JP2010151738A - Method and device for separating magnetic particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method which for quickly and surely separates magnetic particles and is free from the restriction of a system such as a container transportation route or the like, and a device for the same. <P>SOLUTION: The problem is solved by the method composed of: a magnet-collecting process for collecting the magnetic particles on the inner surface of a container by applying the magnetic attraction of a magnet disposed in a magnet-collecting position which is set outwardly; a first suction process for sucking up liquid; a dispensing process for dispensing a cleaning liquid; a movement process for moving the magnetic particles along the inner surface of the container toward the neighborhood of the another magnet-collecting positions where the magnet is disposed; and the second suction process for sucking up the cleaning liquid which is dispensed in the dispensing process. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method and apparatus for separating magnetic particles from a liquid. The apparatus and method of the present invention are used in the case where magnetic particles having an affinity for a specific substance such as an antibody immobilized on the surface are used, and after the specific substance is captured by the magnetic particles, the magnetic particles are separated. Is. More specifically, for example, after a magnetic particle having an antibody or the like having affinity for a measurement object is brought into contact with a blood-derived sample, a component (Bound component) captured by the magnetic particle was not captured. It is used for so-called B / F separation in immunoassay, in which it is separated from the component (Free). In the present specification, the present invention will be described below by taking B / F separation in immunoassay as a specific example.

  In immunoassay using magnetic particles as a solid phase, the ratio of time required for B / F separation in the time required for measurement is high. Therefore, how quickly and reliably the B / F separation can be performed is a point for performing the immunoassay in a short time. In conventional immunoassay, magnetic particles having a particle size of about 1 to 5 μm are often used in order to ensure the efficiency of immune reaction between the antibody immobilized on the particle surface and the specific components in the reaction solution and good magnetic collection. Has been. B / F separation repeats the operation of attracting and separating the reaction liquid while collecting magnetic particles with a magnet, then dispensing the cleaning liquid, dispersing the magnetic particles in the cleaning liquid, and separating again while collecting the magnetic particles with a magnet. However, in order to ensure separation, it is preferable to completely disperse the magnetic particles in the cleaning liquid every time the cleaning liquid is dispensed.

  However, when the magnetic particles having the above-mentioned particle size range are completely dispersed in the solution, it takes time to collect the magnetic particles with a magnet. Therefore, various proposals have been made in the past in order to perform B / F separation quickly while ensuring certainty. For example, in Patent Document 1 and Patent Document 2, magnetic particles collected in a lump shape inside the container side wall by applying a magnetic force from the outside of the container side wall are not completely dispersed, but in a semi-dispersed state, the inner surface of the container A method of moving to the meeting has been proposed. Specifically, in Patent Document 1, a plurality of B / F separation ports are provided in a container transfer path, magnets are alternately arranged in the vicinity thereof, and magnetic particles collected inside the container side wall at a certain port are as follows. The port is designed to move face-to-face. During this movement, the object to be cleaned in the magnetic mass of the magnetic particles is released into the cleaning liquid and separated from the magnetic particles, and the reliability of B / F separation is improved.

  Patent Document 3 is an apparatus including a B / F separation method for collecting magnetic particles on the bottom surface of a container and a means for moving a magnet for collecting magnets in the vicinity of the bottom surface of the container. Patent Document 4 describes a container and a magnet. It is an apparatus provided with the moving means which changes the relative angular position between and continuously.

JP 2001-91521 A JP 2006-112824 A Japanese Patent No. 3213798 Japanese Patent No. 3962789

  As in Patent Document 1, in order to alternately arrange magnets in the vicinity of the B / F separation port provided in the container transfer path, a certain amount of space is required around the container. There is a problem that is limited. When adopting a linear transfer path (for example, a transfer path by a chain) as shown in Patent Document 1 for transferring the container, it is relatively easy to secure the space, but when adopting the turntable method, It is essential that the turntable has a special shape such as a donut shape or a hollow shape. As a result, the structure of the device is complicated and the cost is increased. In both Patent Document 1 and Patent Document 2, since the magnetic particles are moved in the cleaning liquid from one of the inner surfaces of the container to the facing surface, the time until the magnetic particles are collected by the facing magnet even though it is in a semi-dispersed state. Depends on the inner volume of the container, that is, the distance to move the magnetic particles in the solution. As a result, in order to perform B / F separation quickly, it is necessary to use a thin container or a container with a small overall size.

  Patent Document 3 is intended to prevent the displacement of the relative position between the magnet and the container because the displacement of the relative position between the magnet collecting the magnetic particles and the container affects the amount of magnetic particles captured. There is no particular mention of improving the reliability of B / F separation by moving the magnetically collected magnetic particles. Also, the movement of the magnet is limited to the movement at the lower part of the bottom surface of the container. In Patent Document 4, the relative angular position between the container and the magnet is continuously changed using the moving means, and the magnetic particles are moved in the reaction container only to promote the reaction, During the B / F separation, the moving means is stopped.

  The object of the present invention made in view of the problems of the prior art as described above is a magnetic particle separation method that can be carried out quickly and reliably, and is subject to restrictions on the apparatus such as a container transfer path in the implementation. It is to provide an immunoassay in which the time required for all the steps is shortened.

  The present invention made to achieve the above object is a method for separating magnetic particles dispersed in a liquid in a magnetically permeable container from the liquid, wherein a magnet is disposed at a magnetic collection position set outside the container. In the magnetic flux collecting step for collecting magnetic particles on the inner surface of the container by applying a magnetic force from the magnet, the first suction step for sucking the liquid in the vessel, the dispensing step for dispensing the cleaning liquid in the vessel, and the magnetic flux collecting step A magnet is placed at a magnetic flux collection position different from the magnetic flux collection position, and magnetic force is applied from the magnet, and the magnetic force from the magnetic flux collection position in the magnetic flux collection process is stopped to attract the magnetic particles in the vicinity of the magnetic flux collection position in the magnetic flux collection process. Is a B / F separation method comprising a moving step of moving along the inner surface of the container to the vicinity of the different magnetic flux collecting positions, and a second suction step of sucking the cleaning liquid dispensed in the dispensing step. Further, the present invention made to achieve the above object is an apparatus used for separating magnetic particles dispersed in a liquid in a magnetically permeable container from the liquid, container holding means for holding the container, The magnet holding means for holding one or more magnets, the container holding means and / or the magnet holding means are moved, and the magnet is moved to two or more different magnetic collecting positions set outside the container to move the container from each position. B / F separation device comprising: moving means for applying magnetic force of the magnet to internal magnetic particles; suction means for sucking liquid in the container; and dispensing means for dispensing cleaning liquid into the container It is. Hereinafter, the present invention will be described in detail.

  In the present invention, when separating magnetic particles dispersed in a liquid in a magnetically permeable container from the liquid, the magnetic particles collected by applying a magnetic force from a magnet are moved while being slightly dispersed along the inner surface of the container. Is. By moving along the inner surface of the container, the reaction liquid (containing unbound components that are not bound to the magnetic particle surface, which is a solid phase) present in the gap between the magnetic particles is released into the cleaning liquid and separated from the magnetic particles. As a result, the certainty of the B / F separation is improved. Further, according to the present invention, B / F separation is quickly performed while ensuring a minimum dispersion state by moving the magnetic particles along the inner surface of the container while collecting the magnetic particles.

  In the present invention, since the magnetic force of the magnet acts on the magnetic particles dispersed in the liquid in the container from the outside of the container, the container itself is magnetically permeable, or part of the container is magnetically permeable and is external to the container. If the magnetic force of the arranged magnet is sufficient inside, it is possible to collect magnetic particles on the inner surface of the container, and if it is possible to move the magnetic particles collected following the movement of the magnet, There are no restrictions on the size, shape, thickness, material, etc. Examples of conventionally used magnetically permeable containers include containers made of thermoplastic resin such as polypropylene and polystyrene.

  As for magnetic particles, particles having a particle size range of 0.01 to 100 μm can be exemplified. However, if the particle size is too small, it takes a long time to collect the magnetic particles. The efficiency of the reaction trapped by the magnetic particles is undesirably reduced. In general, when carrying out rapid and reliable B / F separation with an easily available magnet, it is preferable to target magnetic particles of about 0.1 to 10 μm. Usually, a substance having affinity for the measurement target substance is fixed to the magnetic particles, but a substance having affinity for such a substance may be fixed. For example, an antibody, an antigen, a receptor, a ligand, biotin, avidin, streptavidin, an enzyme, a substrate, or a nucleic acid can be exemplified. Immobilization of these substances on magnetic particles can be achieved by direct immobilization by chemical bonding, physical adsorption, ionic bonding, etc., affinity of biotin and avidin, biotin and streptavidin, nucleic acid and nucleic acid, or hapten and anti-hapten antibody. A method of indirectly immobilizing using sex can be exemplified. When the present invention is applied for purposes other than immunoassay, for example, for the purpose of purifying or removing a specific substance from a sample, the magnetic particles are made of a substance having affinity for the substance to be purified (target for removal). Fix it.

  In the present invention, first, magnetic particles dispersed and mixed with a reaction solution or sample of immune reaction are collected (magnetization step). For this reason, in a magnetism collection process, a magnet is arranged in a magnetism collection position set up outside the container, and magnetic force acts on the magnetic particles in the container from the magnet. Examples of the type of magnet used in the present invention include neodymium-based and samarium-based rare earth magnets, alnico magnets, ferrite magnets, permanent magnets such as neodybonded magnets, and electromagnets. The shape of the magnet is not particularly limited, and examples thereof include a cylindrical shape, a square shape, a ring shape, and a segment shape. However, a preliminary experiment is performed in consideration of the shape of the container to which the present invention is actually applied. It is preferable to determine. The same applies to the size of the magnet and the magnitude of the magnetic force. The arrangement of the magnets in the magnetic collection position can be carried out by either moving the magnet relative to the container, moving the container relative to the magnet, or moving both the container and the magnet relatively. In carrying out a particularly preferable aspect of the present invention described later, it is particularly preferable to move the magnet with respect to the container. In the case of using an electromagnet, it is possible to collect the magnetic particles by applying a magnetic force to the magnetic particles by simply fixing the electromagnet in a magnetic collection position in advance and simply passing an electric current. In this specification, it is included in the “arranging magnets at the magnetism collecting position” to flow an electric current through an electromagnet whose position relative to the container is fixed in advance.

  In the present invention, the magnetic particles are collected on the inner surface, that is, the side surface or the bottom surface of the container. Particularly preferably, the magnet is disposed on the side surface of the container with the magnetic collection position as the outside of the container side surface. This is because by selecting such a magnetic flux collecting position, it is possible to realize more reliable and quick separation of magnetic particles. That is, in the first suction step and the second suction step to be performed later, in order to separate the liquid component in the container from the magnetic particles as much as possible, the suction pipe is lowered to the bottom of the container to suck the solution. At this time, the magnetic particles can be prevented from being discharged together with the solution to the outside of the container. Further, in the cleaning liquid dispensing step to be performed later, the flow of the dispensed cleaning liquid is maximized at the bottom of the container, which means that the magnetically collected magnetic particles are dispersed and it is necessary to collect the magnetic flux again. Can also be prevented.

  Next, the liquid in the container is sucked in a state where the magnetic particles are collected (first suction step). For suction, a suction means frequently used in B / F separation can be used. The suction means can be constituted by a suction pipe having one end connected to a vacuum pump via a liquid trap bottle, and a moving means for lowering the other end opening of the suction pipe and entering the container. Since the liquid in the container is necessary for reliable B / F separation to be sucked as much as possible and separated from the magnetic particles, the opening is preferably lowered to the bottom of the container.

  Next, the cleaning liquid is dispensed into the container in a state where the magnetic particles are collected (dispensing step). The cleaning solution is not particularly limited except that the measurement target substance or the purification target substance fixed to the magnetic particles is not separated from the magnetic particles, but an interface such as Tween or Triton is used in an appropriate buffer such as Tris or phosphoric acid. The thing etc. which added the activator in a trace amount can be illustrated. The amount of the cleaning liquid to be dispensed is not particularly limited as long as it exceeds the amount of liquid existing in the container, and may be appropriately determined in consideration of the amount of magnetic particles to be separated and the capacity of the container.

  Dispensing means frequently used in B / F separation can be used for dispensing. The dispensing means can be composed of a dispensing pipe whose one end is connected to the liquid pump, and a moving means for lowering the other end opening of the dispensing pipe and entering the container. If magnetic particles are collected on the side of the container when dispensing the cleaning liquid, for example, the cleaning liquid can be dispensed by lowering the dispensing tube to the vicinity of the bottom of the container, but the magnetic particles are collected on the bottom of the container. If it is magnetic, for example, it is preferable that the cleaning liquid is dispensed on the side of the container after the container is slightly tilted.

  The suction means and the dispensing means frequently used in the B / F separation described above are usually configured such that the suction pipe and the dispensing pipe are integrated. For example, these two pipes are connected in parallel and move up and down integrally, or the two pipes are configured as a double pipe (the inside of the inner pipe is a suction pipe, the inside of the outer pipe and the inside of the outer pipe Can function as a dispensing pipe) and can be lifted and lowered integrally.

  Next, a magnet is arranged at a magnetic flux collecting position different from the magnetic flux collecting position in the magnetic flux collecting step, and a magnetic force is applied from the magnet, and the magnetic particles are moved from the vicinity of the magnetic flux collecting position in the magnetic flux collecting step to the vicinity of the different magnetic flux collecting position. Is moved along the inner surface of the container (moving step). The type, size, and the like of the magnet used in the moving process are the same as those used in the magnet collection process. The movement can also be performed by either moving the magnet relative to the container, moving the container relative to the magnet, or moving both the container and magnet relative.

  In order to move the magnetic particles along the inner surface of the container, the magnet itself used in the magnetic collecting process is used so that the magnetic particles move along the inner surface of the container, or the magnet used in the magnetic collecting process It is necessary to use one or more different magnets so that the magnetic particles move along the inner surface of the container.

  In the former case, the movement of the magnet from the magnetic collection position in the magnetic collection process to the magnetic collection position in the movement process is continuous, and the magnetic particles collected in the magnetic collection process constantly generate magnetic force from the same magnet. It will move while receiving. Along with the movement of the magnet, the action of the magnetic force from the magnetic collection position in the magnetic collection process stops, and the magnetic particles do not stop at the position. In the former case, the magnet may be moved relatively along the outer wall of the container so that the collected magnetic particles follow. Therefore, for example, the relative moving speed of the magnet and the container is in the range of 10 to 1000 mm / second, preferably 50 to 500 mm / second, but the followability is the strength of the magnet, the attenuation of the magnetic force in the container, the magnetic particles Therefore, preliminary experiments are preferably performed in advance. In general, it is preferable to use a strong magnet or to use magnetic particles having a high magnetic collection property because the followability of the magnetic particles becomes good. However, if you use a magnet that is too strong considering only the followability, or if you use magnetic particles that are too magnetically collected, dispersion of the moving magnetic particles along the inner surface of the container will not occur and separation will not occur. There is a risk of becoming insufficient. Conversely, if a weak magnet is used or magnetic particles with low magnetism are used, the dispersion of the magnetic particles along the inner surface of the container will be good, but it will take time to collect magnetism in the vicinity of the magnet collection position in the moving process. Since there is a fear that it will be necessary, it is preferable to conduct a preliminary experiment in advance.

  In the latter case, that is, when one or more magnets different from those used in the magnetic flux collecting step are used, these magnets are used so that the magnetic particles move along the inner surface of the container. For example, one or a plurality of intermediate magnetic collection positions are provided between the magnetic collection positions in the magnetic collection process and the magnetic collection positions in the movement process, and the magnets are arranged as if the same magnets are sequentially arranged at the intermediate positions. For example, it is possible to follow the magnetic particles as if they are continuously moved. Further, for example, as will be described in more detail in a later embodiment, it is possible to exemplify preparing a plate-like member and one or more magnets held by the plate-like member and rotating the plate-like member in the vicinity of the container. If one magnet is held, rotation of the plate member causes the magnet to move, for example, from the lower direction toward the container and away from the upper direction. A magnetic collecting process for collecting magnets can be performed, and a moving process can be performed by moving the magnetic particles collected in the same or reverse rotation from the position along the inner surface of the container. Also, in this specification, electromagnets are arranged at the magnetic collection position in the magnetic collection process, the magnetic collection position in the movement process, and an intermediate position between them, and the current magnet is made to flow into a magnet so that the same magnet can be obtained. It is possible to move the magnetic particles along the inner surface of the container by following the magnetic particles as if they are moving continuously. The magnetic particles are moved from the vicinity of the magnetic collection position in the magnetic collection process to the vicinity of the different magnetic collection position along the inner surface of the container. Further, as will be described in detail in a later embodiment, in the present invention, “a magnet is arranged at a magnetic flux collecting position different from the magnetic flux collecting position in the magnetic flux collecting step, a magnetic force is applied from the magnet, and the magnetic particles are collected. To move from the vicinity of the magnetic collection position in the magnetic process to the vicinity of this different magnetic collection position along the inner surface of the container, move the magnetic particles from the vicinity of the magnetic collection position in the magnetic collection process to a different location along the inner surface of the container. After that, it is also included to place the magnet again at the same position as the magnetic collection position in the magnetic collection process and collect magnetic particles in the vicinity of the position.

  Even when two or more magnets (including electromagnets) are used in the present invention, it is not necessary to unify the types and strengths of the magnets in each step. For example, in the magnetic flux collecting process and the moving process, a sufficient magnetic force that can prevent the magnetic particles from being discharged out of the container when the liquid or cleaning liquid in the container is attracted and separated in the first or second suction process. Use a magnet with it. When using multiple magnets in the moving process, use a magnet with sufficient magnetic force as described above as the last magnet, which is finally placed in the magnetic collection position and actually collects magnetic particles. To do. Similarly to these, in the dispensing step, a magnet having a sufficient magnetic force that can prevent the magnetic particles from being dispersed by dispensing the cleaning liquid is used. On the other hand, in the case where a plurality of magnets are used in the moving process, other than the last magnet, for the purpose of increasing the separation efficiency of the magnetic particles, the magnetic particles follow the magnet while being delayed along the inner surface of the container. It is preferable to do so, and for that purpose, a material having a weak magnetic force as compared with the above-described magnetic force is suitable. An example of the strength of the magnet that is actually used is that the strength determined by conducting preliminary experiments in advance is used. When two or more magnets are used in the moving process, the magnetic force from the two magnets is simultaneously applied to the magnetic particles to widen the range of the magnetic force and the dispersibility of the magnetic particles on the inner surface of the container. Can also be raised.

  When moving the magnetic particles collected along the inner surface of the container, in addition to simply moving them linearly, they can be reciprocated on a straight line (the initial position and the moved position are the same), or zigzag May be moved linearly, or may be rotated in a spiral manner, and may be determined in consideration of the size and shape of the magnet, the size and shape of the container, and the like. In particular, it is preferable in terms of the simplicity and reliability of the apparatus configuration that the magnetic particles collected on the inner surface of the container are reciprocated on a straight line including the magnetic collection position. Examples of the reciprocating direction include a horizontal direction and a vertical direction with respect to the measurement container. For such reciprocation, the magnet may be moved along the outer wall of the container, the container may be moved with respect to the magnet fixed by transfer means such as a turntable, chain, rack, etc. You may move both magnets relatively.

  Finally, the cleaning liquid in the container is sucked in a state where the magnetic particles are collected in the vicinity of the magnetic collecting position in the moving step (second sucking step). For suction, a suction means frequently used in B / F separation can be used.

  In the separation method of the present invention, all the steps described above are performed once or more, and in order to increase the separation efficiency, it is preferable to dispense and suck the cleaning liquid three or more times. However, if it is carried out about 10 times, the efficiency will not change even if the washing liquid is further dispensed and sucked, so it is preferable that the upper limit is about 10 times. In repeating all the above steps, it can be exemplified that the second suction step is regarded as the first suction step, and thereafter each step of the present invention is performed in the order of the dispensing step, the moving step, and the second suction step. In this case, in the moving step, the magnetic particles move from the vicinity of the magnetic collection position in the second attraction step to the vicinity of the magnetic collection position in the first attraction step, and from the vicinity of the magnetic collection position in the first attraction step to the second attraction step. It can be exemplified that the movement of the magnetic particles to the vicinity of the magnetic collection position is repeated alternately. In repeating all the above steps, after the second suction step is completed, a dispensing step of dispensing the cleaning liquid is first performed, and then the above-described magnetism collecting step, first suction step, dispensing step, moving step, first step It can also be illustrated that the same operation is repeated in the order of the two suction steps.

  In the separation method of the present invention, the magnetic particles are not dispersed in the cleaning liquid because the magnetic particles are moved along the inner surface of the container by moving the container and / or the magnet (magnet holding means). As a result, rapid separation can be achieved as compared with the conventional method in which dispersion and magnetic collection of magnetic particles are repeated. Also, the apparatus configuration for automation can be simplified and reduced in size including the drive system as shown in this specification.

EXAMPLES The present invention will be described in detail below by examples, but these examples do not limit the present invention.
Reference Example 1 Preparation of Anti-TSH Antibody-Immobilized Magnetic Particles Take 10 mg of commercially available carboxylated magnetic microparticles (trade name Micromer-M, diameter 4.0 μm, manufactured by Micromod) in a 5 mL microtube and 1 mL of 10 mM MES buffer (pH 6. 0), and 1 mL of 10 mM MES buffer (pH 6.0) containing 10% N-ethyl-N ′-(dimethylaminopropyl) carbodiimide (EDC) was added, followed by shaking and stirring at room temperature for 1 hour. . After collecting the supernatant, the supernatant is removed, washed once with 1 mL of 10 mM MES buffer (pH 6.0), sonicated for 5 minutes, then added with 100 μL of 1 mg / mL antibody solution, and incubated at room temperature for 2 hours. And stirred with shaking.

After washing three times with 1 mL of 0.1 M Tris hydrochloric acid buffer (pH 8.0) containing 0.1% sodium azide, 0.1 M Tris hydrochloric acid containing 0.1% BSA and 0.1% sodium azide The solution was blocked with a buffer solution (pH 8.0) and stored in the same buffer solution.
2. TSH zero assay 10 μL of 0.4% dispersion of anti-TSH antibody-immobilized magnetic particles prepared above, 10 μL of TSH zero concentration sample, and 40 μL of purified water containing a surfactant are placed in a cylindrical measurement container (made of polypropylene, 500 μL). The first reaction of the two-step sandwich method was performed at 37 ° C. for 5 minutes. The first washing is carried out by washing method 1 described later using 10 mM Tris hydrochloric acid buffer (150 mM NaCl, 0.05% Tween-20, 1 mM MgCl ₂ , containing 0.1% sodium azide, pH 8.0) as a washing solution. went. Furthermore, 50 μL of 2.8 μg / mL alkaline phosphatase-labeled anti-TSH antibody was added, and the second reaction was performed at 37 ° C. for 3 minutes. Using the same washing solution as the first washing, the second washing is performed by the washing method 1, and finally, 100 μL of 0.4 mM chemiluminescent substrate solution for alkaline phosphatase (Tosoh, JP-A-2005-77142) is added at 37 ° C. The reaction was performed for 5 minutes, and endpoint photometry was performed.
3. B / F separation method 1 (Fig. 1)
As the separation method 1, the separation method of Patent Document 2 was performed. One neodymium magnet (8φ × 8mm) (3) is placed on the outside of the container, one by one, and alternately approached to the container (1) containing the cleaning liquid, and the magnetic particles (2) are in a semi-dispersed state until the inner surface of the container faces. After repeating the moving operation three times, collecting the magnetic particles for 20 seconds and sucking and discharging the cleaning liquid are performed three times in the first separation (the amount of the cleaning liquid is 350 μL each) and 3 to 11 times in the second separation ( The amount of the cleaning solution was 250 μL for the first time, 300 μL for the second time, and 350 μL for the third time.

  Table 1 shows the relationship between the measured chemiluminescence (count / second, cps) and the number of washings. From Table 1, it was found that in the separation method 1, generally good separation efficiency was obtained by 8 second separation operations, and the lowest count value (219 counts / second (cps)) was exhibited by 10 separation operations. Further, the amount of magnetic particles in the container after the above operation was determined by turbidity measurement (600 nm) with a spectrophotometer, and as a result, it was 98% before the cleaning operation.

実施例１
参考例と同じ抗体固定化磁性微粒子を用いたＴＳＨアッセイにおいて、Ｂ／Ｆ分離を以下の分離法２（図２）にて行った。即ち、分離法１と同一の磁石（３）をプレートに固定し、このプレートを容器１の側面の外部に、パルスモータによって水平方向に往復動できるように配置した。洗浄液を入れた状態で、磁石を、その中心を基準に、プラスマイナス１．１ｍｍ、プラスマイナス５．６ｍｍ又はプラスマイナス８．９ｍｍの距離を１１１ｍｍ／秒の速度で３回往復動させた後、直ちに洗浄液を吸引することを第１分離で３回（洗浄液量は各３５０μＬ）、第２分離で８回（洗浄液量は、1回目２５０μＬ、２回目３００μＬ、３回目以降３５０μＬ）行った。

Example 1
In the TSH assay using the same antibody-immobilized magnetic fine particles as in the reference example, B / F separation was performed by the following separation method 2 (FIG. 2). That is, the same magnet (3) as in the separation method 1 was fixed to a plate, and this plate was arranged outside the side surface of the container 1 so as to be reciprocated in the horizontal direction by a pulse motor. With the cleaning solution in the magnet, the magnet was reciprocated three times at a speed of 111 mm / sec with a distance of plus / minus 1.1 mm, plus / minus 5.6 mm or plus / minus 8.9 mm with reference to the center. Immediately aspirating the cleaning solution was performed 3 times in the first separation (the amount of the cleaning solution was 350 μL each) and 8 times in the second separation (the amount of the cleaning solution was 250 μL for the first time, 300 μL for the second time, and 350 μL for the third time and thereafter).

  Table 2 shows the relationship between the measured chemiluminescence (count / second, cps) and the magnet movement distance. From Table 2, in separation method 2, the count value is 232 cps and the magnetic particle recovery rate is 99% under the condition that the magnet is reciprocated plus or minus 8.9 mm with reference to the center, and separation efficiency comparable to that of separation method 1 is obtained. It was.

実施例２
参考例と同一の抗体固定化磁性微粒子を用いたＴＳＨアッセイにおいて、Ｂ／Ｆ分離を以下の分離法３（図３）にて行った。即ち、分離法１と同一の磁石（３）２個をプレートに固定し、このプレートを容器１の側面の外部に、パルスモータによって水平方向に往復動できるように配置した。洗浄液を入れた状態で、磁石を、２個の磁石間の中心を基準にプラスマイナス１．７ｍｍ又はプラスマイナス５．６ｍｍの距離を、１１１ｍｍ／秒の速度で磁石を３回往復動させた後、直ちに洗浄液を吸引するセットことを第１分離で３回（洗浄液量は各３５０μＬ）、第２分離で８回（洗浄液量は、1回目２５０μＬ、２回目３００μＬ、３回目以降３５０μＬ）行った。

Example 2
In the TSH assay using the same antibody-immobilized magnetic fine particles as in the reference example, B / F separation was performed by the following separation method 3 (FIG. 3). That is, two magnets (3) identical to those in the separation method 1 were fixed to a plate, and the plate was arranged outside the side surface of the container 1 so as to be reciprocated in the horizontal direction by a pulse motor. After the magnet is reciprocated three times at a speed of 111 mm / sec with a distance of plus or minus 1.7 mm or plus or minus 5.6 mm with reference to the center between the two magnets in the state where the cleaning liquid is put. Immediately after the first separation, the cleaning liquid was set three times (the amount of the cleaning liquid was 350 μL each), and the second separation was performed eight times (the amount of the cleaning liquid was 250 μL for the first time, 300 μL for the second time, and 350 μL for the third time and thereafter).

  Table 3 shows the relationship between the measured chemiluminescence (count / second, cps) and the magnet moving distance. According to the separation method 3 from Table 3, the count value is 222 cps or 226 cps and the magnetic particle recovery rate is 99 under the condition of reciprocating plus or minus 1.7 mm or plus or minus 5.6 mm with reference to the center between the two magnets. %, Separation efficiency comparable to that of separation method 1 was obtained.

実施例３
参考例１と同一の抗体固定化磁性微粒子を用いたＴＳＨアッセイにおいて、Ｂ／Ｆ分離を以下の分離法４（図４）にて行った。即ち、ネオジウム磁石（５φ×１０ｍｍ）（３）をプレートに固定し、このプレートを容器（１）の外部に、パルスモータを用いて容器側面に沿って上下方向に往復動できるように配置した。洗浄液を入れた状態で、磁石を、その中心を基準にプラスマイナス８．８ｍｍ、移動速度を１７５、２５０、３７５ｍｍ／秒、上端および下端での待ち時間をそれぞれ０．５秒および１．０秒として５回往復移動させた後、直ちに洗浄液を吸引することを第１分離で３回（洗浄液量は各３５０μＬ）、第２分離で５回（洗浄液量は、1回目２５０μＬ、２回目３００μＬ、３回目以降３５０μＬ）行った。

Example 3
In the TSH assay using the same antibody-immobilized magnetic fine particles as in Reference Example 1, B / F separation was performed by the following separation method 4 (FIG. 4). That is, a neodymium magnet (5φ × 10 mm) (3) was fixed to a plate, and this plate was arranged outside the container (1) so as to be able to reciprocate vertically along the side of the container using a pulse motor. With the cleaning solution in the magnet, the magnet is plus or minus 8.8 mm based on the center, the moving speed is 175, 250, 375 mm / second, and the waiting time at the upper and lower ends is 0.5 seconds and 1.0 seconds, respectively. As soon as the reciprocating movement is performed five times, the suction of the cleaning liquid is performed three times in the first separation (the amount of the cleaning liquid is 350 μL each), and five times in the second separation (the amount of the cleaning liquid is 250 μL for the first time, 300 μL for the second time, 3 350 μL after the first time).

  Table 4 shows the relationship between the measured chemiluminescence (count / second, cps) and the magnet movement distance. According to separation method 4 from Table 4, good separation efficiency is obtained when the magnet moving speed is 250 mm / second or more, separation is 230 cps under the condition of 375 mm / second, and the recovery rate of magnetic particles is 99%. Gained efficiency.

実施例４
実施例３における分離法４において、工程に要する時間等を、参考例における分離法１と比較した。ここで、分離法１と分離法２の各工程の時間は、Ｂ／Ｆ分離終了後の磁性粒子回収率が９８％以上となるように設定した。２ステップサンドイッチ法の第１反応後の洗浄回数はともに３回、第２反応後の洗浄回数は実施例４記載の方法においては５回（全洗浄回数８回）、分離法１においては２５０ｃｐｓ以下の洗浄終了後検出シグナルを示す８回（全洗浄回数１１回）として比較した。ただし、分離法１における1回のノズル動作、洗浄液吸排動作、磁石移動に要する時間は測定間で変動するため、実施例３記載の自動化Ｂ／Ｆ分離法において要した時間を用いた。その結果、実施例３記載のＢ／Ｆ分離法によると、全Ｂ／Ｆ分離工程に要する時間は１６１秒となり、分離法１（３４７秒）の２分の１以下となり、実施例３の結果と合わせると、少ない洗浄液置換回数で迅速に良好な洗浄効率が得られることが実証された。

Example 4
In the separation method 4 in Example 3, the time required for the process was compared with the separation method 1 in the reference example. Here, the time of each process of the separation method 1 and the separation method 2 was set so that the magnetic particle recovery rate after the completion of the B / F separation would be 98% or more. In the two-step sandwich method, the number of washings after the first reaction is 3 times, the number of washings after the second reaction is 5 times in the method described in Example 4 (total washing number is 8 times), and the separation method 1 is 250 cps or less. These were compared as 8 times (total number of times of washing 11 times) indicating a detection signal after washing. However, the time required for the automated B / F separation method described in Example 3 was used because the time required for one nozzle operation, cleaning liquid suction / discharge operation, and magnet movement in the separation method 1 varied between measurements. As a result, according to the B / F separation method described in Example 3, the time required for the entire B / F separation step was 161 seconds, which was less than half that of separation method 1 (347 seconds). In combination, it was demonstrated that good cleaning efficiency can be obtained quickly with a small number of cleaning liquid replacements.

  The process in which the method described in Example 3 can reduce the time particularly compared to the separation method 1 includes a standby process at both ends in the reciprocating movement of the magnet, and a standby process from the completion of the movement of the magnetic particles to the suction of the cleaning liquid by the nozzle. It can be seen that it is. When the magnetic particles are moved in the cleaning liquid to the container facing as in the separation method 1, the waiting efficiency is reduced because the cleaning efficiency decreases unless the magnet is moved after waiting for most of the magnetic particles to move to the facing magnet. It is necessary to take enough. Furthermore, if the time until the cleaning liquid is sucked by the nozzle after the movement of the magnet is not set sufficiently longer than the above-mentioned waiting time, the magnetic flux collection becomes slow, and the magnetic particles slightly present in the cleaning liquid may be attracted by the nozzle. There is. Even though the amount of suction at one time is negligible, all 11 washing steps are repeated, so the residual rate of magnetic particles is considered to be non-negligible.

実施例５
図５は、実施例３における分離法４（図４）を自動的に実施するための装置の一例であって、１個の磁石（３）を用いる例を示す図である。磁力透過性容器（１）は、当該容器を垂直に保持する容器保持手段（９）に保持される。磁石保持手段（４）は、ネオジウム磁石（３）（５φ×１０ｍｍ）１個を固定したプレートである。本例の装置では、容器を保持する容器保持手段を固定しておき、磁石保持手段を移動手段（４ａ）によって容器に対して相対的に移動するよう構成してある。具体的には、移動手段であるパルスモータ（４ａ）を駆動することにより、磁石保持手段（４）に保持された磁石（３）を、容器（１）の外部に設定された異なる２個以上の集磁ポジションに移動させる。図５は、磁石（３）を容器外部に設定された集磁ポジションのうちの最上端のものに配置したときの様子を示している。この他、本例の装置では、磁石（３）は容器側面に沿って昇下降するが、その量はパルスモータによって管理することが可能であるから、昇下降動の過程における任意の地点を集磁ポジションとすることが可能である。

Example 5
FIG. 5 is an example of an apparatus for automatically performing the separation method 4 (FIG. 4) in the third embodiment, and shows an example using one magnet (3). The magnetically permeable container (1) is held by container holding means (9) that holds the container vertically. The magnet holding means (4) is a plate on which one neodymium magnet (3) (5φ × 10 mm) is fixed. In the apparatus of this example, the container holding means for holding the container is fixed, and the magnet holding means is moved relative to the container by the moving means (4a). Specifically, two or more different magnets (3) held by the magnet holding means (4) are set outside the container (1) by driving the pulse motor (4a) which is the moving means. Move to the current collecting position. FIG. 5 shows a state in which the magnet (3) is arranged at the uppermost one of the magnetic flux collection positions set outside the container. In addition, in the apparatus of this example, the magnet (3) moves up and down along the side surface of the container. However, the amount can be controlled by a pulse motor, and therefore, any point in the process of moving up and down is collected. It can be in a magnetic position.

The suction means (5) for the liquid in the container and the dispensing means (6) for the cleaning liquid into the container are constituted by a vacuum pump and a pipe connected to the liquid pump (not shown), and are integrally fixed to the plate (7). ing. The plate (7) is configured to move up and down with respect to the container (1) by a moving means for suction / dispensing means comprising a guide rail (8) and a pulse motor (8a). In the suction process, the tip of the suction means (5) is lowered to the vicinity of the bottom surface of the container (1), and in the cleaning liquid dispensing process, the tip of the dispensing means (6) is lowered to the top of the container. In this example, since the magnetic collection position of the magnetic particles (2) is the inner side surface of the container (1), even if the liquid is sucked from the bottom and the cleaning liquid is dispensed from the top to the bottom, Do not disperse.
Example 6
FIG. 6 is an example of an apparatus for automatically carrying out the separation method of the present invention, and includes four low magnetic force neodymium magnets (5φ × 3 mm, 3,810 gauss) (3a) for moving magnetic particles. One high-magnetism neodymium magnet (8φ × 8 mm, 5,050 gauss) (3b) for collecting magnetic particles was held at equal intervals on the outer arc of the magnet holding means (disk-shaped member). It is a figure which shows an example. In the example of FIG. 6, a pulse motor (4b) and a driving system (not shown) are provided as rotating means for rotating the magnet holding means, and the magnet holding means is rotated so that the magnet held there approaches the container (1). , Position it at the magnetic collection position outside the container.

In the magnetic collection process, the magnetic collection magnet 4b is positioned at the magnetic collection position, and magnetic particles in the container are collected in the vicinity thereof. In this state, the suction means (5) is inserted into the container to suck the liquid in the container, and then the cleaning liquid is dispensed from the dispensing means (6). The magnetized magnetic particles can be moved along the inner surface of the container by rotating the magnet holding means, for example, in the direction of the arrow shown in the figure while the cleaning liquid is dispensed. The magnetic particles move up and down along the inner surface of the container without being dispersed in the liquid in the container from the vicinity of the magnetic collection position in the magnetic collection process, and move steps 1 to 7 in the figure as necessary. After the repetition, the magnetic flux is collected in the vicinity of the position by the action of the magnetic flux collecting magnet 3a disposed at the same position as the magnetic flux collecting position in the magnetic flux collecting step (the magnetic flux collecting step 8 in FIG. 6).
Example 7
FIG. 7 shows another example of the apparatus of the present invention using a magnet holding plate. A magnet is held by a disk-shaped magnet holding plate that reaches almost the center of the container, and the magnetic particles collected by the rotation move up and down along the inner surface of the container.
Example 8
FIG. 8 shows an example of the apparatus of the present invention using an electromagnet. It can be exemplified that magnet holding means for holding three electromagnets (3a, 3b and 3c) is provided on the side of the container, and the position of 3b is set as a magnetic collecting position in the magnetic collecting process and the moving process. That is, first, an electric current is applied to the electromagnet 3b to collect magnetic particles in the vicinity thereof, and after attracting the liquid in the container and dispensing the cleaning liquid, the electric current is applied to the electromagnet 3a or 3c. It can be exemplified that the particles move from 3a (3c) through 3b to 3c (3a) along the inner surface of the container without dispersing the particles and then move to 3c (3a), and electricity is passed through the electromagnet of 3b to collect the magnetic flux again in the vicinity thereof. As in the configuration of the sixth embodiment, the magnetism collecting magnet 3b may be stronger than the other electromagnets 3a and 3c. Moreover, when moving a magnetic particle, two electromagnets can also be used simultaneously, such as flowing electricity through both the electromagnets 3b and 3c.

FIG. 1 is a diagram for explaining separation by the separation method 1 of the reference example. FIG. 2 is a diagram for explaining separation by the separation method 2 of the first embodiment. FIG. 3 is a diagram for explaining separation by the separation method 3 of the second embodiment. FIG. 4 is a diagram for explaining separation by the separation method 4 of the third embodiment. FIG. 5 is a diagram showing one configuration example of the apparatus of the present invention described in the fifth embodiment. FIG. 6 is a diagram showing one configuration example of the apparatus of the present invention described in the sixth embodiment. FIG. 7 is a diagram showing one configuration example of the apparatus of the present invention described in the seventh embodiment. FIG. 8 is a diagram showing one configuration example of the apparatus of the present invention described in the eighth embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Container 2 Magnetic particle 3 Magnet 4 Mechanism to move magnet up and down 4 Pulse motor 5 Suction means (tube)
6 Dispensing means (pipe)

Claims (6)

  A method of separating magnetic particles dispersed in a liquid in a magnetically permeable container from the liquid, wherein a magnet is disposed at a magnetic collection position set outside the container, and a magnetic force is applied from the magnet to store the magnetic particles in the container. A magnet is arranged at a magnetic flux collecting position different from the magnetic flux collecting position in the magnetic flux collecting step for collecting the magnetic flux in the inner surface, the first suction step for sucking the liquid in the vessel, the dispensing step for dispensing the cleaning liquid into the vessel, and the magnetic flux collecting step. In addition, the magnetic force is applied from the magnet, and the magnetic force from the magnetic collection position in the magnetic collection process is stopped to move the magnetic particles from the vicinity of the magnetic collection position in the magnetic collection process to the vicinity of the different magnetic collection position along the inner surface of the container. The method comprising: a moving step of moving; and a second suction step of sucking the cleaning liquid dispensed in the dispensing step.   The method according to claim 1, wherein the method uses a single magnet, and the moving step is performed by moving a magnet arranged at a magnetic collecting position in the magnetic collecting step to the different magnetic collecting position. .   The method uses two or more magnets, and the moving step is performed by arranging a magnet different from the magnet arranged at the magnetism collecting position in the magnetism collecting step at the different magnetism collecting position. The method of claim 1.   An apparatus used for separating magnetic particles dispersed in a liquid in a magnetically permeable container from the liquid, a container holding means for holding the container, a magnet holding means for holding one or more magnets, and a container holding Moving means for moving the means and / or the magnet holding means to move the magnet to two or more different magnetic collection positions set outside the container and to apply the magnetic force of the magnet to the magnetic particles inside the container from each position The apparatus comprising: suction means for sucking the liquid in the container; and dispensing means for dispensing the cleaning liquid into the container.   The apparatus according to claim 4, wherein the magnet holding means includes a plate-like member and one or more magnets held by the plate-like member, and the moving means is a rotating means for rotating the plate-like member. .   The apparatus according to claim 4 or 5, wherein the one or more magnets are two or more kinds of magnets having different magnetic forces.

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