JPH07111428B2 - Laser magnetic immunoassay method and measuring apparatus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a laser immunoassay method and measuring apparatus utilizing an antigen-antibody reaction. More specifically, the present invention relates to a laser immunoassay method and a measuring apparatus capable of detecting a specific antibody or antigen from an extremely small amount of sample.

[Prior art and its problems] Development of an immunoassay utilizing an antigen-antibody reaction is currently in progress on a global scale as an early test method for AIDS, new types of viral diseases such as adult T-cell leukemia, and various cancers. There is. This is to detect an antibody or the antigen itself by utilizing the property that an antibody formed when a virus or the like which is an antigen invades a living body, specifically reacts with the above antigen (antigen-antibody reaction). Is. As a microimmunoassay method for this purpose, RIA (radioimmunoassay), EIA (enzyme immunoassay), FIA (fluorescent immunoassay) and the like have been put into practical use. These methods use an antigen or antibody labeled with an isotope, an enzyme, or a fluorophore, and detect the presence or absence of the antibody or antigen that specifically reacts with this.

Of these, RIA is the amount of the sample that contributed to the antigen-antibody reaction,
It is quantified by measuring the radiation dose of the labeled isotope, and at present, the only possible method is ultra-trace measurement on the picogram level. But the RIA
Since radioactive materials must be handled, special equipment is required, and there are restrictions on the time of use, location, etc. from the viewpoint of half-life and waste disposal. Further, in the method using an enzyme or a fluorescent substance, since the presence or absence of an antigen-antibody reaction is confirmed using color development or luminescence, the measurement is semi-quantitative,
The detection limit was also about nanogram. Therefore, there has been a demand for an immunoassay method that has the same detection sensitivity as RIA and is not limited in use.

As a method of using laser light to detect the presence or absence of an antigen-antibody reaction, for the purpose of detecting liver cancer, an antibody against AFP (alpha-fetoprotein) is attached to fine particles of plastic, and the plastics are separated from each other based on the antigen-antibody reaction. A method for investigating the change in mass caused by aggregation from changes in the scattering or transmission state of laser light has been published. In this method, the detection sensitivity is 10 ⁻¹⁰ g, which is 100 times or more that of the conventional method using laser light, but is less than 1/100 of the sensitivity of RIA. Since this method uses changes in Brownian motion of antigen-antibody in an aqueous solution, it is necessary to precisely control the temperature of the aqueous solution containing the sample during measurement, and to avoid the influence of the external environment such as temperature and vibration. There was a drawback that it was easily received.

Further, in the conventional laser light scattering measurement, since only a part of the aqueous solution in which the sample is dispersed is irradiated, there is a limit to the improvement of the detection sensitivity, and a large amount of sample is required.

Under such circumstances, it has been desired to develop an immunoassay method and an assay device which have detection sensitivity and accuracy comparable to those of RIA and have no practical limitation.

[Means for solving problems]

According to the first aspect of the present invention, a magnetic substance-labeled substance obtained by adding magnetic fine particles to a predetermined antigen or antibody as a label,
1st reaction between a magnetic antibody or an antigen and an antigen-antibody reaction
A magnetic field is applied to a solution containing a magnetic substance-labeled analyte complex, which is a complex of the magnetic substance-labeled substance and the analyte after the step and the first step, to induce the magnetic substance-labeled analyte complex to a predetermined position.・
In a laser magnetic immunoassay method including at least a second step of concentrating, a laser is provided at the concentration position where the magnetic substance-labeled analyte complex exists and the non-concentration position of the solution portion where the magnetic substance-labeled analyte complex does not exist. A laser magnetic immunoassay method is provided, which comprises irradiating light simultaneously or in time series and detecting a difference between the received light from the concentrated position and the received light from the non-concentrated position.

According to a modification of the first aspect of the present invention, the second step is performed by using a sample container having an opening at the upper side, and the magnet placed below the sample container and the magnet facing each other. Then, the magnet piece placed directly above the water surface of the sample container induces and concentrates, and the detecting step irradiates the water surface immediately below the magnetic pole piece and the water surface near the magnetic pole piece simultaneously or in time series. Done by

Furthermore, according to another modification of the first aspect of the present invention, the second step is performed using a thin tubular sample container, and the magnetic field at one defined point of the sample container is maximum, The sample container part in which the magnetic field is maximized and guided and concentrated by a magnet configured so that the magnetic field increases toward the maximum point of the magnetic field, and the sample container in the vicinity thereof are simultaneously or time-sequentially. It is performed by irradiating.

In the detection step, any of scattered light, transmitted light, reflected light, interference light and diffracted light may be selected as the light emitted from the sample.

Further, in the detection step, simultaneous irradiation can be performed by decomposing the laser light into two.

Furthermore, in the detection step, time-sequential irradiation can be performed by scanning the laser light between the concentrated position and the non-concentrated position.

It is possible to improve the detection sensitivity by the two-beam method, which is often used in the conventional optical measurement, regardless of whether the laser beam is divided into two or scanned. That is, signal noise unrelated to the sample due to fluctuations in the light source, scattered substances in the air, impurities in the solution, and the like can be removed.

In this case, the detection sensitivity can be improved by quantifying the sample by selectively detecting the emitted light synchronized with the scanning frequency of the laser.

According to a second aspect of the present invention, a sample container containing a sample labeled with magnetic fine particles, and a sample container
A mechanism for guiding / concentrating the magnetic substance-labeled sample complex to a point, and guiding the laser beam to one point where the magnetic substance-labeled body is guided / concentrated in the sample container, and to a total of two points other than the one point. A laser magnetic immunoassay apparatus including at least an incident optical system having a mechanism of (1) and an optical system for receiving laser scattered, transmitted or reflected, or interference light or diffracted light from the two locations in the specimen container, Provided is a laser magnetic immunoassay device comprising a gradient magnetic field generator and a beam splitter or a deflector.

At this time, it is desirable that the beam splitter or the deflector for spatially manipulating the laser beam is installed between the laser light source and the specimen container.

According to a preferred modification of the second aspect of the present invention, the gradient magnetic field generating device includes a permanent magnet or an electromagnet, and magnetic pole pieces arranged so as to sandwich the sample container so as to face the permanent magnet or the electromagnet. ing.

According to another modification of the second aspect of the present invention, any one of the sample container, the permanent magnet or the electromagnet, and the magnetic pole piece is configured to be movable in a horizontal plane.

When the method of driving the magnetic substance-labeled analyte complex inside the solution by the external magnetic force is adopted, the follow-up to the external magnetic force is naturally limited due to the viscous resistance of the solution. Therefore, in order to improve the S / N ratio, the light emitted from the magnetic substance-labeled analyte complex, transmitted light, reflected light, or interference light is repeatedly added.
When the averaging process was performed, there was a problem that the measurement time was long, but in the present invention, background noise from other than the sample that interferes with the signal from the magnetic substance-labeled sample complex can be eliminated, so that it can be performed in a short time. It is possible to measure the S / N ratio.

Needless to say, the magnetic fine particles used in the present invention do not have a problem such as radiation or toxicity, and there is no particular limitation in using them. The magnetic fine particles include various compound magnetic materials such as magnetite and γ-ferrite, and metal magnetic materials such as iron and cobalt, and easily select a stable labeling substance for a sample. be able to.

In the present invention, utilizing that the labeling substance is a magnetic substance,
The labeling substance, specimen or antigen-antibody complex substance can be selectively manipulated by magnetic force. That is, it is easy to separate and remove the unreacted magnetic substance-labeled substance from the sample, and to guide or concentrate the antigen-antibody complex with the magnetic substance-labeled substance to a specific position.

According to these inventions, while using the same laser light,
The limit of the method using AFP can be exceeded. In addition, such a configuration not only contributes to an improvement in detection sensitivity but also makes it extremely easy to automate the measurement.

Hereinafter, the present invention will be described in more detail with reference to the drawings, but the following is merely an example of the present invention and does not limit the technical scope of the present invention.

[Embodiment 1] FIG. 1 shows an example of a laser magnetic immunoassay device of the present invention.

In the figure, reference numeral 1 is a sample container, 2 is a magnetic substance-labeled sample complex, 3
Is a gradient magnetic field generator, 4 is a laser light source, 5 is a beam splitter, 6a and 6b are split laser beams, 7a and 7b are transmitted or diffracted light, 8a and 8b are ND filters, 9a and 9b are photodiodes, 10a and 10b is a scattered light flux, 11a and 11b are slits, 12a and 12b are condenser lenses, 13a and 13b are photomultiplier tubes, and 14a and 14b are electronic circuits.

A cylindrical sample container 1 made of transparent glass that is vertically arranged contains a magnetic substance-labeled sample complex 2 after an antigen-antibody reaction has occurred between the sample and the magnetic substance label. The method described in Japanese Patent Application Nos. 61-224567 and 61-254164 previously filed by the present inventors can be applied to the method for preparing a sample. The gradient magnetic field generator 3 comprises a pair of rare earth magnets 3a, 3a each having a sharp tip shape, facing each other so as to sandwich the sample container. Since the magnetic field on the side of the sample container at the tip of the magnets 3a, 3a is the highest, the magnetic substance-labeled sample complex 2 is guided and concentrated at this position. Incidentally, the magnetic field generator includes a Japanese Patent Application No. 62-152791 filed previously by the present inventors.
It is also possible to use the electromagnet described in.

A He-Ne laser light source 4 and a beam splitter 5 for splitting the laser light emitted from the light source 4 into two are provided on one side of the sample container 1 with an appropriate incident angle on one side of the sample container 1. And photodiodes 9a and 9b for detecting transmitted light or diffracted light corresponding to the respective split laser beams 6a and 6b are disposed on the other side. Further, at another position, two light receiving systems for the scattered light, which are composed of the slits 11a and 11b, the condenser lenses 12a and 12b, and the photomultiplier tubes 13a and 13b, are arranged for the respective divided incident lights. . The scattered light receiving system is preferably arranged so that it can receive scattered light at a right angle to the incident system.
Two photodiodes 9a, 9b and photomultiplier tube 13a, 1
3b is an electronic circuit 14b for processing the obtained outgoing signal,
14a respectively.

The laser light from the laser light source 4 is divided into two via the beam splitter 5 and is incident on the liquid containing the magnetic substance-labeled analyte complex through the tube wall of the analyte container 1. The incident light is preferably incident at right angles to the axial direction of the cylindrical sample container 1. One of the divided laser beams 6a is attracted by the gradient magnetic field generator 3 and passes through the concentration position where the magnetic substance-labeled sample complex existing in the vicinity of the maximum magnetic field point in the sample container 1 and passes as transmitted light or diffracted light. Is emitted. The other split laser beam 6b passes through a non-concentrating position where the magnetic substance-labeled analyte complex does not exist and is emitted as transmitted light or diffracted light. These emitted lights are ND filters
After the dimming is adjusted appropriately by 8a, 8b, the photodiode 9a,
The light is received by 9b, and the difference between the signals is detected by the electronic circuit 14b.

Further, when the particle size of the magnetic substance-labeled analyte complex is large, the split laser beam 6a is diffracted, so that a method of detecting the diffracted light beam can also be used. Furthermore, in addition to the method of detecting transmitted light and diffracted light, scattered light from the concentrated position and the non-concentrated position can be used. In the case of scattered light measurement, one split laser beam 6a is scattered at the concentrated position, the other split laser beam 6b is scattered at the non-concentrated position and emitted as scattered light beams 10a, 10b, respectively, these scattered light beams 10a, 10b. The slits 11a and 11b and the condenser lenses 12a and 12b guide the photomultiplier tubes 13a and 13b, and the electronic circuit 14a detects the difference between the signals.

Incidentally, the laser light source is not limited to He-Ne, but if a short wavelength such as He-Cd laser is used, the sensitivity is improved in the detection of a small magnetic substance-labeled analyte complex, Especially effective.

In the present embodiment, it was possible to instantaneously detect a very small amount of sample on the order of picogram.

Embodiment 2 FIG. 2 shows another example of the laser magnetic immunoassay device of the present invention.

3a is an electromagnet, 3b is a pole piece, 8 is an ND filter, 9 is a photodiode, 10 is a scattered light beam, 11 is a slit, 12 is a condenser lens, 13 is a photomultiplier tube, and 15 is a deflector.

A magnetic substance-labeled sample complex 2 is contained in a sample container 1 having an opening at the top. The sample container 1 is placed on the electromagnet (or permanent magnet) 3a so as to be movable in the horizontal direction directly or via a table, and a magnetic substance-labeled sample complex 2 is guided directly above the sample container 1. The magnetic pole piece 3c for concentration is placed. Since the tip of the pole piece 3c has a sharp shape, the magnetic flux from the electromagnet 3b is concentrated on the tip of the pole piece 3b. Therefore, the magnetic field on the water surface directly under the magnetic pole piece 3b is the highest, and the magnetic substance-labeled analyte complex 2 is concentrated in this portion.

The magnetic pole piece 3c and the electromagnet 3b as described above constitute the gradient magnetic field generator 3. The device 3 is not limited to this example, and may have a configuration using, for example, a rare earth magnet instead of the electromagnet.

Further, above the sample container 1, for example, a He-Ne laser light source 4 and a deflector 15 composed of an acousto-optic device using TeO ₂ that changes the direction of the laser beams 6a and 6b emitted from the light source 4 are magnetic poles. The piece 3c is installed on one side so as to form an appropriate incident angle, and the photodiode 9 for detecting reflected light, interference light, or diffracted light is arranged on the other side. Further, a light receiving system including a slit 11, a condenser lens 12, and a photomultiplier tube 13 for scattered light is arranged at another position. When the scattered light is weak, a photocounting type photomultiplier tube is preferably used.

Incident lights 6a and 6b from the laser light source 4 are made to pass through the deflector 15 and are made incident on the water surface immediately below or in the vicinity of the magnetic pole piece 3c at an angle of 30 ° with respect to the liquid surface of the sample container 1, respectively.
Are emitted as scattered light, reflected light, interference light, or diffracted light in the region where the magnetic substance-labeled sample complex 2 that has been attracted by and gathered near the water surface exists. These emitted lights are detected by the photodiode 9 or the photomultiplier tube 13. In this case, the laser beam is deflected by the deflector 15 and scanned on the liquid surface between the concentrated position where the magnetic substance-labeled sample complex 2 is densely packed and the non-concentrated position where the magnetic substance-labeled sample complex 2 does not exist. By irradiating the light with time, the output light signals from the concentration position and the non-concentration position are respectively divided into the signal from the concentration position and the signal from the non-concentration position detected by the photodiode 9 or the photomultiplier tube 13. The difference is detected by known means. In addition to the method of detecting reflected light and interference light, a method of detecting scattered light can be applied. In this case, the concentrated magnetic substance-labeled analyte complex and the reflected light from the water surface where the magnetic substance-labeled analyte complex does not exist are time-series.
A method of detecting with the photomultiplier tube 13 of the table can be applied.

As in the case of Example 1, it was possible to detect an extremely small amount of picogram-order sample in an instant within 1 second in this Example as well.

As a result of trying to detect influenza virus labeled with magnetic ultrafine particles by using the laser magnetic immunoassay device of this example, in the case of the conventional enzyme immunoassay (EIA), it was detected unless about 100 million viruses were present. While it was not possible, it became clear that the method of the present invention can detect even about 10 viruses.

〔The invention's effect〕

As described above in detail, according to the laser magnetic immunoassay method and apparatus according to the present invention, the most characteristic can be exhibited when the magnetic fine particles are used as the labeling substance, and the RI in a very short time.
Ultrasensitive antigen-antibody reaction test comparable to method A can be performed. Therefore, it becomes possible to widely perform accurate measurement in a general ring, which could not be performed by the RIA method in limited facilities in the past. For example, in a general situation such as mass screening, if accurate measurement such as screening tests for various viruses and cancer can be widely carried out, early diagnosis of cancer or viral diseases etc. becomes possible and effective early treatment is possible. It becomes possible to carry out accurately. Further, the laser magnetic immunoassay method and device according to the present invention is not limited to only the antigen-antibody reaction, various hormones such as peptide hormones or the like that the RIA method has been conventionally applied, or various enzymes,
It can also be applied to the measurement of vitamins and drugs. As described above, the present invention is immeasurable in the medical and medical fields.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram showing an example of the laser magnetic immunoassay device of the present invention, and FIG. 2 is a schematic configuration diagram showing another example of the laser magnetic immunoassay device of the present invention. 1 ... Sample container, 2 ... Magnetic substance-labeled sample complex, 3 ...
Gradient magnetic field generator, 4 ... Laser light source, 5 ... Beam splitter, 6a and 6b ... Split laser lines, 7a and 7b ... Transmitted or diffracted light, 8a and 8b ... ND filter, 9a and 9b
...... Photodiodes, 10a and 10b ...... Scattered luminous flux, 11a
And 11b ... Slit, 12a and 12b ... Condensing lens, 13a
And 13b ... photomultiplier tube, 14a and 14b ... electronic circuit, 1
5 Deflector.

Claims (12)

[Claims] 1. A first step in which a magnetic substance-labeled substance obtained by adding magnetic fine particles as a label to a predetermined antigen or antibody, and an antibody or an antigen as a specimen are subjected to an antigen-antibody reaction, and a magnetic substance label after the first step. A second step of causing a magnetic field to act on a solution containing a magnetic substance-labeled sample complex, which is a complex of a body and a sample, to induce / concentrate the magnetic substance-labeled sample complex at a predetermined position. In the laser magnetic immunoassay method, the concentrated position where the magnetic substance-labeled sample complex is present, and the non-concentrated position of the solution portion where the magnetic substance-labeled sample complex is not present are irradiated with laser light simultaneously or in time series, A laser magnetic immunoassay method characterized by detecting the difference between the received light emitted from the concentrated position and the emitted light from the non-concentrated position. 2. The second step is carried out using a sample container having an opening at the top, and the magnet is placed below the sample container and is placed directly above the water surface of the sample container so as to face the magnet. Induction / concentration is performed by the magnetic pole piece, and the detection step is performed by irradiating the liquid surface immediately below the magnetic pole piece and the liquid surface near the magnetic pole piece simultaneously or in time series. 2. The method for laser magnetic immunoassay according to claim 1. 3. The second step is carried out using a thin tubular sample container, and the magnetic field at one defined point of the sample container is maximum, and the magnetic field increases toward the maximum point of the magnetic field. Patent is characterized in that it is performed by irradiating the sample container part in which the magnetic field is maximized and the sample container in the vicinity thereof simultaneously or in time series, which is induced / concentrated by the magnet configured as described above. The laser magnetic immunoassay method according to claim 1. 4. The laser according to claim 1, wherein the detecting step is performed by detecting scattered light, transmitted light, reflected light, interference light or diffracted light from the detection body. Magnetic immunoassay method. 5. The laser magnetic immunoassay method according to claim 1, wherein in the detection step, simultaneous irradiation is performed by dividing the laser light into two. 6. The method according to claim 1, wherein in the detecting step, time-sequential irradiation is performed by scanning a laser beam between the concentrated position and the non-concentrated position.
Laser magnetic immunoassay method according to the item. 7. The laser magnetic immunoassay according to claim 1, wherein in the detecting step, the quantification of the sample is performed by selectively detecting a signal synchronized with the scanning frequency of laser light. Measuring method. 8. A sample container for containing a sample labeled with magnetic ultrafine particles, a mechanism for inducing and concentrating a magnetic substance-labeled substance at one point in the sample container, and a laser beam in the sample container. An incident optical system having a mechanism for guiding a magnetic substance label to one point where it is guided and concentrated and a total of two points other than the one point,
A laser magnetic immunoassay apparatus including at least an optical system for receiving light emitted from the two locations inside the specimen container, comprising a gradient magnetic field generator and a beam splitter or a deflector. And a laser magnetic immunoassay device. 9. The laser magnetic immunoassay apparatus according to claim 8, wherein a beam splitter is installed between the laser light source and the sample container. 10. The laser magnetic immunoassay apparatus according to claim 8, further comprising a deflector installed between the laser light source and the specimen container for spatially operating a laser beam. 11. The gradient magnetic field generating device comprises a permanent magnet or an electromagnet, and a magnetic pole piece installed so as to sandwich the sample container so as to face the permanent magnet or the electromagnet. The laser magnetic immunoassay device according to any one of claims 8 to 10. 12. The sample container, the permanent magnet or electromagnet, and any one of the magnetic pole pieces are movable in a horizontal plane, according to any one of claims 8 to 10. 2. A laser magnetic immunoassay device according to claim 2.