JP3428746B2 - Immunoassay method and analyzer - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method and an immunoassay apparatus, and more particularly to an immunoassay method and an immunoassay apparatus for determining a test substance in a sample using a chemiluminescent label.

[0002]

2. Description of the Related Art As an example of using magnetic particles as a solid phase in an immunoassay utilizing an antigen-antibody reaction, Japanese Patent Publication No. 62-50
There is issue 784. In this prior art, the magnetic particles and the labeled antibody are allowed to act while the sample is introduced into the duct and circulated, and the antigen and the label in the sample are bound to the magnetic particles. Next, at the location of a magnetic trap provided in the middle of the pipe, the magnetic particles are captured against the flow of the liquid to separate the liquid phase and the solid phase. afterwards,
The magnetic particles are released from the magnetic trap and the radioactivity from the label on the solid phase is measured by a counter while the magnetic particles are flowing downstream. An electromagnet is used for the magnetic trap.

On the other hand, an example of using a chemiluminescent substance as a label for immunoassay is described in Japanese Patent Publication No. 64-500146. Its typical substance is ruthenium bipyridyl R
Ruthenium-containing compounds such as u (bpy) ₃ . In this prior art, an immunocomplex is formed by an immune reaction in which a label made of a chemiluminescent substance is bound to a test component, and the label containing the immunocomplex is chemiluminescent by applying electrochemical energy to a liquid containing the immunocomplex. Is shown.

[0004]

[Problems to be Solved by the Invention] Japanese Patent Publication No. 62-5078
When a chemiluminescent label such as that shown in Japanese Patent Publication No. 64-500146 is applied to the structure of No. 4, it is necessary to emit light because the solid phase to which the label is bound must flow. The efficiency is poor and sufficient measurement sensitivity cannot be obtained.

Therefore, the inventors of the present invention captured the solid phase by a magnetic force in the luminescence chamber having a working electrode and a counter electrode, and electrochemical energy was applied to the label on the solid phase with the solid phase kept in the luminescence chamber. I tried to make it emit light. However, when an electromagnet is used as the magnetic field applying means, it is not only difficult to obtain a magnetic flux density sufficient to capture the solid phase, but it is also found that there is an influence of the residual magnetic field. It was also found that the magnetic field lowers the detection efficiency of the photomultiplier tube as a photodetector when the label is made to emit light while the magnetic field is being applied to the light emitting chamber.

It is an object of the present invention to ensure that the solid phase can be retained in the luminescence chamber and that the luminescence of the label on the solid phase can be detected without being affected by the magnetic field.

Another object of the present invention is to make it possible to bring the magnets close to each other so that the reproducibility of positioning with respect to the light emitting chamber is kept good.

[0008]

The immunoassay method according to the present invention comprises forming an immunocomplex of a test component in a sample and a chemiluminescent label on a solid phase composed of magnetic particles in a container,
In an immunoassay method for measuring luminescence based on a label on a solid phase, a tilt provided on a magnet holding unit holding a permanent magnet.
Surface contacts the inclined surface of the receiving part of the member forming the luminous chamber
In a state in which to approximate permanent magnet emission chamber having a working electrode and a counter electrode by, passed through the reaction product-containing liquid from the container to the emission chamber, to stay in the light emitting chamber of the solid phase in containing liquid by magnetic force And evacuating the permanent magnet away from the light emitting chamber, and then applying a voltage between the working electrode and the counter electrode to cause the label on the solid phase to emit light in the light emitting chamber where the solid phase remains. including.

The immunoassay device according to the present invention comprises a reaction container in which a reaction for forming a combined product of a test substance in a sample and a chemiluminescent label on a solid phase composed of magnetic particles,
A light emitting chamber in which the working electrode and the counter electrode are arranged, a photodetector arranged in the vicinity of the light emitting chamber, and a liquid introduction device for transferring the above-mentioned liquid containing the reaction product from the reaction container so that the liquid contains the reaction product. , A permanent magnet that applies a magnetic field to the light emitting chamber so that the solid phase in the contained liquid is captured when the contained liquid passes through the luminous chamber, and a permanent magnet is placed close to the light emitting chamber when capturing the solid phase. positioning, and a magnet moving device for moving the permanent magnet so as to eliminate the influence of the magnetic field on the optical detector at the time of voltage application between the working electrode and the counter electrode, said magnet moving device is inclined plane
It is equipped with an arm for rotating the magnet holding part,
The member forming the light emitting chamber fits the inclined surface
It is equipped with a receiving part .

[0010]

The biological fluid is used as a sample, and a test component such as a specific antigen contained in the sample is bound to a solid phase composed of magnetic particles by an immune reaction in the container, and the labeled antibody is bound to the test component. This results in the formation of labeled immune complexes on the solid phase. When the fluid containing the reaction product in the sample is introduced so as to pass through the luminescence chamber to which a magnetic field is applied, the liquid containing the unbound reagent of the reaction passes through the luminescence chamber, but the solid phase composed of magnetic particles. Is retained in the light emitting chamber by the magnetic force of the permanent magnet. Thereby, B / F separation of the solid phase and the liquid phase is performed in the light emitting chamber. A number of magnetic particles that make up the solid phase, some bound to the immunocomplex and some unbound, the immunocomplex contain a chemiluminescent label.

Next, when the voltage is applied between the working electrode and the counter electrode in the light emitting chamber by separating the permanent magnet from the light emitting chamber so that the magnetic field does not reach the photodetector, electrochemical energy is given to the label to emit light. . This luminescence is measured by a photodetector located close to the luminescence chamber. The amount of luminescence corresponds to the amount of the label on the solid phase, and therefore has a correlation with the amount of the test component contained in the sample.

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described with reference to FIGS.

In the reaction vessel 25 shown in FIG. 1, first, a predetermined amount of magnetic particles as a solid phase is immunoreacted with a specific antigen as a test component in a sample. Since the antibody capable of binding to the specific antigen is conjugated to the surface of the solid phase, the antigen is bound to the solid phase. Next, an antibody reagent having a chemiluminescent label is added to the reaction container 25 to advance the next immune reaction. Since this labeled antibody is bound to the specific antigen, an immune complex having a label is formed on the solid phase. After such an immune reaction, the suction nozzle 24 is inserted into the reaction container 25, and the fluid containing the reaction product is sucked by the action of the syringe 27 so as to pass through the flow cell 19 functioning as a light emitting chamber. Along with this, the liquid phase containing unreacted reagents and the like flows away from the flow cell 19 downstream, but the solid phase composed of magnetic particles flows into the flow cell 19.
It is retained by the magnetic force of the magnet 6 made of a permanent magnet having a magnetic flux density of 1.5 Tesla. In this state, the buffer solution is sucked from the suction nozzle to wash the inside of the flow cell 19.
The buffer solution contains tripropylamine (TPA) that attracts the excitation of the labeling substance. Ruthenium (II) trisbipyridyl is suitable as the chemiluminescent labeling substance.

A photomultiplier tube 22 as a photodetector is fixedly installed on the flow cell 19. When voltage is applied between the working electrode and the counter electrode while the buffer solution containing the attractant is filled in the flow cell 19, electrochemical energy is given to the label on the solid phase retained in the flow cell 19, The sign emits light. The magnet 6 is moved by the movable arm 3 and approaches the flow cell 19 as necessary, but the positioning error between the working electrode 16 and the magnet 6 when capturing the magnetic particles is configured to be 0.05 mm or less. ing. The distance between the magnetic pole surface of the magnet 6 and the working electrode 16 in the flow cell 19 is set to 0.5 mm with the cell constituent members separated. The magnet 6 is moved by the arm 3 when the sign is caused to emit light.

A mechanism for moving the permanent magnet will be described. A stepping motor 1 is used as a driving means, and a flange 2 having a support shaft 2a extending in the axial direction is fixed to a rotation output shaft 1a thereof by a screw 1b. A ball bearing 9 is attached to a support shaft 2a extending from the flange 2 with an arm 3 integrated with a holder 5 having a stopper having an inclined surface 5a. A magnet 6 is attached to a holder 5 that is integrated with the arm 3. A pulling spring 4 is hung between the support shaft 2a side of the flange 2 and the arm 3, and a pin 7 is fixed to the other side of the support shaft 2a side of the flange 2 perpendicularly to the surface of the flange 2. There is. The pin 7 is passed through the hole 8 of the arm 3. Furthermore, the pin 7 is located at the right end of the hole 8 provided in the arm 3 by the action of the pulling spring 4 that is hung between the flange 2 and the arm 3. Further, the bush 10 is attached to the support shaft 2a.
Is attached and is in contact with the inner ring of the ball bearing 9. Further, a push spring 11, a retaining washer 12, and a screw 13 are attached to the support shaft 2a.

Next, the structure of the flow cell will be described. The flow cell 19 has a cell base 14 having a magnet receiving hole having an inclined surface for positioning the magnet 6 with respect to the light emitting chamber, and a transparent plastic cover 14a that allows light to pass therethrough. The cell base 14 and the cover 14a are integrated via a spacer 15, and a flow chamber 18 in which the reaction solution containing the introduced complex flows is formed near the center of the spacer 15 (FIG. 3). The flow chamber 18 is spindle-shaped when viewed from above, and one end of the flow chamber 18 is located at the flow path inlet 18a and the other end is located at the flow path outlet 18b and is connected to the conduit 31. Further, the working electrode 16 is arranged so as to be exposed at the central lower surface of the spindle-shaped maximum width portion of the flow chamber 18, and the two counter electrodes 17 are arranged so as to be exposed symmetrically on the same plane on both sides of the working electrode 16. ing. The working electrode 16 and the counter electrode 17 extend outside the cell base 14 and are connected to the power supply 23.

A photomultiplier tube 22 is attached on the flow cell 19 via a cover 14a, and a PMT case 20 accommodating a shield tube 21 is installed in the outer periphery thereof to form a detection section.

The magnet drive unit and the flow cell 19 are installed such that the upper surface of the magnet 6 is parallel to the working electrode 16 when the arm 3 of the magnet drive unit is rotated toward the working electrode 16. Holder 5 in position
Cell base 14 is fixed to the substrate plate 29 as I of the inclined surface 14d Gahame case of positioning holes of the conical-like inclined surface 5a and the cell base 14 of the stopper.

The operation of the magnet drive mechanism thus configured will be described. In FIG. 1, two stopped states at the turning end of the arm 3 are shown by a solid line and a two-dot chain line. The solid line corresponds to the magnetic particle capture position, and the chain double-dashed line corresponds to the retracted position. First, in the initial state, the arm 3 is in the retracted position. Next, when trapping the magnetic particles on the working electrode 16 of the flow cell 19, a pulse number slightly larger than the pulse number corresponding to the angle from the retracted position to the trapped position with reference to the position where the protrusion 32 crosses the sensor 30 is used. It is given to the stepping motor 1. Along with this, the flange 2 and the arm 3 simultaneously rotate clockwise so as to approach the working electrode 16 by the action of the pulling spring 4 that is hung on the flange 2 and the arm 3. Since the arm 3 is pressed against the inner ring of the ball bearing 9 by the push spring 11 via the bush 10, the arm 3 can rotate without rattling, and the inclined surface 5a provided in the holder 5 has an inclination of the magnet positioning hole of the cell base 14. Surface 14b
By contact with the upper surface of the magnet 6 and the working electrode 1
The distance from 6 and the center position are determined. Further, the rotation of the stepping motor 1 by the extra number of pulses is absorbed by the extension of the pulling spring 4 that is hung between the flange 2 and the arm 3, so that the working electrode 16 of the magnet 6 is absorbed.
The positioning relative to is maintained. In that state, a syringe 27 is operated by operating a syringe 27 with a liquid such as a reaction liquid containing an immune complex or a reagent that induces electrochemiluminescence, with the electromagnetic valve 26 opened and the electromagnetic valve 28 closed. Two
4 is sucked into the flow chamber 18 of the flow cell 19 to capture the magnetic particles, to which the measurement component and the labeling substance are bound, on the surface of the working electrode 16. Next, the stepping motor 1 is rotated counterclockwise to reversely rotate the arm 3 to the retracted position. By this operation, the magnet 6 returns to the initial position where no magnetic force acts on the photomultiplier tube 22.
Next, a voltage is applied between the electrode composed of the working electrode 16 and the counter electrode 17 to cause a chemical reaction in the captured complex, and the amount of emitted light is measured by the photomultiplier tube 22. After the measurement, with the magnet 6 in the retracted position, the cleaning liquid is sucked from the suction nozzle 24 to wash away the magnetic particles in the flow cell 19. After that, the above operation is repeated for the subsequent samples.

According to the above-described embodiment, the holder to which the magnet is fixed is fitted to the inclined surface provided in the positioning hole of the flow cell, and the arm integrated with the holder is rotated without rattling. Since the magnet is positioned with respect to the electrodes of the flow cell, the positioning of the magnet can be accurately reproduced.

[0021]

According to the present invention, the solid phase can be securely retained in the light emitting chamber by the magnetic force, and the influence of the magnetic field on the photodetector can be eliminated when the label emits light.
This brings an effect that an immunoassay utilizing chemiluminescence can be performed with high measurement accuracy.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of an immunoassay device which is an embodiment of the present invention.

FIG. 2 is a top view of the magnet drive mechanism in the embodiment of FIG.

FIG. 3 is a top view for explaining the configuration near the flow chamber in the flow cell in the embodiment of FIG.

[Explanation of symbols]

1 ... Stepping motor, 3 ... Arm, 4 ... Pull spring,
5 ... Holder, 6 ... Magnet, 9 ... Ball bearing,
14 ... Cell base, 15 ... Spacer, 16 ... Working electrode,
17 ... Counter electrode, 18 ... Flow chamber, 19 ... Flow cell, 22 ... Photomultiplier tube, 24 ... Nozzle, 27 ... Syringe.

─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Hiroyasu Uchida 882 Ichige, Iwaki Katsuta, Ibaraki Prefecture Hitachi Ltd. Measuring Instruments Division (56) Reference JP-A-6-160401 (JP, A) Special Table Flat 10-509798 (JP, A) Special Table Flat 6-508203 (JP, A) (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 33/543 G01N 21/76 G01N 33/553

Claims (2)

(57) [Claims] 1. A in the solid phase consisting of the magnetic particles in the vessel allowed to form an immune complex of the test component and chemiluminescent labels in the sample, the immunoassay method for measuring a light emission based on the label, permanent The tilt provided on the magnet holder that holds the magnet
Connect the slope to the slope of the receiving part of the member that constitutes the luminous chamber.
In a state in which a permanent magnet is brought close to the light emitting chamber having a touch is allowed to working and counter electrodes, passed through the reaction product-containing liquid from the container into the light emitting chamber, wherein the solid phase in the contained liquid by magnetic force Staying in the lighting chamber, and before
After retracting the serial permanent magnet away from the light chamber, the label on the solid phase from the light emitting chamber the solid phase remains by applying a voltage between the said working electrode counter electrode An immunoassay method, which comprises causing light emission. 2. A reaction vessel in which a reaction for forming a bound substance of a test substance in a sample and a chemiluminescent label is formed on a solid phase composed of magnetic particles, and a luminescence chamber in which a working electrode and a counter electrode are arranged. a light detector disposed in the immediate vicinity of the light emitting chamber,
A liquid introduction device which the reaction product-containing liquid from the reaction vessel to transfer the content liquid to pass through the emission chamber, said containing <br/> chromatic solution of the contained liquid within it passes through the light emitting chamber a permanent magnet for applying a magnetic field to the light emitting chamber as the solid phase is captured,
Positioned such that the at the time of solid phase capture proximate the permanent magnet into the light emitting chamber, wherein when a voltage is applied between the working electrode into between the counter electrode so as to eliminate the influence of the magnetic field to said detector
A magnet moving device for moving the permanent magnet, said magnet moving device causes rotation of the magnet holding portion having an inclined surface
It has a closing arm and constitutes the light emitting chamber.
An immunoassay device characterized in that the member has a receiving portion that fits the inclined surface .