JP2002509255A - Method and apparatus for performing a number of similar biological / chemical tests on a microanalytical scale - Google Patents

Abstract

(57) Abstract: To perform a number of similar chemical / biological tests on a microanalytical scale with a large number of compounds from a compound library, put those compounds into recesses in a test plate, The plates are tested in their wells by processing at various processing stations (12, 12.1, 20). The plates are stored in a storage container (10) as a plate stack (1). The plate stack (1) is stored using the stack operating means (31) and removed from its storage location. Each of the plate stacks is placed on a transport means (12) and transported individually by the transport means (12) along a rail network (33/34/35) to a processing station (12, 12.1, 20). The plates are separated from the stack for processing when they reach the processing station (12, 12.1, 20) and then re-entered into the plate stack (1). Each processing station (12, 12.1, 20) includes at least one plate separation element (32) to perform this separation function. Each plate stack (1) has a plurality of plates and a coating plate stacked on one another. Advantageously, all stacks have substantially the same size.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The invention relates to the method according to the preamble of the first independent claim, which belongs to the field of automated microanalysis. The method is useful for performing a number of similar biological / chemical tests (assays) on a microanalytical scale. To perform such an assay, a library of test compounds is used, and all or a selection of the compounds present in the compound library are subjected to the same test. The method can also be used to select compounds from a compound library. The invention also relates to a device according to the preamble of the corresponding independent claim.

[0002]

[Prior art]

A compound library, for example, a library of synthetic organic molecules, is a large number of plates, each with multiple wells, each well containing one of the compounds dissolved in a standard solvent in an amount sufficient for a large number of trace tests. Known to be included. In the following description, a plate having a well containing such a large amount of compound is referred to as a “library plate”. To perform a series of tests, use the compounds in the library plate, remove the test amounts of each compound from the wells in those library plates, and transfer them to the empty wells in the new plate. Prepare a "plate". Test plates containing only one compound in each well and test plates containing a mixture of compounds in each well can be prepared in substantially the same manner. Library plates and test plates can be stored for longer periods of time if they prevent or at least greatly reduce contact with air or oxygen, respectively.

[0003] Assaying compounds in a compound library in a manner as described immediately above, for example, helps to find compounds with particular pharmacological effects. To perform these tests, each test plate is typically processed in a plurality of successive processing steps at a plurality of processing sites. This treatment is carried out, for example, in three steps: adding one or several liquid reagents to cause a reaction with the test compound; incubating for a given time in a given atmosphere and a given temperature. Adding additional liquid reagents to stop the reaction and / or for the color reaction; measure certain properties (color, fluorescence, radiation, etc.) of the reaction mixture. Steps (eg, filtration or centrifugation) for separating the reaction mixture and removing a portion of the reaction mixture may be provided.

Assays such as those described above typically use standardized plates (eg, 96 (8 × 12) wells) (micro test plates) that stabilize the plates when stacked. Means (e.g., protruding edges on the upper surface of the plate and corresponding grooves on the lower surface of the plate). The processing stations required to fill the wells of the plate and perform the assay and the means of transporting the plate from one processing station to the next are controlled by a central control unit. The processing path from the first processing station to the last processing station that each plate must handle for the particular test to be performed may vary from test to test. Typically, each plate is moved back and forth along the processing path so that there are multiple plates along the processing path at any one time.

[0005] The transport of individual plates from the reservoir, through the first processing station and subsequent stations, and possibly back to the reservoir, is usually carried out using a suitably controlled robot arm. All processing stations and storage vessels are located within the reach of the same. This robotic arm, which transports all plates on a processing path, performs a number of different transport steps per unit of time, each of which transports a plate along a portion of the processing path (eg, one Transported from one processing station to the next. Obviously, the robot arm must perform a series of extremely complex movements and it must perform a number of empty movements (movements without plates) during the actual transport phase. This robot arm proves to be the part of the test system that limits the speed of the assay.

[0006] The number of compounds contained in one of the above compound libraries needs to be extremely large and constantly increasing in order to make it sufficient for comprehensive testing. Compound libraries containing tens of thousands of compounds are very common and these are being further expanded. With the library, the number of tests in a series of tests also increases.
This means that it is becoming increasingly important to have a test system that allows as high a throughput as possible to perform such a large number of tests in a practical time. There is a need for means and methods that can not only gradually increase the efficiency of each scheme, but also bring it up to different dimensions.

[0007]

[Means for Solving the Problems]

For the reasons set out above, it is an object of the present invention to make it possible to carry out a considerably larger number of tests than can be carried out with known methods and devices, and without substantially increasing the number of devices (processing stations). It is an object of the present invention to provide a method and a device which enable an increase in efficiency. The method and the device according to the invention allow the use of a known micro-test plate with 96 cavities and without the use of a parallel working station, one day per day.
It is possible to achieve a throughput of about 000 tests. However, because the method and the apparatus are not limited to this particular type of standardized plate, a further increase in throughput can be achieved by using a plate with more depressions. Furthermore, it is possible to carry out the method using known processing stations.

[0008] The basic concept of the present invention is substantially to treat stacked plates (stacks of plates) rather than to treat individual plates as has been done in the past, wherein the stack of plates to be treated is Advantageously, all have the same size (ie, all contain the same number of plates). In the method of the present invention, all plates (test plates, empty plates and library plates) are stored as stacks, operated as stacks and transported as stacks. The plates are separated from the stack only for processing at the processing station, processed as individual plates, and re-inserted after processing. Such stack management simplifies plate traffic on the processing path and significantly increases throughput compared to known plate management.

A stack of plates includes, for example, ten plates. In this stack, each plate covers the underlying plate, and at the top of the stack is a cover plate that covers the top plate. This cover plate has no other functions. Most of the time, the plates in the stack cover each other during the assay, so no separate lid is needed on the plates. This is a further advantage of the method of the invention.

In order to carry out the method according to the invention, a plurality of processing stations are provided, each processing station having at least one means for separating the plates from the stack, ie removing one plate from the stack and processing the plates. Equipped with a means to place and place the plate back into the stack after processing. In addition, a plurality of transports, each of which can individually transport at least one of the stacks of plates along the processing path, a stack of plates removed from the storage container and placed on the transport, Stack operating means for unloading the stack from the transport means and returning the stack to the storage location. The device of the present invention, comprising a processing station with plate separation means, a stack transport means, a storage container and a stack manipulation means, allows the assay to be performed fully automatically, as is often the case with similar devices with plate management. Controlled by a central computer.

[0011] Advantageously, any particular plate has a particular position in a particular stack and is returned to that position after any separation. It is also advantageous that a particular stack of plates (library plates) used several times each have a particular storage position in the storage container, and that stack is always returned to that position. Separation means are designed so that any plate can be removed from the stack and returned to its position so that the series of tests can be performed without loss of freedom compared to performing tests by plate management. Should be. Furthermore, the storage container and the stack operating means should be designed such that each stored stack can be removed from or put into a storage location independently of the other stacks present in the storage location.

However, the condition that the position of the plates in the stack is substantially unchanged and that the position of the stack in the storage is unchanged is not essential for the method according to the invention. With the help of appropriately complex management software, it is also possible to locate ever-changing "disordered" plates and stacks and extract them for processing.

In any case, the storage container has arbitrary access to the entire stack, and is completely free to select the stack from the storage container when loading the compounds into the plates and performing the tests; It should be designed to be completely independent of storage order. This does not apply to individual plates in the storage area. For example, if only one plate in a stack is to be processed, the entire stack is removed, transported, and only the plates to be processed are separated at the processing station. This means that any access to the individual stacks must be guaranteed for storage and retrieval, and any access to the individual plates for separation of the plates from the stack. I do.

For storage of the stacks, for example, storage containers having draw-out shelves are used, on which shelves are pulled out with the aid of stack operating means comprising corresponding graspers. The stacks are placed apart from each other so that they can be removed from the shelves and / or each stack can be placed in an empty position on such shelves. For transporting the stack, for example, individual transport means are provided which can be moved on a rail network comprising controlled points. These vehicles are loaded with at least one stack by means of a stack operating means, which transports the one stack in a predetermined order, which is predetermined for a specific series of tests. Transport along the test path leading to the processing station.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the method and the device of the present invention will be described in more detail with reference to the accompanying drawings.

FIG. 1 shows a stack 1 of plates 2 with depressions 3 as used in the method of the invention. The stack 1 holds a coating plate 4 at its top, which has no depressions as shown or has empty depressions, the sole function of which is that of the second plate from the top. Is to cover. The covering function for all other plates in the stack is taken over by the plate immediately above in the stack.

The plate 2 is provided with stacking means that are compatible with each other on its upper and lower surfaces (for example with a tongue-shaped edge 5 on the upper surface and a corresponding groove (shown) so that the stack is relatively stable. No) on the bottom). The surface 6 on which the stack is placed,
That is, for example, the shelves of the storage containers, the support areas of the transport means and the processing positions of the processing stations are advantageously equipped with the same stacking means so that the stack can be easily and accurately placed in a defined position. It is. Also, for the bottom plate of each stack, there may be different positioning means that work together with the corresponding means of the support surface, or a special bottom positioning where each stack has no other function as well as the covering plate. Plates can also be included.

FIG. 2 is a highly schematic representation of an example of a test performed according to the method of the present invention. The movement of the stack or plate to be performed in successive phases a-i is indicated using arrows, and a series of phases may be repeated. The individual aspects are as follows: a. In a storage container 10 having a shelf 11, the stack 1 of test plates is stored together with the other stacks of test plates. To test the test compound in the wells of the test plate of Stack 1, storage container 10 is opened and shelf 11 is withdrawn; b. Stack 1 is grasped by stack operating means (not shown) and lifted from shelf 11; c. Stack 1 is loaded on transport means 12 and transported to processing station 12; d. One plate of the stack 1 (e.g. immediately below the coating plate) is placed at the processing position 13 of the processing station 12 and processed with the aid of a plate separating element (not shown). E. The plate is removed from the processing station with the aid of a plate separating element (not shown) and returned to the stack 1; if necessary, repeat steps d and e for the other plates in the stack 1; f. Stack 1 is transported further; if necessary, repeat steps d, e and f at the next processing station; g. The stack is transported back to the storage container 10 and returned to the shelf 11 using stack operating means (not shown); h. Shelf 11 is pushed back into storage container 10; i. The storage container 10 is closed.

FIG. 2 shows a processing path of only one stack 1. However, when performing a continuous test, multiple stacks may be used to ensure that the stack is constantly being removed from or returned to one or more storage bins, and that the plates are processed one after another at the processing station. The stack is moving continuously along the processing path, and the loaded vehicle can be held in a buffer ahead of the processing station. The unloading vehicle is reloaded with stacks from the same or different storage containers.

FIG. 3 illustrates a process involving two plates or two stacks of plates at once, such as the preparation of a library plate (new generation) in which compounds selected from a mother library are placed on that plate, The preparation of a test plate using a library plate is schematically illustrated as in FIG. The removal of the stack of plates from the storage container and the return of the stack of plates to the storage container are not shown. This is the same as described above with respect to FIG.

In FIG. 3, the stack and plate movements performed in the following successive phases k to p are indicated by arrows: k. The stack of library plates 1.1 and the stack of empty plates 1.2 are transported to the processing station 20 on transport means 12, respectively. The processing station 20 is equipped for the removal of liquid from the well and for the addition of liquid to the well (liquid operating station), and the processing station 20 has two plate separating elements (not shown). Is equipped; l. One library plate is removed from stack 1.1 and placed in a processing position, and the compound is removed from at least one well; m. The library plate is returned to stack 1.1; n. One empty plate from stack 1.2 is removed and placed in the processing position, and at least one compound removed from the library plate is placed in the well, the empty plate becoming a test plate; o. The test plate is returned to stack 1.2; if necessary, repeat steps l-o with plates removed from stack 1.1 and / or stack 1.2; p. Each stack is transported therefrom, and stack 1.1 is returned, for example, for storage, and stack 1.2 is processed directly at a processing station along a processing path, or also returned and stored. Is done.

FIG. 3 may give the impression that the movement of stacks 1.1 and 1.2 takes place simultaneously. This is not at all a requirement for the method of the invention. For example, to prepare a second generation library plate containing only selected compounds from the mother library, for example, compounds from several mother library plates may be transferred to a single empty plate. The movement of the stack to, from, and from the processing station is not simultaneous.

FIG. 4 is a highly schematic representation of an example of an apparatus for performing the method of the invention as described in connection with FIGS. The apparatus includes a storage area on the left and a processing area on the right.

The storage area is provided with a plurality of storage vessels 10 having draw-out shelves, which can be selectively moved, for example, along rails 30 (dashed lines) to the area of the stack operating means. . The stack operating means is shown as a double-headed arrow connecting the storage area with the processing area. It is expedient to provide a plurality (for example two) of stack operating means 31 so that the plates removed from several storage containers can be operated simultaneously. In the area of the stack operation means 31, a means for opening the storage container 10 and pulling out the shelf 11 is also provided.

The processing area comprises a processing station 12 having one plate separating element 32,
A plurality of processing stations 20 having the above-described plate separating elements 32 are included. The plate separating element is shown as a double-headed arrow connecting the stack transport system with the processing station 12 or 20. The stack transport system consists of a rail network 33 (dashed lines) with branches with controlled points of control. This rail network is advantageously closed by itself and leads to the area of the stack operating means 31 and the plate separating element 32, and it is convenient to design a rail network having a detour 43 in all these areas. It is.

The stack transport means 12 can be moved individually along the rails of the rail network. Rail networks can be used for transportation in only one direction (eg, clockwise), in which case the possible processing paths differ in that they bypass specific processing stations. However, the rail network may include a transverse link 35 and have areas used in both directions, for example to allow for a reduced processing path having a different processing order or a reduced return of stacks of library plates. .

Most of the tests performed involve, as mentioned above, the step of incubating at a given temperature under a given atmosphere. It is particularly advantageous to incubate the plates as a stack using the method of the invention. However, because the stack of plates is relatively bulky, when the stack is placed in an environment at a nominal temperature, the delay time for different locations within the stack to reach this nominal temperature is quite different.
Therefore, it is proposed to preheat the stack before incubation. This is done by successively preheating the separated plates, for example by placing the plates on a metal surface at a nominal temperature one after another for a short time. The preheated plate is returned to the stack, which is then transported to the incubator. The incubator is advantageously a continuous incubator, through which at least part of the transport system passes.

In FIG. 4, one of the treatment stations is equipped as a preheating station (12.1), downstream of which is a continuous incubator 36, in which a plurality of stacks on a transport means are provided with a predetermined temperature. When the dwell time has passed, the stacks pass at such a speed that they reach the exit.

In another embodiment, a processing station for preheating or preservation includes an inlet plate separating element and an outlet plate separating element, and an internal plate transport means for transporting individual plates from the inlet to the outlet. May be equipped. In such a case, each plate is individually fed to the internal transport by the first separating element, transported from the inlet to the outlet while preheated or incubated, and then individually transported by the second separating element at the outlet. To be stacked again.

FIG. 5 is a schematic diagram of an example embodiment of a plate separation element. This is schematically illustrated in FIG. 4 by a double-headed arrow 32, in order to transport the stack 1 from the transport device to the working position of the processing station, to remove the plate 2 from the stack 1 and to remove it from the processing station. It is used in the device according to the invention for placing it in position 13, returning it to the stack 1 after processing, and loading the stack 1 again on the transport device. The plate separation element is shown in three different functional aspects, called a, b, c.

The plate separating element includes a stack shovel 40 and a stack sled 41. These can be moved to the following positions by the corresponding drive and control means: a. The stack shovel 40 is moved at a transfer position 42 beneath the stack 1 being prepared on a vehicle (not shown). Stack shovel 40 moves stack 1 from transfer position 42 to work position 43 (horizontal double-headed arrow); b. Stack excavator 40 is moved to a height such that the plate removed from the stack is at the same height as stack sled 41 (vertical double-ended arrow); c. The plate 2 to be removed is moved to the processing position of the processing station with the help of the plate sledge 41 (horizontal double-headed arrow).

To remove the plate 2 from the processing position 13, reassemble the plate into the stack 1, and place the stack on the transport, the steps shown above are performed in reverse.

FIG. 6 shows a means for cooperating with the vertical movement of the stack shovel in the working position to arrange the stack 1 placed on the stack shovel 40 for removal or to return the plate 2 ′. I have. FIG. 6 is a view of the working position 43 of the stack shovel 40 shown in FIG. 5 as viewed from the left side. Stack 1 to remove plate 2 '
Those means for interacting with the stack shovel include, in addition to the stack shovel, two upper and lower rotating support component pairs 50 and 51, which are individually activated to rotate from the holding position to the rest position. When rotated to the holding position, each support piece of each pair 50 or 51 is placed on the stack 1 from the opposite side below a plate 2 or 2 ′ having a corresponding height on the stack shovel 40.

The lower support component pair 51 is arranged at such a height that the plate placed thereon can be grasped by a plate sled (not shown).

FIG. 6 shows two functional aspects (referred to as a and b) of the support component pair 50 and 51. They are as follows. a. The stack shovel 40 lifts the stack 1 so that the plate 2 ′ is at the same height as the upper support component pair 50. The upper support component pair 50 is rotated from the rest position to the holding position, while the lower support component pair 51 remains in the rest position. b. The stack shovel 40 is lowered slightly so that there is a gap between the plate on the upper support component pair 50 and the lower part of the stack. The lower support part pair 51 is then rotated into the support position such that the plate 2 'to be removed rests on these support parts and the stack shovel is lowered slightly. Thus, the plate 2 'to be withdrawn can be removed from the stack 1 by a plate sled (not shown) and separated from the adjacent plate in such a way that it can be re-integrated into the stack after processing. .

Using the means shown schematically in FIG. 6, it is also possible to create a gap in the stack to put the plates therein, or to close the gaps in the stack caused by removing the plates. Is possible.

The stack shovel 40, the plate sled 41, and the pair of support components 50 and 51 shown in FIGS. 5 and 6 are driven by appropriate driving and control means according to their functions. Those skilled in the art will be able to ascertain the necessary drive and control means without difficulty, and need not be described in detail herein.

[Brief description of the drawings]

FIG. 1 illustrates a stack of plates as used in the method of the present invention.

FIG. 2 is a schematic diagram illustrating tests performed according to the method of the present invention.

FIG. 3 is a schematic diagram illustrating the preparation of a test plate from a library plate or the preparation of a new generation library plate from an old generation library plate according to the method of the present invention.

FIG. 4 is a schematic view showing an embodiment of the apparatus of the present invention for performing the method of the present invention.

FIG. 5 is a schematic diagram of an embodiment of a plate separating unit.

FIG. 6 is a diagram showing details of a plate separating means.

[Procedure amendment]

[Submission date] September 19, 2000 (2000.9.19)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW [Continued from summary] with multiple plates and coated plates You. Advantageously, all stacks have substantially the same size.

Claims (20)

[Claims] 1. A large number of different compounds are placed in the depressions (3) of the plate (2), the compounds are stored in the depressions (3) of the plate (2) and the plates (2)
By transporting the compound to a series of processing stations (12, 12.1, 20) and treating the compounds in the depressions (3) of the plate (2) at the processing stations (12, 12.1, 20). A method for performing a number of similar chemical / biological tests wherein the compound in the cavity (3) of (2) undergoes a test reaction and measurement, wherein the plate (2) is a plate stack (1). Stored and operated as plate stacks (1) and transported as plate stacks (1), where each plate stack (1) is a stack of several plates (1) and a top plate (1) A coating plate (4) placed thereon, and a processing station (
12, 12.1, 20) wherein the individual plates (2) are removed from the stack (1) and returned to the stack (1) after processing. 2. Each plate (2) removed from a plate stack (1)
Are returned to the same location of the same stack (1) after processing. 3. The method according to claim 1, wherein a specific storage location in the storage container is assigned to each stack. 4. The method according to claim 1, wherein the plate (2) is a standardized micro test plate. 5. A processing station (12, 12.1, 2) from a storage area along a rail (33/34/35) and a tissue network at a point of inversion.
0) and from one processing station (12, 12.1, 20) to the next processing station (12, 12.1, 20) by means of individually driven transport means (12). The method according to any one of claims 1 to 4, characterized in that it is characterized in that: 6. The method as claimed in claim 1, wherein the plates are separated for preheating and are stored immediately after the preheating as a stack of plates. the method of. 7. The plate stack (1) for incubating comprises a continuous incubator (36).
7. The method of claim 6, wherein the method is transported through. 8. A means for storing plates (2), a plurality of processing stations (
12, 12.1, 20) and plate (2) at processing station (12, 12.1).
, 20) at the processing site (13), and the plate (2) from the storage location to the processing stations (12, 12.1, 20) and to each processing station (12, 12.1, 20).
, 12.1, 20), means for transporting between the two.
An apparatus for carrying out the method according to item 1, wherein the means for storing the plates (2) is a stack storage in which the stacks (1) of plates can be removed and moved in a freely selectable order. The means for transporting (2) is a stack transport system for individually transporting the plate stacks (1), the device removing the stack from the storage location, storing the stack again, and transporting the stack by transport means (
12) further comprising at least one stack operating means (31) for loading and unloading from it, and each processing station (12, 12.1, 20).
) Removes the freely selectable plate (2) from the plate stack (1) and arranges at least one plate separating element (32) therefor for returning the plate (2) to the plate stack (1). ). 9. The means for storing the plate stack (1) is a drawer-type shelf (
9. Device according to claim 8, characterized in that it comprises a storage container with 11), on whose shelves (11) the plate stacks can be arranged separately from one another. 10. Device according to claim 9, characterized in that a plurality of storage containers (10) are arranged on the rail (30) so that they can be moved along the rail. 11. A transport system in which the stack transport system is a track network with controlled points of control (33/34/35) along which it is individually driven.
Device according to any of claims 8 to 10, characterized in that is moved. 12. The device according to claim 11, wherein the rail network includes a track (33) that is closed itself. 13. A rack (33/33) which can be moved from above towards the withdrawn shelf (11) and is equipped for grasping the plate stack.
34/35) The stack operating means (31) characterized in that the stack operating means (31) comprises grippers which can be moved over and lowered onto the transport means placed on it.
The device according to any one of claims 12 to 12. 14. At least one plate separating element (32) is arranged at each processing station (32), the plate separating means removing a freely selectable plate (2) from the plate stack (1), Means for placing (2) at a freely selectable site in the plate stack (1), and a stack (1)
Means for placing a plate (2) removed from a processing station in a processing position (13) of a processing station. 15. The plate separating element (32) is equipped for removing the stack (1) from the vehicle located in the transfer position (42) and can be moved to a working position, and the working position Device according to claim 14, characterized in that it comprises a stack shovel (40) that can be lifted to different heights with a shovel. 16. An upper support component pair (50) and a lower support component pair (51) cooperating with a stack shovel (40) in a working position (43).
16. The device according to claim 15, characterized in that the support parts are rotatable to a rest position or a holding position. 17. The apparatus according to claim 16, wherein the plate separating element comprises a plate sled movable between a processing station and a working position. 18. At least one processing station comprises a preheating station (
12.1) and a thermostat (36) downstream of its preheating station (12.1).
18. Apparatus according to any one of claims 8 to 17, characterized in that: 19. The device according to claim 18, wherein the incubator (36) is a continuous incubator and at least one track passes through the incubator. 20. At least one of the processing stations includes a plate transport means leading from the entry point to the exit point, a first separation element for placing individual plates at the entry point, and for removing individual plates from the exit point. A second separation element is disposed at said one of the processing stations.
An apparatus according to any one of claims 9 to 13.