JP2000083650A - Automatic test equipment for enzyme reaction test - Google Patents

Abstract

(57) [Summary] [PROBLEMS] In an enzyme reaction test, operations such as reagent dispensing, management of reaction time, and extraction of a reaction sample are automatically performed to eliminate variations in reaction conditions and reaction times, thereby improving accuracy. To be able to perform high-quality tests. In addition, it reduces the exposure of human-derived tissues to the experimenter. An automatic dispensing device (1) capable of moving a dispensing probe (19) in each of X, Y, and Z directions and a control device (13). Vial racks 3 and 4 on processing table 2
, A thermomixer 5, a cooler unit 6, and a sample container 7 are arranged. A reagent solution, a common reagent, a degrading enzyme solution, and the like are stored in vials 10, 11, and 12 of each device. The procedure for dispensing and transferring is determined based on the test parameters input to the control device 13, and reagents and the like are dispensed in accordance with the procedure to extract a reaction sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for automatically performing an enzyme reaction typified by a drug metabolism test, and more particularly, to reacting a reagent with a decomposing enzyme under predetermined test conditions and for a predetermined time. The present invention relates to an apparatus for stopping a reaction every time a time elapses and automatically generating a group of metabolic reaction samples to be used for subsequent analysis.

[0002]

2. Description of the Related Art Metabolic tests of this kind are frequently performed during the development of new drugs, and most of the test operations are performed manually. The test procedure for each sample is
All are common. Therefore, the operator simply repeats the dispensing operation of the reagent and the decomposing enzyme solution and the like, and the transfer operation of the reacted sample. However, in most cases, a plurality of test reagents are simultaneously tested while shifting the time, so that work such as dispensing must be performed while controlling the time for each sample.

[0003] In the test apparatus of the present invention, the dispensing operation of reagents and reagents is performed by an automatic dispensing apparatus having arms reciprocating in the X, Y and Z axes. The product itself is commercially available and known (Gilson, Liquid Handler, Model No. 215).

[0004]

As described above, conventionally, an enzymatic reaction test is performed by a large number of workers. Therefore, one test requires much time. Since a researcher or a skilled worker having the same technology as the researcher is indispensable, human costs are high and the throughput per unit time / day is limited. Since it is a manual operation, variations in the dispensed amounts of reagents, variations in the time until the reaction is stopped, and habits of individual workers cannot be avoided, so that the test results tend to vary. There is room for human error, and in such cases, the test itself may have to be repeated from the beginning. When a reagent is dispensed or the like, an operator may be exposed to a living tissue derived from a human.

An object of the present invention is to automatically perform an enzyme reaction test, thereby reducing the cost and time required for the enzyme reaction test, and reducing the cost for new drug development and the like. Another object of the present invention is to eliminate variations in reaction conditions and reaction times by automating operations such as reagent dispensing and time management, and to always obtain constant test results, and thus to obtain highly accurate enzyme reactions. An object of the present invention is to provide an automatic test device that can perform a test. Another object of the present invention is to perform an enzymatic reaction test in an unmanned state as required, so that the enzymatic reaction test can be performed efficiently, and the test is carried out using a degrading enzyme in which contact or exposure is a problem. Even in such a case, an object of the present invention is to provide an automatic test apparatus capable of performing an enzyme reaction test safely and reliably.

[0006]

The automatic test apparatus of the present invention comprises an automatic dispensing device 1 and a control device 13 for controlling the operation of the automatic dispensing device 1. The automatic dispensing device 1 includes a probe moving unit that moves the dispensing probe 19 to a predetermined position, and a positive pressure or a negative pressure applied to the probe 19,
Main body 15 that controls suction holding and discharge of sample etc.
And At least one vial rack 3.4 for standing and holding a group of vials 10 and 11 and a thermomixer 5 for shaking and stirring while keeping a plurality of reaction vials 9 at a predetermined temperature state, in the vicinity of the automatic dispensing apparatus 1.
Then, the cooler unit 6 for keeping the plurality of reagent vials 12 cool at a predetermined temperature state and the sample container 7 are stereoscopically arranged. While moving the probe 19 according to a predetermined procedure based on the test parameters previously input to the control device 13,
A sample is dispensed and transferred between each vial 9, 10, 11, 12 and the sample container 7.

Specifically, the probe moving means XY
X-axis arm 16 and Y-axis arm 1 that move in the Z axis directions
7, a plurality of vial racks 3, 4, a thermomixer 5, a cooler unit 6, and a sample container 7 are arranged on the processing table 2.

[0008]

In the enzyme reaction test, a reagent vial 12 containing a necessary reagent solution and a decomposing enzyme solution is placed in a cooler unit 6.
The process is started by placing the reaction vial 9 and the other vials 10 and 11 in the upper predetermined position and loading them into the thermomixer 5 and the respective vial racks 3 and 4 and storing the required amount of sample in the sample container 7. This preparation is performed manually. next,
Activate the automatic dispensing device 1 and dispense the sample into each vial 9.1
Dispense into 0, 11, and 12, and complete the pre-stage processing. In this state, the decomposing enzyme solution is added to the reaction vial 9, the thermomixer 5 is operated, the reaction sample in the reaction vial 9 is mixed, and the first-stage reaction (temperature equilibration) is performed for a certain time. Thereafter, a start reagent is added to start a metabolic reaction. When a certain time has elapsed, a part of the reaction sample in the reaction vial 9 is taken out and transferred to the vial 11 in which the reaction stopping reagent has been dispensed in advance. Thereafter, the metabolic reaction sample group can be generated for each elapsed time by repeatedly performing the transfusion of the reaction solution every time a fixed time elapses.

Procedures such as dispensing and transfer are determined in advance when test parameters are input to the control device 13,
The time management of each reaction vial 9 and the procedure setting for avoiding mutual interference between dispensing operations are controlled by a program incorporated in the control device 13.

[0010]

As described above, according to the automatic test apparatus of the present invention, the need for human labor is limited to only the initial preparation work.
A series of metabolic reaction tests can be performed automatically. Automatic dispensing device 1
Since the dispensing and transfer are performed mechanically using the method, and the timing is precisely defined by the controller 13, there is no variation in test conditions and reaction time, and there is no room for erroneous operation. Only the accuracy of the enzyme reaction test can be improved, and the reliability of the test result can be improved. After the preparation work is completed, a series of metabolic reaction tests can be performed automatically without the need for human intervention, so that a series of tests can be performed efficiently, significantly reducing the cost required for testing, and reducing contact and exposure. Enzyme reaction tests can be performed safely even when dealing with degrading enzymes that pose a problem.

[0011]

1 to 3 show an embodiment of an automatic test apparatus according to the present invention. In FIG. 1, an automatic test apparatus includes an automatic dispensing apparatus 1 and a plurality of vial racks 3.4, a thermomixer 5, a cooler unit 6, a sample container 7 and the like, each of which is localized on a processing table 2.
Four groups of vials 9 standing upright by these devices 3 to 7
10. The control unit 13 controls the operation of the automatic dispensing device 1 and the like.

The automatic dispensing apparatus 1 includes a main body 15 disposed adjacent to one side end of the processing table 2, an X-axis arm 16 protruding from the main body 15 along a rear edge of the processing table 2. ,
A Y-axis arm 17 reciprocally operated by the X-axis arm 16 left and right, and a Z-axis arm 18 reciprocated by the Y-axis arm 17 back and forth.
A hollow needle-like dispensing probe 19 is arranged at the lower end of the front surface of a Z-axis arm 18 that reciprocates up and down with respect to the axis arm 17. Inside the main body 15, a syringe pump and a three-way volve for dispensing or sucking a sample accurately from the tip of the probe 19, washing the inside of the probe 19, or supplying distilled water are arranged. By controlling the operation of these devices, the probe 19 can suck and hold a sample or the like, or discharge the held sample or the like. The probe 19 and the main body 1
5 is connected via a flexible tube, but is not shown in FIG. Each of the above arms 16
The probe 19 can be moved to an arbitrary position on the processing table 2 by linking the 17 and 18 to operate, and dispensing and transfer between the vials 9 to 12 and the sample container 7 can be performed.

The vial rack 3 adjacent to the thermomixer 5 among the two vial racks 3 and 4 contains a compound solution, that is, a vial 1 containing a solution of a reagent.
A group of 0 is loaded. The vial rack 4 located on the right side of the processing table 2 is loaded with a group of vials 11 for receiving a metabolic reaction sample, which is transferred from the reaction vial 9 every predetermined time. The number of vials is prepared by multiplying the number of kinds of reagents by the number of metabolic reaction samples taken out of the reaction vial 9 at different reaction times, and the number of vials therein is for stopping the reaction of the degrading enzyme. Of the stop reagent (acetonitrile) has been dispensed in advance. For example, for three kinds of reagents, 0 minutes, 5 minutes, 30 minutes, and 6 minutes from the start of the metabolic reaction.
When two reaction samples are to be taken out every 0 minutes and 120 minutes, 30 vials 11 are prepared.

The thermomixer 5 is a commercially available product, and a plurality of reaction vials 9 are loaded in a reaction rack 21 at the upper part of the main body. The upper end of the reaction vial 9 is closed with a rubber cap 9a, but the probe 19 can pierce the rubber cap 9a to dispense a sample or the like into the reaction vial 9, or to transfer the metabolic reaction sample in the reaction vial 9 Can be taken out. The reaction vial 9 is kept at a predetermined temperature by a heater in the reaction rack 21,
Makes a circular motion along a fixed locus, whereby the reaction sample in the reaction vial 9 can be shaken and stirred.

As described above, the probe 19 moves the sample and the reaction sample into and out of each reaction vial 9, but stops driving the reaction rack 21 only during that time. However, since the position when the reaction rack 21 stops is not always constant, the specific reaction vial 9
, It becomes difficult to put a sample and a reaction sample in and out. In order to cope with such a situation, the reaction rack 21 is driven along a circular locus having a diameter value slightly smaller than the opening diameter of the reaction vial 9, and the shift amount of the center of the reaction vial 9 when the reaction rack 21 stops. Is smaller than the radius value of the opening with respect to the reference value. That is, only by lowering the probe 19 at the reference position, a sample or the like can be reliably moved into and out of the reaction vial 9 arranged at that position.

The cooler unit 6 includes a container body 6a containing a cold storage material and a lid 6b for standing and holding the reagent vial 12. The cooler unit 6 cools the reagent and the decomposing enzyme solution contained in the reagent vial 12 at a low temperature. Keep cool. Each reagent vial 12 contains a common reagent such as a phosphate buffer, a glucose 6-phosphate solution, a glucose 6-phosphate dehydratase, a reaction initiation reagent, and a decomposing enzyme solution. The sample container 7 contains a stop reagent.

The control device 13 is composed of a general-purpose computer system, and incorporates a program for controlling the operation procedure of the automatic dispensing device 1 as time passes. Before conducting the test, the type of reagent and its storage location,
By inputting test parameters such as the time to be sampled, the storage position of each sample, and the dispensed volume using, for example, a keyboard, the procedure of the metabolic reaction test is determined and stored. Simultaneously with the input of the parameters, vials accommodating the respective sample groups and the degrading enzymes are arranged at predetermined positions.

[0018]

[Test Example] Using the above automatic test equipment, cytochrome P
Warfarin, one of the 450 representative substrates
falin) metabolic reaction test. As the microsome to be used, a solution of rat liver microsome (concentration: 0.5) was used.
mg / ml) was used. The test is roughly classified into a pre-process in which each sample is dispensed, and a reaction process in which a reaction sample is taken out of the reaction vial 9 every time a predetermined time elapses to stop the metabolic reaction.

(Pre-stage processing) As described above, after the preparation work and the input of the test parameters are completed, the automatic dispensing apparatus 1 is operated to dispense a sample or the like in the following procedure.
The concentration of the sample and the like was adjusted as follows. Phosphate buffer ... 1 mol / l Glucose 6-phosphate ... 40 mmol / l Glucose 6-phosphate dehydratase ... 1000 units / ml Reagent solution ... ... 12 μmol / l reaction-initiating reagent ..... Mixture of a solution of reduced nicotinamide adenine dinucleotide (5.5 mol / l) and a solution of nicotinamide amide adenine dinucleotide phosphate (8 mol / l) liquid.

980 μl of the reaction stopping reagent (acetonitrile) is taken out of the sample container 7 and dispensed into each vial 11 loaded in the right vial rack 4. From the vial 12 on the cooler unit 6, 120 μl of the phosphate buffer was taken out and the reaction vial 9 was removed.
Dispense into Further add 490 μl of distilled water to each vial 9
Inject into 100 μl of glucose 6-phosphate solution is taken out from another vial 12 on the cooler unit 6 and dispensed into each reaction vial 9. 10 μl of glucose 6-phosphate dehydratase solution is taken out from another vial 12 on the cooler unit 6 and dispensed into each reaction vial 9. From the vial 10 loaded in the vial rack 3 on the left side, 100 μl of the reagent solution is taken out and dispensed into each reaction vial 9. Thus, the first-stage processing ends. The probe 19 is washed with water as needed.

(Reaction Treatment) Microsomes (decomposing enzyme solution) are taken out from the reagent vial 12 on the cooler unit 6 by 120 μl and dispensed into each reaction vial 9. Thereafter, the thermomixer 5 is started to start stirring. The reaction vial 9 is kept at 37 ° C. by the heater in the reaction rack 21.

From the reagent vial 12 on the cooler unit 6, 200 μl of the reaction starting reagent solution was taken out, and
Dispense every reaction vial 9 where 5 minutes have passed since the end of the above dispensing. In each reaction vial 9, after dispensing the reaction starting reagent solution, the probe 19 is put on the cap 9 a.
Stir for several seconds with the probe removed.
Is pierced into the reaction vial 9, 200 μl of the reaction sample is aspirated, and 100 μl is dispensed into two vials 11 on the right vial rack 4. This is the reaction sample 0 minutes after the start of the test, and the number of reaction samples twice as many as the number of the reagents is obtained on the vial rack 4.
The probe 19 is washed with water for each reaction vial 9.

At 10 minutes after the addition of the reaction starting reagent solution (described above), 200 μl of the reaction sample was aspirated from each reaction vial 9 in the same manner as above, and the two vials on the right vial rack 4 were aspirated. 100 μl for 11
Dispense each. Since a reaction stopping reagent is dispensed in each vial 11 in advance, the enzymatic decomposition of the reaction sample is stopped. The same sampling is performed for each reaction vial 9. 30 minutes, 60 minutes, 12 minutes from the addition of the reaction start reagent solution
Every time 0 minutes elapse, the same sampling as described above is performed, and the enzyme reaction test is completed.

Thereafter, the remaining amount of the test substance contained in the obtained reaction sample is measured by using a high performance liquid chromatography and a mass spectrometer to determine the degree and rate of metabolism of the test substance by the decomposing enzyme. And so on. Note that the dispensing procedure of the pre-stage process is not necessarily in the order described, and can be changed depending on the vial arrangement or the positional relationship with the reaction vial. The test parameters such as the volume of the reagent to be dispensed, the number of times the reaction sample is collected, and the reaction time can be arbitrarily changed according to the purpose and conditions of the test.

In order to evaluate the reliability of the automatic test apparatus, the present inventors conducted tests only by hand by a skilled researcher,
The test results were compared when the same test was performed by an automatic test apparatus. As a result, it has been confirmed that the test results obtained by the apparatus of the present invention do not vary in the metabolic tendency of each reaction sample, and that a highly accurate test can be performed.

In the above embodiment, the X-axis arm 16 is
Although the automatic dispensing apparatus 1 protruding orthogonally from FIG. 5 has been illustrated, it is not necessary. For example, the X-axis arm 16 may be housed inside the main body 15. The layout of each device 3, 4, 5, 6, 7 on the processing table 2 can be arbitrarily changed. In addition, the number of vial racks 3 and 4 can be increased as necessary, and the adjacent boundaries between the vials 10 and 11 loaded in both racks 3 and 4 do not need to be provided for each rack.

[Brief description of the drawings]

FIG. 1 is a perspective view of an automatic test apparatus.

FIG. 2 is a perspective view of a thermomixer.

FIG. 3 is a plan view showing an arrangement of devices on a processing table.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Automatic dispensing apparatus 2 Processing table 3 Vial rack (left) 4 Vial rack (right) 5 Thermomixer 6 Cooler unit 7 Sample container 9 Reaction vial 10 Vial 11 Vial 12 Reagent vial 13 Control device 15 Main body 16 X-axis arm 17 Y axis arm 18 Z axis arm 19 Probe

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G058 AA09 BB02 BB07 BB09 BB12 CC08 EA02 EA04 EA08 ED02 FA03 FA07 GE06 4B029 AA07 AA27 BB16 BB20

Claims (2)

[Claims] 1. An automatic dispensing device 1 and a control device 13 for controlling the operation of the device 1. The automatic dispensing device 1 is a probe transfer means for moving a dispensing probe 19 to a predetermined position. And a main body 15 for applying a positive pressure or a negative pressure to the probe 19 to control suction holding and discharge of a sample or the like, and a group of vials 10. 1
At least one vial rack 3
4, a thermomixer 5 for shaking and stirring while keeping a plurality of reaction vials 9 at a predetermined temperature state, a cooler unit 6 for keeping a plurality of reagent vials 12 at a predetermined temperature state, and a sample container 7 in a stereoscopic arrangement. Dispensing a sample between each vial 9, 10, 11, 12 and the sample container 7 while moving the probe 19 in a predetermined procedure based on the test parameters previously input to the control device 13. , And an automatic test apparatus for enzyme reaction test, which performs transfusion. 2. An X-axis arm 16, a Y-axis arm 17, and a Z for moving the probe moving means X, Y, and Z in respective axial directions.
Consisting of a shaft arm 18, a plurality of vial racks 3, 4, a thermomixer 5, a cooler unit 6, and a sample container 7
2. The automatic test apparatus for an enzyme reaction test according to claim 1, wherein is disposed on the processing table 2.