JPH07104355B2 - Method for fluorescent labeling of sugars - Google Patents

Description

TECHNICAL FIELD The present invention relates to a sugar chain analysis pretreatment method for attaching a fluorescent label to a sugar chain,
In particular, it relates to a method of performing such pretreatment using an automated device.

2. Description of the Related Art Conventionally, in order to attach a fluorescent label to a saccharide for analysis, it is said that a pyridyl aminating agent and hydrochloric acid are added to the reducing end of the saccharide or sugar chain to form a Schiff base, which is then reduced with a reducing agent. Methods have been used. In order to improve the low reaction yield and instability by the conventional method, the present inventors have previously proposed a method using a substantially anhydrous acid (preferably acetic acid) instead of the hydrochloric acid in the pyridyl amination. developed.

Problems to be Solved by the Invention In this case, when the test saccharide is a complex carbohydrate such as glycoprotein or glycolipid, after isolating the sugar chain from these substances,
Since the above-mentioned pyridyl amination is performed, there are drawbacks that a large number of reagents and the like are required for each reaction, and that the operation is complicated and takes a very long time.

The present invention is intended to solve the problems in the pretreatment (isolation and fluorescent labeling) of the sugar chain analysis as described above by automating the steps.

[Means for Solving the Problems] Therefore, according to the present invention, a large number of sample tube receiving holes arranged in an annular shape for supporting a top-end open type sample tube containing a sugar sample and an annular arrangement of the sample tube receiving holes are provided. A sample-side turntable having a heater and a cooling channel disposed in close proximity to the sample-side turntable, and an array of lid units for the respective sample tubes that can be raised and lowered immediately above the sample-side turntable, and each lid unit , A reaction station including an elevating lid mechanism having an inert gas branch supply line opened on the exposed surface into the sample tube and a vacuum branch suction line, and isolation of the sugar chain of the sample and its sugar chain Each reagent such as hydrazine, N-acetylating agent, aminopyridine, substantially anhydrous acid, and reducing agent necessary for fluorescent labeling of Corresponding to a plurality of reagent tube receiving holes annularly arranged for supporting the upper end opening type test tube containing each azeotropic agent used when evaporating and removing the excess reagents, and the plurality of reagent tube receiving holes. And a plurality of pipette receiving holes arranged concentrically in an annular manner, and the pipette receiving hole has a reagent-side turntable configured to support a pipette having a pointed end at the lower end, and the receiving hole at the tip. An arm having a chuck / air inlet / outlet engaging with a supported pipette upper end opening is provided, which is different from the pipette whose chuck / air inlet / outlet is supported at a predetermined azimuth position of the reagent side turntable. Corresponds to each upper end opening of the reagent tube supported at a predetermined azimuth position and each upper end opening of the sample tube supported at a predetermined azimuth position on the sample side turntable. Each sample tube supported on the reaction-side turntable is used by using a sugar chain labeling reaction device equipped with a pipette arm mechanism capable of rotating and elevating at each position within a range including a position. The reagent or azeotropic agent is supplied to the reagent-side turntable by holding and carrying one of the pipettes supported by the reagent-side turntable by the chuck and air inlet / outlet at the tip of the arm, and the tip of the pipette in the corresponding test tube. After injecting the reagent or azeotropic agent into the sample tube, the reagent or azeotropic agent is sucked into the sample tube, and then the sample or the azeotropic agent is introduced into the sample tube supported at a predetermined azimuth position on the sample side turntable. When heating or cooling to the processing temperature necessary for the reaction accompanying the reagent injection into the sample tube or the azeotropic removal of excess reagent after injection of the azeotropic agent, the sample The heater and the cooling channel built in the side turntable are activated, and when the reagents and the like are azeotropically removed, the sample tube on the sample side turntable is sealed with an inert gas branch to the lid unit. This is a method for fluorescent labeling of sugars, characterized in that the supply pipeline and the vacuum branch suction pipeline are activated to perform vacuum suction while feeding an inert gas into the reaction tube.

Action According to the present invention, in the automation of the above process,
Almost no complicated manual operation is required in the middle, time can be shortened by substantially full automation, and stable results can be realized by executing a reliable reaction process.

Example FIG. 1 is a schematic diagram showing the entire structure of an automated apparatus for carrying out the method of the present invention. In FIG. 1, (1) is a sample side turntable, (2) is a lift mechanism located directly above the turntable, and (3) is arranged on the same plane as the sample side turntable (1). Reagent side turntable (4) is this reagent side turntable (3)
A support shaft that is a pipette arm for inhaling and collecting a reagent or the like from an upper reagent tube (described later) and injecting it into the sample tube (5) on the sample side turntable (1), the rear end of which can be raised and lowered and rotated. It is supported on the upper end of (6). (7) is a bearing for each shaft (6), and (8) is a transmission gear meshed with a gear (6a) fixed to the shaft (6). A thread groove or a rack (not shown) is formed in the bearing (7) of the shaft (6) and the lower portion (6b) of the gear (6a) to engage with a suitable transmission mechanism (9) so that it can be moved up and down. Has become.

The turntables (1), (3) and the support shaft (6) of the pipette arm (4) are each driven by a step motor. That is, the step motor (SM1) rotationally drives the sample side turntable (1), the step motor (SM2) rotationally drives the arm support shaft (6), and the step motor (SM3) drives the support shaft (6). Lifting and driving, step motor (SM
4) is for rotating the reagent side turntable (3). Two air cylinders are used as an accessory lifting mechanism for the device. That is, (10) is an air cylinder for driving the lifting lid mechanism (2) up and down, and (11) is an electromagnet piece (12) for holding and releasing the lid of the reagent tube on the reagent side turntable (3). An air cylinder for raising and lowering. Further, the chuck / air inlet / outlet (13) at the tip of the pipette arm (4) is adapted to be supplied and exhausted from the cylinder pump (14). (15) is a motor for driving the piston of the cylinder pump (14). The step motors (SM1) to (SM4), the drive motor (15) for the cylinder pump (14), the compressed air source connected to the air cylinders (10) and (11), and the like have a control interface circuit (16). The drive is controlled by a computer (17) via the. In addition, (18a) in the base cabinet part of the computer (17),
(18b) is a control card or other insertion port, and the line (1) drawn in relation to the sample side turntable (1)
9) is a cooling water circulation pipe.

FIG. 2 is a schematic plan view for explaining the schematic functions of the sample side turntable (1) and the reagent side turntable (3), which are the main parts of the apparatus configuration shown in FIG. On the upper surface of the sample side turntable (1), a large number of sample receiving holes (21) arranged in an annular shape for receiving and supporting the upper opening type sample tube (20) are formed, and the upper surface of the reagent side turntable (3). A plurality of annularly arranged reagent tube receiving holes (23) for receiving and supporting the upper end open type reagent tube (22) are formed in the tube, and the reagent tube receiving holes (23) are concentrically provided. A plurality of pipette receiving holes (24) arranged in an annular shape are formed. The tip chuck and air inlet / outlet of the arm (4) are pipettes in one pipette receiving hole (24) (described later).
Take out the reagent from one of the corresponding reagent tubes (22) into the pipette, and then rotate it to the position shown by the broken line so that the pipette rushes into the sample tube (20) at that position, and its lower end. The reagent and the like are discharged and injected by air pressure from the tip. The arm (4) vertically moves and intermittently supplies air (reagent injection) at this position, and in combination with the intermittent operation of the turntable corresponding thereto, all the sample tubes (20) supported on the turntable (1). The reagent and the like are added to the sugar sample in ().

In addition, a bracket-shaped pipette removing member (2 having a U-shaped notch in relation to the reagent side turntable (3)
5) is located.

FIG. 3 shows a preferable environment and atmosphere for carrying out a reaction by a reagent injected in a sample tube (20) supported on a sample side turntable (1) or azeotropic removal of the reagent after the reaction. It shows a structure that contributes to the formation. That is, (26) shown in FIG.
A water cooling jacket as a cooling channel connected to the cooling water circuit (19) shown in the figure, which includes a sample tube (2
It is doubled along the inner circumference and the outer circumference of the annular arrangement of (0). Further, a heater (27) is embedded at a position further inside the inner circumference water cooling jacket (26), and the turntable (1) main body is made of a material having good thermal conductivity, so that it is necessary for processing steps such as reaction. Heating to temperature and cooling can be done quickly. At the time of processing, each of the lid units (2a) annularly arranged in the lifting lid mechanism (2) is airtightly fitted into the upper end opening of the sample tube (20). The lid unit (2a) is provided with an inert gas supply port (28) such as N ₂ or argon, which opens toward the inside of the sample tube (20), and a vacuum suction port (29). These gas supply port and vacuum suction port (28), (2
9) is for vacuum suction while supplying an inert gas into the sample tube (20) when azeotropically removing the excess reagent after the reaction.

FIGS. 4 to 6 relate to a pipette (30) in which the chuck / air inlet / outlet (13) at the tip of the arm (4) is received and supported in the pipette receiving hole (24) on the turntable (3) by the arm operation. And a mechanism for allowing it to be lifted up, and further to be released after being inhaled and withdrawn from the upper end openings of the reagent tube (22) and the sample tube (20) to inhale and expel reagents and the like. Is. As shown in FIG. 4, a ring (31) made of a resin having suitable elasticity and chemical resistance, for example, polytetrafluoroethylene (trademark “Teflon”) is provided on the outer periphery of the thin cylindrical chuck / air inlet / outlet port (13). ) Is fitted. When the chuck / air inlet / outlet (13) located directly above the pipette (30) in the turntable (3) enters the pipette (30) as the arm (4) descends, the fitting ring (31) causes the fitting ring (31) to move. When the arm (4) is lifted again by engaging with the inner peripheral surface of 30) by pressing, the pipette (30) is pulled out from the pipette receiving hole (24) of the turntable (3),
It is possible to lift. When the lifted pipette (30) finishes its functions such as inhalation of reagents and discharge to the sample tube as described above according to the arm operation,
The arm (4) returning to the turntable (3) side is further rotated until it engages with the pipette removing member (25) as shown in FIG. As shown in FIG. 6, the engagement state of the arm (4) with the pipette removing member (25) is such that the chuck / air inlet (13) is a semicircular or U-shaped cut in the upper end bracket portion of the pipette removing member (25). When the lower surface of the bracket portion of the removing member faces or contacts the upper surface of the flange (32) of the pipette (30) in the state of being in contact with the (25a) and the arm (4) rises in this state, the chuck and the air inlet / outlet ( The fitting ring (31) of 13) slides out of the inner peripheral surface of the pipette (30) locked to the bracket of the removing member (25), and thus, the pipette (30).
Is released and falls. An appropriate pipette collector is placed at this drop position.

In the embodiment, a lid element is fitted to the reagent tube (22) supported on the reagent side turntable (3) except when the pipette is inserted to prevent contamination by dust or the like. FIGS. 7 and 8 show the operation of the lid adsorption mechanism for releasing the test drug (22) by lifting such a lid element (33) in preparation for pipette insertion, and re-engaging after the pipetting operation. Is shown. That is, the lid suction mechanism (34) is composed of the air cylinder (11) and the electromagnet piece (12) described above, and the test tube (22a) to be used next in the sequence operation is the electromagnet piece (1).
When it reaches the position just below 2), the air cylinder (11) operates and the electromagnet piece (12) provided at the tip of the piston moves down to a position close to the lid element (33a) of the reagent tube (22a). Stop. The electromagnet piece is magnetized here so that the lower end surface has one polarity. All lid elements (33) applied to the reagent tubes (22) have permanent magnets embedded so that the upward pole faces are of a polarity that is attracted to the electromagnet pieces (12), and thus Lid element (33
a) is adsorbed by the electromagnet piece (12) and opens the reagent tube (22a). The electromagnet piece (12) rises from this position by the operation of the air cylinder (11), and the other (downward) magnetic pole of the lid element (33a) attracted to this moves to the position just below this by the rotation of the turntable. Then, the lid element (33) for the next reagent tube is lifted to a position where it does not exert a magnetic influence such as suction or movement. Further, the electromagnet piece (12) has the turntable (3) again in its original position after the reagent in the reagent tube (22a) has been sucked into the pipette, that is, the reagent tube (22a) is in the electromagnet piece (12). When returning to the position immediately below, it descends again to bring the lid element (33a) close enough to the upper end opening of the reagent tube (22a),
In this state, the lid element (33a) is excited by the opposite polarity to exert a repulsive force on the lid element (33a), and the lid element (33a) is fitted again to the reagent tube (22a).

In the sugar chain analysis pretreatment method of the present invention, the reaction apparatus configured as described above is used. First, each sample tube (2) supported by the sample side turntable (1)
To supply a reagent, an azeotropic agent, or the like to (0), one of the pipettes (30) supported by the reagent side turntable (3) is connected to the arm (4) and a chuck / air inlet / outlet port (13) at the tip of the arm. The sample is held by the above operation and brought to the corresponding reagent tube (22), and the tip of the pipette (30) is projected into the reagent tube to inhale the reagent or azeotropic agent, and then the sample. It is carried out by bringing the sample into a sample tube (20) supported at a predetermined azimuth position on the side turntable (1) and sequentially injecting the suction liquid into each sample tube. Next, the reaction accompanying the injection of the reagent into the sample tube (20) or the heating or cooling to the processing temperature necessary for azeotropic removal of the excess reagents after the injection of the azeotropic agent is performed on the sample side turntable (1). It is performed by using a heater (27) and a water cooling jacket (26) built in the. further,
At the time of azeotropic removal of the reagents and the like, the sample tube on the sample side turntable (1) is supplied with an inert gas to the lid unit (2a) which is closed and vacuum suction is performed to remove the same. It promotes.

Example of processing sequence (1) Decomposition of complex sugars For the sugar sample contained in each sample tube (sample part volume: maximum diameter 7 mmφ, depth 7 mm conical), hydrazine (liquid) 100 with the first pipette. After ~ 200μ is added,
Heat at 90-100 ° C for 10 hours. In this case, the sample tube is covered with a lid unit after the addition of hydrazine, and an inert gas such as N ₂ is introduced through the lid unit and pressurized. Then, by gradually heating at a rate of about 10 ° C./hour, the hydrazine decomposition reaction is accelerated at the temperature of 90 to 100 ° C. The decomposition reaction raises the pressure in the sample tube to 2 to 4 atmospheres.

(2) Removal of unreacted hydrazine (suction by high vacuum) In order to remove unreacted hydrazine by evaporation under reduced pressure,
100 μl of toluene is introduced as an azeotrope with a second pipette and volatilized at a low temperature. The pressure of the sample tube is a 10 ^-3 ~10 ^-5 P _a by connecting the high vacuum source to facilitate the evaporation scattering is given appropriate vibration. Centrifugal force due to rotation of the turntable (1) is preferably used for this. Such evaporation promotion is performed at room temperature of 30 ° C. for 10 minutes. Then, this non-reacting hydrazine azeotropic removal step is repeated four times. In this case, 4 identical pipettes
For repeated use, wash it each time.

(3) N-Acetylation 100 μm of an alkaline aqueous solution (ammonium acetate) as an N-acetylating agent is added by a third pipette, and further 5 μm of acetic anhydride is added by a fourth pipette. The sample tube is capped in this state, and at about 30 ° C
The acetylation reaction is carried out for 30 minutes. Also in this case, appropriate vibration is applied to the sample tube to accelerate the reaction. This acetylation step is performed twice, but the pipette is replaced each time to prevent contamination.

(4) Removal of unreacted acetylating agent (suction by high vacuum) Acetic acid 50μ was introduced by the fifth pipette, and after covering the sample tube, N ₂ was circulated through this lid unit and vacuum suction was performed. The unreacted N-acetylating agent is removed by the evaporation operation under reduced pressure. Furnace pressure was 10 ^-3 ~10 ^-5 P _a, to facilitate the removal, vibration is given again. This acetylating agent removal process is 30 ℃
For 30 minutes.

(5) Pyridyl amination By a sixth pipette, 40 μ of 2-aminopyridine is added. The pyridyl amination reaction is carried out at 90 ° C. for 1 hour, and appropriate vibration is also given to accelerate the reaction. This temperature rise to 90 ° C is performed at a high speed of about 100 ° C / min.

(6) Removal of unreacted 2-aminopyridine (suction by high vacuum) Triethylamine was added as an azeotropic agent with a seventh pipette, and in this state, the sample tube was capped and vacuum sucked while flowing N ₂ , the sample tube pressure and 10 ^-3 P _a, 20 min 90 ° C.
In order to accelerate the liquid removal, a suitable vibration is applied. This step is performed 4 times.

(7) Reduction 2 μ of borane complex was added as a reducing agent by the 8th pipette, followed by heating at 90 ° C. for 1 hour,
The Schiff base formed on the sugar chain in the sample is reduced.
As a result, the end of the fluorescent reagent attached to the sugar chain is stabilized.

(8) Removal of reducing agent A process of adding 100 µ of water with a ninth pipette and heating at 90 ° C for 5 minutes is repeated 4 times.

As a result, when the reduction is performed, the lifting lid mechanism is activated to lift all the lids on the sample tube, and an alarm indicating the end of the reaction is issued. The total time required for the pretreatment of the sugar chain by the method of the present invention was 14 hours and 25 minutes.

Since the above embodiment is carried out automatically in the apparatus configuration as shown in the figures and described above, it is possible to quickly set the reaction environment (temperature, etc.), to supply the inert gas, and to use the vacuum suction in combination to cause the excess of azeotrope. By reliably removing the reagents, etc., it is possible to shorten the overall time and stabilize the reproducibility of the reaction.

EFFECTS OF THE INVENTION As described above, according to the present invention, it is possible to perform the sugar chain analysis pretreatment step in a stable and high reaction yield by shortening the time by substantially fully automating the step and executing the reaction process reliably. It is what

[Brief description of drawings]

FIG. 1 is a diagram showing an entire apparatus configuration example for use in the present invention, FIG. 2 is a plan view of relevant parts, FIG. 3 is a cross-sectional view of the sample turntable and lid lifting mechanism, and FIG. The figure shows a partially broken side view showing the cooperative relationship between the pipette arm and the pipette. Fig. 5 is a partial plan view showing the arrangement relationship between the pipette arm and the pipette removing member. Fig. 6 shows the relationship between the pipette arm and the pipette removing member. FIG. 7 is a schematic view showing a lid holding mechanism for adsorbing and lifting the lid element of the reagent tube, and FIG. 8 is a partial view showing the lid element adsorbing and holding state of the lid holding mechanism. is there. (1) …… Sample side turntable (2) …… Lifting lid mechanism (3) …… Reagent side turntable (4) …… Pipette arm (5) …… Sample tube (6) …… Support shaft (7) …… Bearing (8) …… Transmission gear (9) …… Elevation transmission mechanism (10), (11) …… Air cylinder (12) …… Electromagnet piece (13) …… Chuck and air inlet (14) …… Cylinder pump (15) …… Motor (16) …… Control interface circuit (17) …… Computer (18a), (18b) …… Control card insertion slot (19) …… Cooling water circulation pipe (20) …… Upper end Open sample tube (21) …… Sample receiving hole (22) …… Upper end open type reagent tube (23) …… Reagent tube receiving hole (24) …… Pipette receiving hole (25) …… Pipeette removing member (26) …… Water cooling jacket (27) …… Heater (28) …… Inert gas supply port (29)… Vacuum suction port (30) ...... pipette

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ikunoshin Kato 3-4-1, Seta, Otsu City, Shiga Prefecture

Claims (1)

[Claims] 1. Pyridylamino is prepared by adding hydrazine to a glycoconjugate such as glycoprotein or glycolipid to decompose it, and then acetylating to isolate the sugar chain, and adding aminopyridine and substantially anhydrous acid to the sugar chain. In the method of labeling with a reducing agent, the sample is provided with a plurality of annularly arranged sample tube receiving holes for supporting an upper-end open type sample tube containing a sugar sample, and the sample tube receiving hole. And a sample-side turntable having a heater and a cooling channel arranged in the vicinity of the annular array, and an array of a lid unit for each of the sample tubes that can be moved up and down immediately above the sample-side turntable. Ascending / descending by arranging an inert gas branch supply line and a vacuum branch suction line opened on the exposed surface of each lid unit into the sample tube A reaction station including a lid mechanism, and reagents such as hydrazine, N-acetylating agent, aminopyridine, substantially anhydrous acid, and reducing agent necessary for isolation of the sugar chain of the sample and fluorescent labeling of the sugar chain And a plurality of reagent tube receiving holes arranged in an annular shape for supporting upper end-opening type reagent tubes respectively accommodating respective azeotropic agents used when removing excess reagents after each reaction by evaporation, and the plurality of reagents A plurality of pipette receiving holes that are arranged concentrically in an annular shape corresponding to the pipe receiving holes, and a reagent-side turntable adapted to support a pipette having a tip at the lower end of the pipette receiving hole; The tip includes an arm having a chuck and air inlet / outlet that engages with the pipette upper end opening supported by the receiving hole, and the arm is provided with the chuck / air inlet / outlet at a predetermined azimuth angle position of the reagent side turntable. At a position corresponding to each upper end opening of the reagent tube supported at another predetermined azimuth position and the upper end opening of the sample tube supported at the predetermined azimuth position on the sample side turntable. Within a range including, and using a sugar chain labeling reaction device equipped with a pipette arm mechanism that can be rotated and moved up and down at each of the positions, to a sample tube supported on the reaction side turntable. For supply of reagents or azeotropic agents, one of the pipettes supported by the reagent side turntable is held and transported by the chuck and air inlet / outlet at the end of the arm, and the tip is inserted into the corresponding test tube. After inhaling the reagent or the azeotropic agent, the sample tube is held on a sample tube supported at a predetermined azimuth position on the sample side turntable. Then, by injecting the inhalation liquid into the tube, heating or cooling to a processing temperature necessary for reaction accompanying reagent injection into the sample tube or azeotropic removal of excess reagent after injection of an azeotropic agent. At that time, the heater and the cooling channel built in the sample side turntable are operated, and when the reagents and the like are azeotropically removed, the sample tube on the sample side turntable is not covered by the lid unit closed. A fluorescent labeling method for sugars, characterized in that an active gas branch supply pipeline and a vacuum branch suction pipeline are operated to perform vacuum suction while feeding an inert gas into the reaction tube.