JP2010286297A - Immunoassay method and immunoassay device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunoassay method and an immunoassay device for quickly gathering reactive conjugates in an inspection solution without using any nozzles. <P>SOLUTION: The immunoassay method includes: a reaction process for producing reactive conjugates by allowing a luminescent label, a magnetic body, and a substance to be analyzed to react with one another in a vessel; a first reactive conjugate capturing process for capturing the reactive conjugates while the vessel passes a first magnetic field generation section; a second reactive conjugate capturing process for capturing the reactive conjugates at a second magnetic field generation section after the first reactive conjugate capturing process; and a measurement process for measuring the substance to be analyzed after the second reactive conjugate capturing process. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an immunoassay method and an immunoassay device.

  As a conventional immunoassay method using an antigen-antibody reaction, there is a fluorescence immunoassay method (Patent Document 1). In this measurement method, first, a fluorescent substance bound to an antibody and an antigen (analyte) are reacted in a test container, and a magnetic complex having an antibody bound to the reaction product is added and reacted to produce a reaction complex. Thereafter, the fluorescence intensity emitted when the reaction complex is irradiated with light is measured to determine the amount of the analyte. In the measurement of the analyte, the reaction complex collected by the magnet is irradiated with laser light, and the fluorescence intensity generated from the phosphor of the reaction complex is detected. Using the fact that the fluorescence intensity is proportional to the amount of the analyte, the analyte is quantitatively measured from the detected fluorescence intensity.

  In addition, a technique for shortening the time for collecting magnetic particles with a magnet has been proposed. In Patent Document 2, the number of reaction complexes that pass through a magnetic field created by a magnet is increased by discharging a liquid or gas from a stirring suction nozzle into a cuvette and blowing up magnetic beads that existed at the bottom of the cuvette. There has been proposed a method of gathering magnetic particles in a test solution with a magnet in a short time.

JP-A-5-249114 JP 2001-116752 A

  In the fluorescence immunoassay described above, when the reaction complex enters an area where the magnetic force is exerted by the magnet, it is attracted to the magnet. The force with which the reaction complex is attracted to the magnet is proportional to the amount of the magnetic substance contained in the magnetic particles constituting the reaction complex, that is, the volume of the magnetic substance.

  On the other hand, the viscous resistance that the reaction complex receives from the solution is proportional to the contact area between the reaction complex and the solution, that is, the surface area of the reaction complex. Since the reaction complex is usually small and has a small volume, the viscous resistance becomes larger than the force attracted by the magnet even in the vicinity of the magnet.

  Therefore, there is a problem that it takes a very long time to collect the magnetic particles in the cuvette with a magnet.

  Further, in the technique as disclosed in Patent Document 2, since the nozzle is put in the sample, there is a problem that the nozzle needs to be cleaned when the sample is continuously measured.

  The present invention has been made in view of the above, and an object of the present invention is to provide an immunoassay method and an immunoassay device that can assemble reaction complexes in a test solution in a short time without using a nozzle. To do.

  In order to solve the above-described problems and achieve the object, the immunoassay method according to the present invention includes a reaction step of reacting a luminescent label, a magnetic substance, and an analyte in a container to produce a reaction complex; A first reaction complex capturing step in which the container passes through the first magnetic field generation unit and captures the reaction complex, and a second reaction in which the reaction complex is captured by the second magnetic field generation unit after the first reaction complex capturing step. It is characterized by comprising a complex capturing step and a measuring step for measuring the analyte after the second reaction complex capturing step.

  In the immunoassay method according to the present invention, the first reaction complex capturing step includes a magnetic field passing step in which the container passes through the first magnetic field generating unit, a magnetic field changing step in which the magnetic field line distribution changes in the passing direction of the reaction complex, It is preferable to provide.

  In the immunoassay method according to the present invention, it is preferable that in the first reaction complex capturing step, the container passes through the first magnetic field generator while rotating around the direction along the passing direction.

  The immunoassay device according to the present invention includes a test container containing a reaction complex generated from a luminescent label, a magnetic substance, and an analyte, a first magnetic field generator, a second magnetic field generator, and an analyte. And a measuring means for measuring a substance.

  In the immunoassay device according to the present invention, it is preferable that the first magnetic field generating unit has a magnet arranged on the circumference so as to surround the cuvette.

  In the immunoassay device according to the present invention, it is preferable that the first magnetic field generator is arranged so that adjacent magnets have different poles.

  In the immunoassay device according to the present invention, it is preferable that the first magnetic field generator has a plurality of magnets arranged at substantially equal intervals.

  In the immunoassay device according to the present invention, it is preferable that the magnetic force line distribution created by the first magnetic field generator changes with respect to the passing direction of the reaction complex.

  The immunoassay method and the immunoassay apparatus according to the present invention have an effect that the reaction complexes in the test solution can be collected in a short time without using a nozzle.

It is a top view which shows the structure of the immunoassay apparatus which concerns on 1st Embodiment, Comprising: It is a figure which shows a part with a block diagram. (A) is a diagram schematically showing a luminescent label to which an antibody is bound, (b) is a diagram schematically illustrating an antigen as an analyte, and (c) is a schematic diagram of magnetic particles to which an antibody is bound. The figure which shows, (d) is a figure which shows typically the reaction complex 155 produced | generated from these. It is a perspective view which shows the structure of a 1st magnetic field generation part. It is sectional drawing which shows the positional relationship of the passage area | region of a test container, and a 1st magnetic field generation part and a 2nd magnetic field generation part. It is a top view which shows the example of arrangement | positioning of the magnetic pole of the magnet which comprises a 1st magnetic field generation part. (A) is a perspective view which shows the structure of the 1st magnetic field generation part of the immunoassay apparatus which concerns on 2nd Embodiment, (b) is a top view which shows the structure of a 1st magnetic field generation part, (c) is (a). Sectional drawing in a VIC-VIC line, (d) is sectional drawing in the VID-VID line of (a). (A) is a perspective view which shows the structure of the 1st magnetic field generation part of the immunoassay apparatus which concerns on 3rd Embodiment, (b) is a figure which shows the state which the strong magnetic field generate | occur | produced in the position VIIA, (c) is (b) (D) is a diagram showing a state where a strong magnetic field is generated at a position VIIC different from (b) and (c).

  Embodiments of an immunoassay method and an immunoassay device according to the present invention will be described below in detail with reference to the drawings. In addition, this invention is not limited by the following embodiment.

(First embodiment)
FIG. 1 is a top view showing the configuration of the immunoassay device according to the first embodiment, and is a diagram showing a part in a block diagram. In FIG. 1, the first magnetic field generation unit located above the second magnetic field generation unit is not shown.
The immunoassay device 100 includes a cuvette 110, a first magnetic field generation unit 120, a second magnetic field generation unit 130, and measurement means.

  In the cuvette 110, a reaction complex generated from a luminescent label, a magnetic substance, and an analyte is accommodated. The inspection container 110 is preferably a plastic container that can store a microliter to milliliter sample. For example, an Eppendorf tube or a microtube can be used. The material of the cuvette 110 may be any material as long as it allows magnetic force to pass, and may be made of glass or the like.

  FIG. 2 is a diagram schematically showing an antibody 151, a luminescent label 152, an antigen 153 as an analyte, a magnetic particle 154 as a magnetic substance, and a reaction complex 155. FIG. The bound luminescent label 152 is shown, (b) shows the antigen 153 as the analyte, (c) shows the magnetic particles 154 to which the antibody 151 is bound, and (d) shows that these react with the antigen-antibody. The resulting reaction complex 155 is shown.

  The substantially Y-shaped object in FIGS. 2 (a), 2 (c), and 2 (d) is an antibody 151, and the antibody 151 specifically corresponds to the antigen 153 as the analyte shown in FIG. 2 (b). Antigen-antibody reaction.

  The luminescent label 152 emits fluorescence or Raman scattered light by light irradiation. As the luminescent label 152, for example, fluorescent microbeads, quantum dots, or SERS nanotags are used.

  As the fluorescent microbead, for example, a probe (flow cytometry alignment bead) (manufactured by Molecular Probes) that is excited by light at 488 nm and emits fluorescence at 515 nm to 660 nm can be used.

The SERS nanotag is a bead having a diameter of several tens to several hundreds of nanometers that emits strong Raman scattered light by light irradiation. For example, US Pat. No. 7,192,778 discloses the emission of Raman scattered light having a wavelength of 800 to 1800 cm ⁻¹ , and when irradiated with excitation light (laser light) having a wavelength of 633 nm, the Raman scattered light having a wavelength of 670 nm to 710 nm is disclosed. There are examples that occur. The structure of the SERS nanotag is, for example, a structure in which gold nanoparticles to which a Raman active reporter molecule of about 60 nm is attached are shielded with an encapsulating shell containing polymer, glass, or any other dielectric material. Here, “SERS” is an abbreviation of “Surface Enhanced Raman Scattering”.

  The luminescent label is desirably chemically stable, has an absorption band capable of emitting light at the wavelength of the light source unit 141, and is made of a material that does not cause nonspecific adsorption between the luminescent label and the magnetic particles.

The magnetic particle 154 as a magnetic body is a bead having a diameter of several hundred nm to several μm, in which a polystyrene core in which a magnetizable substance (Fe ₂ O ₃ ) is uniformly distributed is covered with a hydrophilic polymer, For example, Dynabeads (trademark) (manufactured by Dynal) can be used.

  FIG. 3 is a perspective view showing the configuration of the first magnetic field generator 120. The first magnetic field generator 120 is composed of four magnets 122 arranged so as to surround the cuvette passage region 121. These magnets 122 are preferably magnets having a strong magnetic force (for example, neodymium magnets, electromagnets). When the cuvette passage region 121 is formed in a cylindrical shape, the magnets 122 are equiangularly spaced on the circumference around the central axis. It is good to arrange. The number of magnets constituting the first magnetic field generation unit 120 can be arbitrarily set, and may be 1 to 3 or 5 or more.

FIG. 4 is a cross-sectional view showing the passing relationship of the cuvette 110 and the positional relationship between the first magnetic field generator 120 and the second magnetic field generator 130.
The first magnetic field generation unit 120 is disposed on the upstream side of the path through which the cuvette 110 passes from the position where the cuvette 110 and the second magnetic field generation unit 130 face each other. Accordingly, the cuvette 110 reaches the position facing the second magnetic field generator 130 after passing through the cuvette passage region 121 of the first magnetic field generator 120.
A magnetic field is formed inside the magnet by the first magnetic field generator 120.

FIG. 5 is a plan view showing an arrangement example of the magnetic poles of the magnet 122 constituting the first magnetic field generation unit 120.
As shown in FIG. 5, in the first magnetic field generation unit 120, four magnets 122 are sequentially arranged so that adjacent magnets have different magnetic poles facing the central axis side of the cuvette passage region 121. In general, the lines of magnetic force exit from the north pole and enter the south pole and do not cross or overlap each other. Therefore, as shown in FIG. 5, when the magnets 122 are arranged so that different magnetic poles are adjacent to each other toward the center of the cuvette passage region 121, a magnetic force line 123 is formed between the adjacent magnets, and a magnetic force line crossing the cuvette 110. The number of 123 increases. When the magnetic lines of force 123 are formed in this way, the reaction complexes 155 are attracted along the lines of magnetic force 123, so that a large number of reaction complexes 155 can be collected by the first magnetic field generator 120. As a result, the reaction complex 155 further aggregates to a large particle size, and is less susceptible to surface resistance than when not aggregated. Therefore, when the cuvette 110 reaches the second magnetic field generation unit 130 and is arranged in the measurement unit, the reaction complex 155 can be collected in the second magnetic field generation unit 130 in a short time.

  The second magnetic field generator 130 is arranged to collect the reaction complex in the cuvette 110 at a predetermined position. This predetermined position is a position at which the cuvette 110 is held by the cuvette holder 131, and is a position at which the measuring device irradiates the reaction complex with predetermined light and detects light from the reaction complex. . The second magnetic field generator 130 is preferably a magnet having a relatively strong magnetic force, such as a neodymium magnet or an electromagnet.

  The measuring means includes a light source unit 141 as an incident optical system for irradiating light to the cuvette 110, and a light detection unit 142 as a detection optical system for detecting light from the reaction complex in the cuvette 110. And an operation unit 143.

  For the light source unit 141, for example, a laser, a laser diode, or an LED can be used. For the light detection unit 142, for example, a spectroscope, a photomultiplier tube, a photodiode, or photon counting can be used. In the detection of light from the reaction complex, if it is necessary to remove the excitation light, it is preferable to place an interference filter in front of the light detection unit 142 to eliminate the influence of the excitation light.

  In the light detection unit 142, optical signals from the luminescent label 152 and the magnetic particles 154 of the reaction complex 155 are measured. The output of the light detection unit 142 is input to the calculation unit 143 of the data processing computer and used for data accumulation / analysis.

  The light emitted from the light source unit 141 is condensed into the measurement region of the cuvette 110 by the excitation light irradiation lens 144. The lens 144 preferably has a large numerical aperture (NA). This is because when the NA is large, the light source spot diameter can be set as small as about 10 to 100 μm. In other words, the light from the light source unit 141 is irradiated to the narrow region in order for the second magnetic field generation unit 130 to collect the reaction complex in a predetermined narrow region.

  On the other hand, light emission from the non-reactive luminescent label 152 becomes background noise. Therefore, when the NA of the excitation light irradiating lens 144 is large, only the luminescent label 152 constituting the reaction complex 155 can be efficiently excited, and the excitation of the unreacted luminescent label 152 not bound to the magnetic particles is suppressed. be able to.

  Fluorescence or Raman scattered light emitted from the reaction complex 155 is collected by the signal detection lens 145 and collected by the light detection unit 142. The lens 145 preferably has a large numerical aperture (NA). When NA is large, fluorescence emitted from the reaction complex 155, Raman scattered light, or the like can be efficiently collected. If it is necessary to remove the excitation light, an interference filter can be placed in front of the detector to eliminate the influence of the excitation light.

Here, the tip position of the second magnetic field generator 130, the focal position of the excitation light irradiation lens 144, and the focal position of the signal detection lens 145 substantially coincide with each other.
In the above configuration, the reaction complex 155 and the magnetic particles in the cuvette 110 are applied to the tip of the second magnetic field generator 130 by the action of the magnetic force of the magnetic particles 154 and the magnetic field generated by the second magnetic field generator 130. 154 is collected.

Next, the immunoassay method according to the first embodiment will be described.
The immunoassay method of the first embodiment includes a reaction step, a first reaction complex capturing step, a second reaction complex capturing step, and a measuring step.

  First, in the reaction step, the reaction complex 155 is produced by reacting the luminescent label 152, the magnetic particles 154, and the antigen 153 in the cuvette 110.

  After the completion of the reaction process, the cuvette 110 containing the reaction complex 155 passes through the magnetic field gradient formed in the cuvette passage region 121 by the first magnetic field generation unit 120 and is then transported to the measurement unit (the first unit). Reaction complex capture step). In the first reaction complex capturing step, when the cuvette 110 enters the magnetic field created by the first magnetic field generation unit 120, the reaction complex 155 including the magnetic particles 154 gathers along the lines of magnetic force generated by this magnetic field. Furthermore, as the cuvette 110 passes through the first magnetic field generator 120, the reaction complex 155 is collected by the magnetic field, and the aggregate of the reaction complex 155 gradually increases.

  Subsequently, in the second reaction complex capturing step, when the cuvette 110 is held by the cuvette holding unit 131 and placed in the measuring unit at a predetermined position for measurement, the second magnetic field generating unit 130 creates the cuvette. Due to the magnetic field, the reaction complex 155 including the magnetic particles 154 in the cuvette 110 gathers in the vicinity of the second magnetic field generator 130. At this time, the reaction complex 155 is aggregated by the first reaction complex capturing step to have a large particle size. Therefore, compared with the case where the reaction complex 155 is not aggregated, it is less affected by the surface resistance. For this reason, when the cuvette 110 is arranged in the measurement unit, the reaction complex 155 can be collected on the second magnetic field generation unit 130 side in a short time.

(Second Embodiment)
6A is a perspective view showing the configuration of the first magnetic field generation unit 220 of the immunoassay apparatus according to the second embodiment, FIG. 6B is a plan view showing the configuration of the first magnetic field generation unit 220, and FIG. Sectional drawing in the VIC-VIC line of (a), (d) is sectional drawing in the VID-VID line of (a).
In the immunoassay device according to the second embodiment, the configuration of the first magnetic field generation unit 220 is different from that of the first magnetic field generation unit 120 according to the first embodiment. Other configurations are the same as those of the immunoassay apparatus according to the first embodiment, and the same reference numerals are used for the same members.

  As shown in FIGS. 6A and 6B, the first magnetic field generation unit 220 includes a plurality of magnets 222 arranged in a spiral around the central axis of the substantially cylindrical cuvette passage region 221. The plurality of magnets 22 are arranged so that the magnetic poles facing the central axis side of the cuvette passage region 221 in the adjacent magnets are different from each other.

When the magnet 222 is arranged in a spiral like this, as shown in FIGS. 6 (c) and 6 (d), the distribution of lines of magnetic force across the cuvette 110 changes depending on the location of the first magnetic field generator 220. Therefore, in the first reaction complex capturing step, the reaction complex 155 not only aggregates along the lines of magnetic force but also moves as the cuvette 110 moves in the cuvette passage region 221 and the magnetic field line distribution changes. As a result, it is possible to bind to the reaction complex 155 in another place in the cuvette 110, and the particle diameter becomes larger. Therefore, when the cuvette 110 is placed in the measurement unit, the reaction complex 155 can be collected in the second magnetic field generation unit 130 in a short time.
In addition, about another structure, an effect | action, and an effect, it is the same as that of 1st Embodiment.

(Third embodiment)
FIG. 7A is a perspective view showing the configuration of the first magnetic field generator 320 of the immunoassay device according to the third embodiment, and FIG. 7B is a plan view showing the configuration of the first magnetic field generator 320 at the position VIIA. The figure which shows the state in which the strong magnetic field generate | occur | produced in (c) is a top view which shows the structure of the 1st magnetic field generation part 320, Comprising: The figure which shows the state which the strong magnetic field generate | occur | produced in the position VIIB different from (b), (D) is a top view which shows the structure of the 1st magnetic field generation part 320, Comprising: It is a figure which shows the state which the strong magnetic field generate | occur | produced in the position VIIC different from (b) and (c).

  In the immunoassay device according to the third embodiment, the configuration of the first magnetic field generator 320 is different from that of the first magnetic field generator 120 according to the first embodiment. Other configurations are the same as those of the immunoassay apparatus according to the first embodiment, and the same reference numerals are used for the same members.

  As shown in FIG. 7, the first magnetic field generator 320 includes a plurality of electromagnets 322 arranged around the central axis of the substantially cylindrical cuvette passage region 321. An alternating current having a phase shift is applied to each electromagnet 322 by a control unit (not shown).

  In the first magnetic field generation unit 320, by passing an alternating current through each electromagnet 322, the magnetization intensity generated in the first magnetic field generation unit 120 changes with time. For example, at the time shown in FIG. 7A, the position where the magnetic field is strong is the position of VIIA, and at another time shown in FIG. 7B, the position where the magnetic field is strong is the position of VIIB. At a further point in time, the strong magnetic field is at the position of VIIIC.

In this way, by applying an alternating current having a phase shift to each electromagnet 322 and moving the position where the magnetic field is strong, the reaction complexes 155 at different positions in the cuvette 110 can be coupled to each other, Further, the reaction complex 155 has a larger particle size. Therefore, when the cuvette 110 is placed in the measurement unit, the reaction complex 155 can be collected in the second magnetic field generation unit 130 in a short time.
In addition, about another structure, an effect | action, and an effect, it is the same as that of 1st Embodiment.

Next, a modification of the above embodiment will be described.
In the first embodiment, when the cuvette 110 is moved while being rotated, the reaction complexes 155 at different positions in the cuvette 110 can be combined with each other with a simple apparatus configuration. A reaction complex 155 having a diameter can be generated.

  In the second embodiment, when the cuvette 110 is moved while being rotated, the reaction complexes 155 at different positions in the cuvette 110 can be combined with each other while having a simple device configuration. A reaction complex 155 having a diameter can be generated. Furthermore, it is preferable that the arrangement direction (spiral direction) of the magnet 222 and the rotation direction of the cuvette 110 are opposite. Thereby, the rotational speed of the reaction complex 155 in the cuvette 110 can be increased, and the reaction complex 155 having a large particle size can be generated in a short time.

  In each embodiment, it is preferable to use a non-magnetized material (for example, stainless steel) for the member that carries the cuvette 110. Since a non-magnetized material is used, the cuvette 110 can be easily transported in the first and second reaction complex capturing steps.

  As described above, the immunoassay apparatus and immunoassay method according to the present invention are useful for immunoassays that require a reaction complex in a test container to be assembled in a short time.

DESCRIPTION OF SYMBOLS 100 Immunoassay apparatus 110 Test container 120 1st magnetic field generation part 121 Test container passage area 122 Magnet 123 Magnetic field line 130 2nd magnetic field generation part 131 Test container holding part 141 Light source part 142 Light detection part 143 Calculation part 144, 145 Lens 151 Antibody 152 Luminescent label 153 Antigen 154 Magnetic particle 155 Reaction complex 220 First magnetic field generator 221 Test container passage area 222 Magnet 320 First magnetic field generator 321 Test container passage area 322 Electromagnet

Claims (8)

A reaction step of producing a reaction complex by reacting a luminescent label, a magnetic substance, and an analyte in a container;
A first reaction complex capturing step in which the container passes through the first magnetic field generator and captures the reaction complex;
A second reaction complex capturing step of capturing the reaction complex in a second magnetic field generation unit after the first reaction complex capturing step;
A measuring step of measuring the analyte after the second reaction complex capturing step;
An immunoassay method comprising: The first reaction complex capturing step includes:
A magnetic field passing step in which the container passes through the first magnetic field generation unit;
A magnetic field changing step in which a magnetic field line distribution changes with respect to a passing direction of the reaction complex;
The immunoassay method according to claim 1, comprising: The first reaction complex capturing step includes:
2. The immunoassay method according to claim 1, wherein the container passes through the first magnetic field generator while rotating around a direction along a passing direction. A cuvette containing a luminescent label, a magnetic material, and a reaction complex generated from the analyte;
A first magnetic field generator;
A second magnetic field generator;
A measuring means for measuring the analyte;
An immunoassay device comprising:   The immunoassay device according to claim 4, wherein the first magnetic field generation unit has a magnet arranged on the circumference so as to surround the cuvette.   6. The immunoassay apparatus according to claim 5, wherein the first magnetic field generation unit is arranged so that adjacent magnets have different poles.   The immunoassay apparatus according to claim 5 or 6, wherein the first magnetic field generation unit includes a plurality of magnets arranged at substantially equal intervals.   The immunoassay device according to any one of claims 5 to 7, wherein the magnetic field line distribution created by the first magnetic field generator changes with respect to a passing direction of the reaction complex.

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