 and the unused compound A.

  Hereinafter, similarly to the steps [4] to [6], the case where the labeled sugar chain is purified by means such as centrifugation or filtration using the reaction vessel 100 will be described.

  [7-1] First, as shown in FIG. 8A, the labeled sugar chain obtained in the step [6-2] and dried in the reaction vessel 100, and the polymer particle 20, Add lysate 26.

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

  In addition, when the polymer particle 20 is not dried by heating the compound-containing liquid 25 in the step [6-2], the addition of the solution 26 into the reaction vessel 100 in the present step [7-1] is omitted. can do.

  Further, when a fluorescent substance composed of an aromatic amine is used as the compound A, the sugar chain labeled with the compound A obtained in the above steps [6-1 ′] to [6-4 ′] Since it is not dried, also in this case, the addition of the solution 26 into the reaction vessel 100 in this step [7-1] can be omitted.

  [7-2] Next, the processing tool 300 in which the reaction vessel 100 is mounted on the outer vessel 200 is centrifuged using a centrifuge (see FIG. 8B).

  Thus, as shown in FIG. 8C, the solution 26 is selectively transmitted from the reaction vessel 100 in a state where the polymer particles 20 remain in the reaction vessel 100 by allowing the solution 26 to pass through the filter 105. Can be stored in the outer container 200, and the labeled sugar chain dissolved in the solution 26 can be moved to the outer container 200. As a result, the labeled sugar chain and the polymer particle 20 are separated.

  At this time, since the unused compound A is usually soluble in the solution 26, it moves to the outer container 200 side in a state of being dissolved in the solution 26.

  [7-3] Next, in place of the filter 105, a container 100 ′ in which the silica gel 106 is disposed on the lower side of the main body 101 is prepared. obtain.

  And as shown in FIG.8 (d), in the said process [7-2], the solution 26 containing the labeled sugar chain isolate | separated to the outer side container 200 is diluted with nonaqueous solvents, such as acetonitrile, It supplies in container 100 'provided with the silica gel 106. FIG.

  As a result, as shown in FIG. 8 (e), the solution 26 that has passed through the silica gel 106 flows out into the outer container 200 as droplets through the through-hole 104.

  At this time, since the solution 26 obtained by diluting the silica gel 106 with a non-aqueous solvent such as acetonitrile permeates, the labeled sugar chain and the unused compound A contained in the solution 26 are adsorbed on the silica gel. The labeled sugar chain has higher adsorption power to silica gel than the unused compound A. Therefore, by supplying a nonaqueous solvent such as acetonitrile into the container 100 ′ and washing the silica gel 106, at least a part of the unused compound A can be removed from the silica gel. Thereafter, the labeled sugar chain can be purified (separated) in a state where mixing of unused compound A is accurately prevented or suppressed by adding water into the container 100 ′ and allowing the silica gel to permeate.

  The sugar chain labeled with Compound A can be purified through the above steps.

  In addition, when it is not necessary to remove the unused compound A, this step [7] may be omitted.

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

  In particular, when the sugar chain is labeled with N-aminooxyacetyl-tryptophanyl (arginine methyl ester) represented by the chemical formula (2), high-sensitivity analysis can be performed using MALDI-TOF MS.

  Now, in such a method for purifying sugar chains, by using means such as centrifugation and filtration using a supplementary carrier (polymer particles 20) and a processing tool 300, the steps are compared with the conventional methods described above. Therefore, cost and time waste can be reduced.

  And in order to refine | purify sugar chain with the more outstanding precision, as demonstrated in each said process, the particle | grain dispersion liquid 22, the sugar chain containing liquid 23, the washing | cleaning liquid 24, the compound containing liquid 25, and the solution 26, respectively, Since it is configured to be supplied to the reaction vessel 100 (main body portion 101), it is required to supply these liquids to the main body portion 101 with high accuracy.

  However, in the reaction vessel 100, as described above, since the inner diameter of the main body portion 101 and the mouth portion 102 is larger in the mouth portion 102, a step 107 is formed at the boundary portion between them. Therefore, for example, when the liquid is supplied to the main body portion 101 along the inner peripheral surface (inner wall) of the mouth portion 102, the liquid remains in the step 107. Therefore, if each step is performed in such a state where it remains, the accuracy of sugar chain purification is reduced.

  In particular, in the sugar chain purification method, the liquid supplied to the main body 101 by centrifugation is moved to the outer container 200 side, but even if such centrifugation is performed, If the reaction vessel 100 has a step 107, the liquid remains in the step 107.

The treatment tool of the present invention is used to solve such a problem.
<Processing tool>
Hereinafter, although the processing tool of the present invention will be described, the present invention has a feature in the configuration of a reaction vessel (processing tool main body) provided in the processing tool. Therefore, the reaction vessel will be described in detail below.

<< First Embodiment >>
First, 1st Embodiment of the reaction container with which the processing tool of this invention is provided is described.

  Drawing 1 is a longitudinal section (a) and a top view (b) showing a 1st embodiment of a reaction vessel with which a processing tool of the present invention is provided. In the following description, the upper side in FIG. 1 is referred to as “upper” and the lower side is referred to as “lower”.

  A reaction vessel 10 shown in FIG. 1 has a main body 1 capable of storing a liquid and a mouth 2 provided on the opening side (upper side) of the main body 1.

  The main body 1 has a bottomed cylindrical shape as a whole, and both the inner diameter and the outer diameter are substantially constant in the height direction.

  The main body 1 has a bottom surface 3, a through hole 4, and a filter 5, and has the same configuration as the main body 101 of the reaction vessel 100 described above.

The mouth portion 2 is provided on the upper side of the main body portion 1 and has a cylindrical shape as a whole.
The inner diameter of the mouth 2 is substantially constant from the lower end toward the upper end in the height direction, but gradually increases from the middle toward the upper end. That is, the inner diameter is substantially the same as the inner diameter of the main body 1 at the lower end of the mouth portion 2, but gradually increases from the middle toward the upper end with respect to the inner diameter of the main body 1.

  In other words, the inner peripheral surface (inner surface) of the mouth portion 2 has a funnel shape as a whole, and the upper inner peripheral surface is inclined from the outer peripheral surface side of the mouth portion 2 toward the inner peripheral surface side. An inclined surface (tapered portion) 6 is formed.

  By configuring the mouth portion 2 as described above, the inclined surface 6 forms an area gradually decreasing portion in which the opening area of the mouth portion 2 gradually decreases from the mouth portion 2 toward the main body portion 1 side. Thereby, unlike the reaction container 100 described above, the step 107 is not formed at the boundary between the main body portion 101 and the mouth portion 102. Therefore, even if liquid such as the particle dispersion liquid 22, the sugar chain-containing liquid 23, the cleaning liquid 24, the compound-containing liquid 25, and the solution 26 is supplied to the inner peripheral surface of the mouth part 2, The liquid can be supplied into the main body 1 without the liquid droplets remaining on the inner peripheral surface of the body 2. Therefore, the inclined surface 6 (area gradually decreasing portion) functions as a movement promoting means for promoting the movement of the droplet supplied to the inner peripheral surface of the mouth portion 2 into the main body portion 1.

  Therefore, the liquid supplied to the inner peripheral surface of the mouth portion 2, that is, the inclined surface 6, when the processing tool 300 (reaction vessel 100) is centrifuged using a centrifuge, along the inclined surface 6 and to the lower side thereof. It moves toward the end and is finally guided into the main body 1. In other words, the inclined surface 6 promotes the movement of the liquid adhering to the inner peripheral surface of the mouth portion 2 to the main body portion 1.

  For this reason, the sugar chain separated from the glycoprotein can be reliably and reliably supplied into the main body 1. Therefore, since no waste occurs in this way, the sugar chain can be purified with better accuracy. That is, the yield of sugar chains can be improved.

  The inclined surface 6 is not particularly limited, but is inclined so that the angle θ2 with respect to the central axis of the mouth portion 2 is preferably about 15 to 65 °, more preferably about 25 to 55 °. Thereby, when the processing tool 300 including the reaction vessel 100 is mounted in the hole 531 of the centrifuge 500 as described above, the difference between the angle θ1 and the angle θ2 can be reduced. That is, the inclination angle of the inclined surface 6 with respect to the support plate 511 can be reduced. Therefore, when the processing tool 300 is centrifuged using the centrifuge 500, a larger centrifugal force can be applied to the liquid (droplet) adhering to the inclined surface 6. Therefore, the liquid supplied to the inclined surface 6 can be reliably guided in the main body 1.

  The inclined surface 6 is preferably subjected to an adhesion preventing process for preventing or reducing the adhesion of liquid. By performing such a process, the liquid can be more reliably moved to the main body 1 along the inclined surface 6 by centrifugal force.

The adhesion preventing process is not particularly limited, and examples thereof include a liquid repellent process for forming a liquid repellent film on the inclined surface 6 and a smoothing process for reducing the unevenness of the inclined surface 6 to form a smooth surface.
The movement promoting means is not limited to the case of the inclined surface 6 as shown in FIG. 1, and includes, for example, a curved concave surface, a curved convex surface, or an inclined surface 6, and any two or more of these are included. You may be comprised by what combined.

  On the other hand, the outer diameter of the mouth portion 2 is substantially constant along the height direction, and the size thereof is larger than the outer diameter of the main body portion 1. Thereby, compared with the main-body part 1, the opening part 2 will protrude on the outer side in the periphery shape. Therefore, when the reaction container 10 is attached to the outer container 200, the mouth part 2 functions as an engaging part that engages with the opening edge 201 of the outer container 200.

  The constituent material of the reaction vessel 10 is not particularly limited, and examples thereof include polypropylene, polyethylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, and the like, and one or more of these are combined. Can be used.

  In the present embodiment, the inner diameter of the mouth portion 2 is constant at the lower end, but is configured to gradually increase from the middle toward the upper end, that is, the inclined surface 6 is formed from the middle of the mouth portion 2 to the upper end. Although it was set as the structure, it is not limited to this structure, The inclined surface 6 may be provided from the lower end of the opening part 2 to the upper end. In other words, the area gradually decreasing portion may be provided over the entire axial direction of the mouth portion 2. By setting it as such a structure, the advantage that the setting of the angle of the inclined surface 6 can be performed easily is acquired.

<< Second Embodiment >>
Next, 2nd Embodiment of the reaction container with which the processing tool of this invention is provided is described.

  FIG. 2 is a plan view showing a second embodiment of a reaction vessel provided in the processing tool of the present invention. In the following description, the upper side in FIG. 2 is referred to as “upper” and the lower side is referred to as “lower”.

  Hereinafter, although the second embodiment will be described, the description will focus on the points different from the first embodiment, and the description of the same matters will be omitted.

  In 2nd Embodiment, the structure of the opening part 2 differs, and other than that is the same as that of the said 1st Embodiment.

  As shown in FIG. 2, a groove 7 is formed in the mouth part 2 along the vertical direction (axis method) of the mouth part 2 on the inclined surface 6.

  In the present embodiment, eight grooves 7 are formed at equal intervals along the circumferential direction of the inclined surface 6. That is, each groove 7 is located radially about the axis of the mouth 2.

  With such a configuration, when the processing tool 300 (reaction vessel 100) is centrifuged using a centrifuge, the liquid supplied (attached) to the inclined surface 6 moves along the circumferential direction of the inclined surface 6. It is possible to reliably prevent this. Therefore, each groove 7 functions as a restricting means for restricting the movement of the droplet along the circumferential direction of the inclined surface 6.

  In addition, although the edge of the cross section (cross section in the direction perpendicular to the longitudinal direction) of the groove 7 of the present embodiment has a U shape (rectangular shape), other examples include, for example, a semicircular shape and a U shape. The shape may be a V shape or the like.

<< Third Embodiment >>
Next, 3rd Embodiment of the reaction container with which the processing tool of this invention is provided is described.

  FIG. 3 is a plan view showing a third embodiment of a reaction vessel provided in the processing tool of the present invention. In the following description, the upper side in FIG. 3 is referred to as “upper” and the lower side is referred to as “lower”.

  In the following, the third embodiment will be described, but the description will focus on differences from the first embodiment, and description of similar matters will be omitted.

  In 3rd Embodiment, the structure of the opening part 2 differs, and other than that is the same as that of the said 1st Embodiment.

  As shown in FIG. 3, a ridge (rib) 8 is formed in the mouth portion 2 along the vertical direction of the mouth portion 2 on the inclined surface 6.

  In the present embodiment, eight ribs 8 are formed at equal intervals along the circumferential direction of the inclined surface 6. That is, the ribs 8 are located radially about the central axis of the mouth 2.

  With such a configuration, when the processing tool 300 (reaction vessel 100) is centrifuged using a centrifuge, the liquid supplied to the inclined surface 6 moves along the circumferential direction of the inclined surface 6. Can be reliably prevented. Therefore, each rib 8 functions as a restricting means for restricting the movement of the droplet along the circumferential direction of the inclined surface 6.

  Note that the rib 8 of the present embodiment has a U-shaped (rectangular) edge in the transverse cross section (cross section in the direction perpendicular to the longitudinal direction). The shape may be a V shape or the like.

<< Fourth Embodiment >>
Next, 4th Embodiment of the reaction container with which the processing tool of this invention is provided is described.

  FIG. 4 is a plan view showing a fourth embodiment of a reaction vessel provided in the processing tool of the present invention. In the following description, the upper side in FIG. 4 is referred to as “upper” and the lower side is referred to as “lower”.

  Hereinafter, the fourth embodiment will be described, but the description will focus on the differences from the second embodiment, and the description of the same matters will be omitted.

  In 4th Embodiment, the structure of the opening part 2 differs, and other than that is the same as that of the said 2nd Embodiment.

  In the present embodiment, the eight grooves 7 are curved in the longitudinal direction of the groove 7 instead of being formed linearly along the vertical direction of the mouth portion 2.

With such a configuration, as in the second embodiment, when the processing tool 300 (reaction vessel 100) is centrifuged using a centrifuge, each groove 7 has a droplet in the circumferential direction of the inclined surface 6. Although it functions as a restricting means for restricting the movement along, it also functions as a movement promoting means because the movement of the liquid to the main body 1 can be promoted by being curved in the longitudinal direction.
In addition, the groove | channel 7 may be formed helically besides the case where it curves in a longitudinal direction.

  Further, as described in Embodiments 2 to 4, when the mouth 2 is provided with the groove 7 or the protrusion 8, the lid 2 is attached to the mouth 2 to seal the reaction vessel 100. It may be. In this case, since the groove 7 or the ridge 8 functions as a locking portion that locks the lid portion, it is possible to reliably prevent the lid portion from falling off.

  As mentioned above, although the processing tool of this invention was demonstrated based on embodiment of illustration, this invention is not limited to these. For example, the processing tool of the present invention may be a combination of any two or more configurations (features) of the above-described embodiments.

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

DESCRIPTION OF SYMBOLS 1 Main-body part 2 Mouth part 3 Bottom face 4 Through-hole 5 Filter 6 Inclined surface 7 Groove 8 Projection 10 Reaction vessel 20 Polymer particle 21 Pure water 22 Particle dispersion liquid 23 Sugar chain containing liquid 24 Cleaning liquid 25 Compound containing liquid 26 Solution 100 Reaction Container 100 ′ Container 101 Main body 102 Portion 103 Bottom surface 104 Through hole 105 Filter 106 Silica gel 107 Step 200 Outer container 201 Opening edge 300, 300 ′ Processing tool 500 Centrifuge 501 Casing 511 Support plate 520 Driving means 521 Motor 522 Rotating shaft 530 Rotating body 531 Hole 532 Central axis

Claims (9)

A processing tool mounted on a centrifuge and used.
A processing tool main body comprising a bottomed cylindrical main body capable of storing a liquid and a cylindrical mouth provided on the opening side of the main body;
Movement of the liquid adhering to the inner surface of the mouth into the main body when the processing tool is mounted on the centrifuge and centrifuged while the liquid is stored in the main body of the processing tool main body. A processing tool characterized in that a movement promoting means for promoting the movement is provided in the mouth.   The processing tool according to claim 1, wherein the movement promoting means is configured by an area gradually decreasing portion formed by gradually decreasing the opening area of the mouth portion toward the main body portion.   The processing tool according to claim 2, wherein the area gradually decreasing portion is provided over the entire axial direction of the mouth portion.   The processing tool according to claim 1, wherein the movement promoting unit has a function of regulating movement of the attached liquid along a circumferential direction of the mouth.   The processing tool according to claim 4, wherein the movement promoting means is configured by a ridge or a groove along the axial direction of the mouth portion. A through hole is formed at the bottom of the main body,
The processing tool according to claim 1, wherein the processing tool main body further includes a filter provided so as to close the through hole.   Furthermore, when the said processing tool is mounted | worn with the said centrifuge and it centrifuges, it has an outer container which stores the said liquid in the said main-body part which passed the said through-hole, when it mounts | wears with the said processing tool main body. Processing tools. The outer container is composed of a cylindrical member whose one end is closed,
The said processing tool main body is a processing tool of Claim 7 provided with the engaging part engaged with the opening edge part, when the said outer side container is mounted | worn near the boundary of the said main-body part and the said mouth part.   Furthermore, the processing tool in any one of Claim 1 thru | or 8 which has a cover part with which the said opening | mouth part of the said processing tool main body is mounted | worn and sealed.

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