JP3239999B2 - Measuring device by specific binding of magnetic particles - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a measuring device and an adapter used for the measuring device. The present invention is particularly suitable for an automatic immunoassay system.

[0002]

BACKGROUND OF THE INVENTION Solid-phase immunoassays are usually performed in one container,
The analyte to be measured, which will be contained in the sample, first reacts with the separation reagent bound to the solid phase, and the other steps required for the measurement are also performed in the same container. The complication here is that a large amount of reagent input and liquid removal must be performed. If several different measurements are taken, it may also be necessary to store large quantities of the different reagents.

[0003] There is also known a system in which a solution to be used in each stage of measurement is previously placed in a container in each stage. The solid phase is a disposable pipette spout (pippet
formed by the inner surface of the te jet). At each stage, the pipette spout is directed into the corresponding vessel, the solution is pumped into the spout and a reaction takes place, after which the spout is emptied and transferred to the next vessel. During that phase, the solution will go through the spout. The device has a plurality of suction cylinders with pumps, so that a plurality of measurements are taken in parallel. No precision dosing is required with this device.
This device does not require any solution reservoirs either. The disadvantage, however, is that the sample is connected through the gas phase to the cylinder of the device, which cannot be washed automatically. This is a contamination
Can cause danger. Also, the liquid will remain at the pipette spout and move to the next stage.
In addition, piston pumps wear out easily and unpredictably,
For this reason, it is often necessary to check the condition of these pumps. Another problem is the seal between the pipette spout and the wicking cylinder. Given all the considerations, this device can cause many problems. Moreover, the available solid surface area is limited only to the internal surface of the pipette spout.

[0004]

The measuring device defined in claim 1 has been invented. Its advantageous applications are indicated in the other claims.

[0005] The measuring device for specific binding of magnetic particles according to the present invention comprises reacting a sample with an analyte separation reagent bound to a solid phase separated from a container in a medium contained in the container, thereby obtaining an analyte. Measuring the analyte from a sample that will contain the analyte by forming a separate reagent complex, performing intermediate steps if necessary, and then determining any complexes formed. And a reaction vessel (2) installed in the apparatus.
And magnetic particles (8) for the reaction vessel having a separation reagent bound to a solid phase on the outer surface; a measurement vessel and a measuring instrument for detecting any formed complex;
One or more containers for the intermediate stage performed in the medium, whereby the reaction container, the measuring container and the container for the intermediate stage are arranged in a straight line to form a plate (1) And one or more containers for the intermediate stage, forming a cassette (14) movable in the longitudinal direction of the plates, by virtue of the presence of several parallel plates, and the particles from the containers. A mover for removing and moving to another container (3 / 3.1 / 3.
2) and its actuator, whereby a magnet (10) is connected to the mover to attach the particles to the mover after the reaction, and when the mover moves relative to the vessel, A mover (3 / 3.1 / 1), wherein at least one container comprises a medium used for working in the measuring stage, said mover comprising magnetic gripping means (11) capable of vertically moving inside the mover. 3.2) and its actuator and a detection head (1) movable laterally of the plate
7) and an arm for installing a mover for each plate, and the upper part of the inner space of the container is enlarged.
Part is formed, and the lower edge of the enlarged part is directed downward.
It is characterized by being open like a funnel .

[0006] In order to adhere the particles to the mover as a dense mass, the surface of the mover is contoured (3).
6/13).

[0007] In order to create a liquid flow, the surface of the mover is essentially everywhere a downward slope, preferably also having a sharp point (5) at the bottom of the mover. preferable.

It is preferable that the transfer device (3.1 / 3.2) functions as a stirrer.

The reaction vessel and at least another vessel are required for the measurement, preferably all vessels of the plate required for the measurement are connected to one vessel unit (1).

[0010] Preferably, the container contains all the media required for the measurement.

Preferably, at least one container, preferably all containers, are closed with a pierceable film (28).

[0012] Preferably, at least some of the closed vessels contain an inert gas phase.

[0013] As used herein, a separation reagent refers to a substance that reacts with an analyte and binds to the analyte in a solid phase. In immunoassays, the separation reagent is usually an antigen or an antibody. Here, medium generally means a solution used in a certain measuring step, such as a reaction solution or a washing solution.

In the apparatus of the present invention, the outer surface of the solid particles separated from the reaction vessel is used as a solid phase, and the measuring step is performed in two or more vessels. Particles are transported to a specific mobile device (re
transfer from one container to another using a mover). The particles are held in a container containing the sample and a separation reaction takes place. Then, other necessary steps are performed in another container, and finally the particles are transferred to the measuring container. The medium required for the measurement has been charged in the container in advance.

[0015] The particles are preferably magnetic particles, whereby the mover preferably contains a magnet movable in association with the mover.

[0016] The plurality of containers are preferably formed as one unit. However, in principle, several steps, in particular the measurement of the reactants formed, can, if desired, be carried out outside the unit of the vessel. An external measuring container can be used especially when the complex is measured directly from the solid phase, for example by fluorimetry or radiometry.

On the other hand, several steps, for example washing, can be carried out in the same vessel. The media can also be loaded into or removed from certain containers. Separate dosing could possibly be done without the need for precise dosing, for example at the stage where the same medium is used in several different measurements. In particular, washing is probably such a stage. However, in general, units of containers are more convenient, such that all different media are provided in different containers.

At least the washing is usually performed in the middle of the measuring step. In addition, the resulting reaction complex usually binds at an intermediate stage with the tracer detected at the measuring stage.
A tracer can be one that can be detected directly, or one that releases a detectable compound from a particular substrate. Normal,
Detection can be fluorescent, luminescent, absorbant, or radiological.

In the device of the present invention, there is no risk of contamination since no sample is drawn into the device from the plate container. In addition, the device according to the invention can be an automatic device which performs a simple and very reliable operation.

The present invention relates to, for example, immunological measurement, DN
A-Suitable for hybridization or hormone measurement.

The surface of the mover is preferably shaped such that the liquid runs off the surface as completely as possible.
It is also preferable that the moving device has a tip at the lower end. The bottom of the reaction vessel is conveniently designed to be of the same shape as the mover in order to use as little medium as possible.

By using solid particles remote from the mover, a very large solid surface area is obtained.
The most advantageous is the so-called micropar
ticle). Magnetic particles that are easily attached to the mover with the aid of magnets are preferably used.

If non-magnetic separating particles are used, the mover is provided with, for example, a grid or filter for separating the particles from the medium.

It is preferred to stir the medium during the reaction in order to speed up the bulk transport and, for example, the required reaction time. This is preferably done by moving the mover. It is particularly advantageous to move the mover vertically, so that the medium must flow through the gap between the container and the mover, and such mixing is very effective. For more effective mixing, the mover is made wider so that a suitably narrow gap is formed between the container and the mover. With proper design of the container and the transfer device, stirring can also be facilitated.

The unit of the container forms a plate for use in one measurement. The mover can be placed in a container in the plate. The containers used at different stages may be of different sizes.

The container is preferably closed with a film, which is perforated in performing the measurement. The film may be pierced using a mover, but alternative piercing tips may be used. The point may be provided with a sharp blade that forms a strip that breaks in a controlled manner. The drilling point may be mounted on the same actuator as the mover in the device. The tip of the container preferably has an extension so that a small piece of perforated film can rest. The closed container may contain an inert gas phase to improve durability.

[0027] The device may also be equipped with a safety device system to confirm the presence of the medium in the container before the stage is started. A mobile may conveniently operate as an indicator of such a system based on electrical conductivity measurements.

If desired, in particular in the reaction vessel in which the sample is placed, some suitable substance may be immobilized on the wall of the vessel or on the remaining solid phase away from the vessel, and such a substance may be subsequently measured. Capture substances from the sample or from the complexes formed that may interfere with the step.

In the present invention, the plate containers are aligned so that the mover need only be moved along a straight path in the horizontal plane of the plate. The containers for the different stages may be located in any order with respect to each other. Preferably, the container is permanently fixed to another container. The plate may be made from a suitable material, preferably plastic.

The plate advantageously has a detent and the device has a corresponding part so that the plate cannot be misplaced by mistake.

[0031]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Some examples of the present invention will be described below with reference to examples.

In the figures described above, FIG. 1 is an explanatory view showing an example of a magnetic particle mover in a reaction vessel for use in the measuring apparatus of the present invention, and FIGS. 2 (a) and 2 (b) FIG. 3 is an explanatory view showing an example of using a magnetic particle and another moving device in the measuring apparatus of the present invention, FIG. 3 is an explanatory view showing a set of apparatuses usable in one embodiment of the present invention, and FIG. FIG. 5 is an illustration of another embodiment of another set of devices having a larger capacity, FIG. 5 showing a third set of reactors located in a reaction vessel usable for the measurement device of the present invention. FIG. 6 is an explanatory view showing a magnetic particle mover, and FIG. 6 is a plan view of the mover of FIG.

According to FIG. 1, wells 2 positioned linearly
An immunoassay is carried out using a moving device 3 having a plate 1 made of a material and a cylindrical sheath 4. The mover 3 has a sharp point 5 at its lower end, the lower end being formed as a cone 6. The mobile has at its upper end a handle 7 which is convenient for robotics, so that the mobile can be informed about the precise control of its horizontal and vertical position.
The well also includes magnetic particles 8 coated with a separation reagent that reacts with the analyte to form a complex. The mover has a hole 9 for receiving a movable pin 11 with a magnet 10.

The magnet 10 of FIG. 1 has two magnets, one on the other, such that the same poles are in opposite directions (SN-NS). In this way, a strong change in the magnetic field at the junction of the magnets and a favorable condition for attracting particles to this point on the surface of the mover 3 are also created. Correspondingly, the magnetic field outside the two magnets becomes weaker in the vertical direction, so that the particles are more likely to plug only in that position on the magnets. It is also possible for a plurality of magnets to be located behind each other. This is advantageous when a narrow structure is required.

The test sample is first introduced into the first well 2 of plate 1, optionally containing a suitable diluent,
Further, the magnetic particles 8 coated with a desired separation reagent and the moving device 3
Is introduced into well 2. At this stage, the pin 11 is in the upper position, so that the particles are free to move through the well. The analyte that would be contained in the sample thereby reacts with the separating reagent to form an immune complex. During the reaction, the mover moves through the wells to promote mixing. After incubation, the magnet moves to the lower position,
The particles thereby collect on the surface of the mover and can be transferred to the second well. In the second well, the particles are released again, for example to perform a wash or tracer reaction, after which they can be collected again and proceed. Finally, the measurement required for the measurement is performed in the final well.

During the reaction and washing, the transfer device 3 is connected to the well 2
Is moved up and down, thereby efficiently mixing the media.

The plate 1 can be made of a suitable plastic material.

No liquid transfer is required during the measurement, so that a safe, simple and reliable system can be constructed.

The diameter of the cover 4 is chosen to match the diameter of the well 2, so that an efficient flow around the cover can be obtained when moving the cover up and down. When separating particles, multiple up and down movements are preferably performed using the cover of the mover in a well (eg, 1).
About 20 times in 0 second). The weakly adhered particles fall, but they will probably adhere better later.

FIGS. 2 (a) and 2 (b) show an embodiment of the method of the present invention using a differently designed mover.

The first well 2 of the plate 1 contains magnetic particles 8 coated with a separation reagent for the analyte to be measured and, if necessary, a diluent.

The mover 3.1 has a hole 9 starting from its top.
The hole can receive a pin 11 having a magnet at its lower end. The mover has a drop-like extension 12 at its lower end and a sharp cusp 5 at its tip. In addition, an annular recess 13 is provided on the surface of the extension near the lower end.

The sample is placed in the first well 2, after which the mover 3.1 is pushed into the well 2 with the pin 11 in the upper position. When the mover is moved, the medium and the particles 8 mix efficiently and form a suspension. Upon completion of the incubation, the pins are pushed down,
Thereby, particles are collected on the surface of the extension portion 12 pulled by the magnet 10 to form a dense mass in the concave portion 13 (see FIG. 2B). The mover is then moved to the next well and the pins are lifted, thereby causing the particles to mix again with the media. The particles comprising a second well containing a first wash, a third well containing a second wash,
It is then transferred to a fourth well containing the enzyme conjugate that attaches to the immune complex. After incubation with the tracer, the transfer device is further equipped with three reaction and wash wells for counting in the last well containing the substrate for the enzyme, from which the enzyme releases a detectable mixture by fluorimetry. Via. After the substrate reaction, the transfer device is moved aside and the fluorescence measurement is performed in such a way that both excitation radiation and emission radiation are guided through the well entrance.

In the measurement, light need not be guided through the walls of the well. For this reason, the cheapest materials possible and the simplest manufacturing techniques possible can be used.
The material is preferably opaque to reduce background emission.

Measurement by luminescence can be performed in a similar manner.

When the reaction result is measured by an absorptiometric method, light emission is caused by the measurement container being transparent or by special preparation from the measurement container to the detector (for example, a bottom surface that reflects light). Must be obtained.

FIG. 3 shows a set of devices capable of making ten measurements simultaneously.

The measuring plate 1 is located in the cassette 14. At the last well-wise end of each plate is a code 15 that informs the instrument about the measurement being considered. In addition, the code may be used to provide other data, especially elapsed time.

The cassette 14 is pushed into the apparatus in the longitudinal direction of the plate, so that one end of the cord first passes through the opening 16, where the cassette is automatically moved. The device has a movable detection head 17 in the transverse direction of the plate, with an identifier 18 for reading the code and a measuring device 19 for establishing the reaction result. When a unit for perforating a film that closes a moving device and a well is used, or when a magnet pin moving unit is used, all the units are installed on the arm 20. The equipment is
It also has a heater with a thermostat to keep the temperature of the plate at the desired temperature.

The mover for each sample plate is mounted on the arm 20. The sample is loaded into the first well of plate 1 in cassette 14 and the cassette is pushed inward. When it has moved to its extreme position, the identifier 18 reads the code 15, whereby the control unit receives the information necessary to make the measurement. The mover is dropped into the first well. After incubation, the mover is lifted and the plate advances one step to perform the second step. In this way, the process proceeds from one well to the next,
Finally, measurements are taken in the last well. The measurement result of each plate is shown on the display 21.

All measurements may be different, provided that the measurements are performed on the number of wells available in the plate. In all measurements, not all wells may be required, in which case they do not contain any media.

Of course, it is also possible to use a device in which both the detection head and the mover are mounted on the same arm.

FIG. 4 shows a set of modular sets which can handle six cassettes simultaneously.
of equipment).

In the plate 1 used in this apparatus, the cord 15 is located at the end in the first well direction. The cassettes 14 are pre-warmed in the incubator 22 and their cord ends are first pushed into the device through the supply opening 16. The required mover for each cassette is located on the arm 20 at a fixed location on the corresponding plate.

The device has one common detection head 17, which can move laterally and comprises an identifier 18 and a measuring device 19. The identifier reads the code 15 on each plate and the cassette moves inward to its extreme position, where the sample and possibly also the diluent is also loaded into the first well. Dotted line 23 indicates the operation course of the thrower. The cassette is moved outward, the first well is located below the arm 20 with the mover and the first stage is performed. The cassette then moves in each step until the last well reaches the meter.

FIG. 4 shows a power supply unit 2 in the apparatus.
4, a diagram of the control unit 25, the sample input pump 25, the airing and dilution unit 26 and the tip cleaning well 27 is shown.

The plate 1 of FIG. 5 is closed by a film 28, which is pierced by the mover 3.2.
At the entrance of the well 2 there is an enlarged part 29 on which the perforated film is applied. On the top surface of the plate there is a gap 30 between the wells. It indicates a leak point that may be between wells and prevents the transfer of liquid from one well to another through such a leak point.

The hole in the mover 3.2 accommodates a movable pin 11 with a magnet 10 at the lower end.

The mover 3.2 has an extension 12.2 at its lower end.
Having. Its lower end is in the shape of a cone with a sharp point 5. In this way, the magnetic particles 8 are protected in a sheath over the extension since the extension can be used to pierce the film 28. The extension also functions as an effective stirring piston. The lowermost part of the well 2 has a sharp cone shape corresponding to the extension.

The edge 31 at the extension 12.2 is sharp to minimize the adhesion of droplets. In response,
The lower edge 32 of the enlargement of the well 2 has an appropriate zigzag downwardly to remove any remaining droplets from the mover 3.2 when the mover 3.2 is removed from the well. To open (flare).

The upper end of the mover 3.2 is provided with a cone-shaped inlet extension 33 which is easily positioned about the sheath in the well 2. A plug 34 for closing the well inlet is located above the inlet extension.

The surface of the mover 3.2 on the extension 12.2 is provided with a vertical ridge 35. The magnetic particles 8 are located in grooves 36 between these ridges and are protected during movement. The bottom of the groove is formed in a plane suitable for promoting dissociation into a liquid. The protective ridge may be like a thread (eg, one thread with two ends).

[0063]

According to the apparatus of the present invention, there is no risk of contamination since the sample is not drawn into the apparatus from the plate container. In addition, the device according to the invention can be an automatic device which performs a simple and very reliable operation. Therefore, the present invention is particularly suitable for an automatic immunoassay device.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing an example of a magnetic particle mover in a reaction vessel for use in the measurement device of the present invention.

FIG. 2 is an explanatory diagram showing an example in which magnetic particles and another moving device are used in the measuring device of the present invention.

FIG. 3 is an explanatory diagram showing a set of devices that can be used in an embodiment of the present invention.

FIG. 4 is another embodiment of the measuring device of the present invention,
FIG. 4 is an explanatory diagram showing another set of devices having a larger capacity.

FIG. 5 is an explanatory view showing a third magnetic particle mover located in a reaction vessel usable for the measuring device of the present invention.

FIG. 6 is a plan view of the moving device of FIG. 5;

Continuation of front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/543 G01N 33/553

Claims (8)

(57) [Claims] An analyte-separation reagent complex is formed by reacting an analyte-separation reagent bound to a solid phase remote from a container with a sample in a medium contained in the container to form an analyte-separation reagent complex, if necessary. An apparatus for measuring an analyte from a sample that will contain the analyte by performing an intermediate step and determining any complexes that subsequently formed, the apparatus comprising: A reaction vessel (2) installed in the apparatus and a magnetic particle (8) for the reaction vessel having a separation reagent bound to a solid phase on the outer surface, and a detector for detecting any complex formed. A measuring container and a measuring device, and one or more containers for an intermediate stage performed in the medium, whereby the reaction container, the measuring container and the container for the intermediate stage are arranged in a straight line. Play by One or more containers for the intermediate stage, forming a cassette (14) forming a plate (1) and having several parallel plates so as to be movable in the longitudinal direction of said plates; A mover (3 / 3.1 / 3.2) and an actuator thereof for removing the particles from the container and transferring the particles to another container, whereby the particles are attached to the mover after the reaction. A magnet (10) is coupled to the mover, wherein when the mover moves relative to the container, at least one container includes a medium used to work in a measurement stage, the mover being associated with the mover. A moving device (3/3...) Provided with magnetic gripping means (11) capable of vertically moving the inside.
1 / 3.2) and its actuator, and a detection head (17) movable laterally of the plate
If, Toka arm for installing a translator for each plate
An enlarged portion is formed in the upper part of the internal space of the container,
The lower edge of the enlarged portion opens like a funnel toward the lower side
And it has apparatus. 2. The apparatus according to claim 1, wherein the surface of the mover has a contoured area (36/13) for adhering the particles to the mover as a compact mass. 3. In order to create a liquid flow, the surface of the mover is essentially everywhere a downward slope, preferably also having a sharp point (5) at the bottom of the mover. The device according to claim 1. 4. The device according to claim 1, wherein the transfer device (3.1 / 3.2) functions as a stirrer. 5. The reaction vessel and at least another vessel are required for the measurement, preferably all vessels of the plate required for the measurement are in one vessel unit (1)
5. The device according to claim 1, 2, 3 or 4, wherein the device is connected to a. 6. The apparatus according to claim 1, wherein said container contains all media required for the measurement. 7. The device according to claim 1, wherein at least one container, preferably all containers, are closed with a pierceable film (28). 8. The apparatus according to claim 7, wherein at least some of the closed vessels contain an inert gas phase.