JP2509227B2 - Laser magnetic immunoassay device - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunoassay device that utilizes an antigen-antibody reaction. More specifically, the present invention relates to a laser magnetic immunoassay device capable of quantitatively detecting a specific antibody or antigen from an extremely small amount of sample.

[Conventional technology]

The development of immunoassays using antigen-antibody reactions as an early test method for new viral diseases such as acquired immunodeficiency syndrome, adult T-cell leukemia, and various cancers is currently being promoted worldwide. I have.

Radioimmunoassay (hereinafter referred to as RIA method), oxygen immunoassay (EIA), fluorescent immunoassay method, and the like have already been put into practical use as conventionally known microimmunoassay methods using a primary reaction. These methods are methods in which the presence or absence of an antibody or an antigen that specifically reacts with an antigen or antibody to which an isotope, an enzyme, or a fluorescent substance is added as a label, respectively.

The RIA method quantifies the amount of the sample that contributed to the antigen-antibody reaction by measuring the radiation dose of the labeled isotope, and is currently the only method capable of measuring an ultratrace amount of picogram. However, since this method uses a radioactive substance, special equipment is required, and attenuation of the labeling effect due to half-life and the like must be taken into consideration, so that there is a large limitation in its implementation. Furthermore, considering the current situation where radioactive waste treatment is a social issue, its implementation is naturally limited.

On the other hand, the method of using an enzyme or a fluorescent substance as a label is a method of detecting the amount of a sample that has contributed to the antigen-antibody reaction by observing color development or luminescence, and there is no limitation in practice such as the RIA method. However, it is difficult to accurately quantify the color development or luminescence, and the detection limit is about nanogram.

Further, as a method of detecting the presence or absence of an antigen-antibody reaction using laser light, there is a method using AFP (alpha-fetoprotein) developed mainly for the purpose of detecting liver cancer.

This method is a method in which an antibody against AFP is added to plastic microparticles and the mass change caused by aggregation of plastic particles due to an antigen-antibody reaction is examined, and 10 ^-10 g
The detection sensitivity of is achieved. This is more than 100 times more sensitive than the conventional method using laser light, but is less than 1/100 of that of the RIA method. Furthermore, since this method utilizes the change in Brownian motion of the antigen-antibody complex in the aqueous solution, the temperature of the aqueous solution containing the antibody, the influence of the fluctuation, or the influence of the impurity particles mixed in the aqueous solution is extremely susceptible, In principle, it is unexpected that the detection sensitivity is further increased.

As described above, in the above-mentioned immunoassay method, the RIA method having high detection sensitivity has many restrictions on its implementation because it uses a radioactive substance, while an enzyme immunoassay method that is easy to perform, Fluorescent immunoassays and the like have low sensitivity and cannot perform precise quantitative measurement.

Therefore, the present inventors have provided Japanese Patent Application No. 61-224567 "Laser Magnetic Immunoassay" as an immunoassay that eliminates the above-mentioned drawbacks.

In this method, first, magnetic ultrafine particles are used as a label, and a specific or unknown antigen or antibody is attached with this label to obtain a magnetic substance-labeled body. Next, the antibody or antigen as a specimen is allowed to undergo an antigen-antibody reaction with a known solid-phased antigen or antibody, or the antibody or antigen as a specimen is directly solid-phased to cause an antigen-antibody reaction with the magnetic substance-labeled body. Let After that, the unreacted magnetic substance-labeled body is removed, and then the sample is dispersed in the liquid phase. In this case, when the sample is an antigen or an antibody that causes a specific antigen-antibody reaction with the magnetic label, the magnetic label remains in the liquid phase containing the sample, and in other cases , There is no magnetic substance label in the liquid phase. Therefore, it is possible to identify and quantify the sample by knowing the presence or absence and the amount of the magnetic substance-labeled substance in the liquid phase. The presence or absence and the amount of the magnetic substance-labeled substance can be known by measuring the scattering of laser light by the sample dispersed in the liquid phase and the change in the intensity of transmitted light.

According to this method, there is an advantage that the detection sensitivity and accuracy are comparable to those of the RIA method, but there is no practical limitation.

Furthermore, the inventors of the present invention have proposed a laser magnetic immunoassay method and apparatus that embody the above-described measurement method in Japanese Patent Application No. 61-2524.
Offered in No. 27. In the laser magnetic immunoassay device according to this application, the concentration of the magnetic substance-labeled substance and the control of scattered light of the specimen are performed mechanically by using a permanent magnet, or by using an electromagnet or the like having a plurality of arrangements. It was done like this.

In addition, the inventors of the present invention have conducted the above-mentioned measurement method (Japanese Patent Application No. 61-2245).
Japanese Patent Application No. 62-22062 is also provided as a laser magnetic immunoassay method and apparatus which is a more specific version of the Japanese patent No. 67). The laser magnetic immunoassay device according to this application is also one in which the concentration of the specimen labeled with the magnetic ultrafine particles and the control of the scattered light from the magnetic substance labeled body are performed by a general electromagnet or a permanent magnet.

[Problems to be solved by the invention]

The present invention has the following problems with the above-mentioned laser magnetic immunoassay device.

That is, in the apparatus of Japanese Patent Application No. 61-252427, the mechanism is complicated by the mechanical method using a permanent magnet, and the structure of the electromagnet is not suitable for generating a strong magnetic field by the method using an electromagnet. Therefore, there is a drawback that it takes a long time to locally concentrate the magnetic label using a magnetic field.
Also, in the apparatus of Japanese Patent Application No. 62-22062, it is difficult to locally generate a strong magnetic field because a general electromagnet or a permanent magnet is used. For example, in order to concentrate the sample, it is necessary to generate a high gradient magnetic field on the surface of the sample container, but when the magnetic core diameter of the electromagnet is reduced, the magnetic flux in the magnetic core is saturated. Therefore, there is a drawback that it takes a long time to concentrate the sample.

Therefore, an object of the present invention is to provide a laser magnetic immunoassay device having a simple structure and capable of locally concentrating a sample in a short time.

[Means for solving problems]

The present invention provides a test container having an opening above which houses a sample labeled with magnetic ultrafine particles, a laser light source that guides laser light to the liquid surface of the sample in the test container, and scattered light of the laser light by the sample. A light receiving system for receiving the light, an electronic circuit section for processing the output of the light receiving system, a concentration mechanism for concentrating the magnetic substance labeled sample just below the liquid surface of the sample, and a periodic drive of the magnetic substance labeled sample after concentration. And a DC power source for exciting the electromagnet, wherein the concentrating mechanism and the drive mechanism are arranged such that the concentrating mechanism and the driving mechanism sandwich an electromagnet and a magnetic core of the electromagnet so as to sandwich the inspection container. And an intermittent pulse power source, and the electronic circuit section is configured to selectively detect only scattered light that is periodic in the intermittent pulse, and repeatedly add and average the scattered light signals. Characterized by A.

According to a preferred aspect of the present invention, the magnetic core of the electromagnet and the magnetic pole piece are made of a high-permeability material having a small residual magnetization, and the magnetic pole piece having a needle-like tip portion is installed immediately above the liquid surface of the sample, The laser light source is attached so as to irradiate the liquid surface in the inspection container just below the magnetic pole piece.

In addition, the direct charging source and the intermittent pulse power source continuously output a large direct current for a predetermined time, and then the cycle is from 0.05 Hz.
It is controlled to output intermittent pulses within the range of 20 Hz and within DC offset.

Further, a mechanism is provided for moving either the inspection container or the electromagnet and the pole piece in a horizontal plane.

〔Example〕

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

FIG. 1 is a schematic configuration diagram of a laser magnetic immunoassay device for explaining an embodiment of the present invention.

1 is an inspection container, 2 is a sample container in the inspection container, 3 is an electromagnet, 4 is a magnetic core of the electromagnet, 5 is a magnetic pole piece, 6 is a laser light source, 7 is a laser incident optical axis, and 8 is a scattered light detection axis. , 9 is a slit, 10 is a condenser lens, 11 is a photomultiplier tube, 12 is the pole piece holding component, 13 is a guide groove for moving the inspection container, 14 is the electromagnet support, and 100 is an electronic circuit section. is there.

The sample container 2 of the inspection container 1 is open upward, and the sample container 2 contains, for example, a magnetic substance-labeled sample after an antigen-antibody reaction. The inspection container 1 and the method for adjusting the sample are described in Japanese Patent Application No. 62-2206 previously invented by the present inventors.
The inspection container and the sample preparation method described in No. 3 are preferable. Since the inspection container 1 can move in one direction in the horizontal plane along the guide groove 13, it is possible to continuously measure a plurality of samples in the same container.

The electromagnet magnetic core 4 and the pole piece 5 are preferably made of a high magnetic permeability material having a small residual magnetization, for example, pure iron having high purity is recommended. The diameter of the electromagnet core 4 is equal to the specimen container 2 of the inspection container 1.
It is essential that the diameter of the magnetic pole piece 5 is sufficiently larger than the diameter of the sample container 2 and the diameter of the magnetic pole piece 5 is sufficiently smaller than the diameter of the sample container 2 of the inspection container 1. For example, when the diameter of the sample container 2 is 10 mm, the diameters of the magnetic core 4 and the pole piece 5 are 50 mm and 2 mm, respectively.
Furthermore, it is preferable that the pole piece 5 has a sharp tip such as a needle on the side facing the magnetic core 4. The magnetic pole piece 5 is screwed to the magnetic pole piece holding component 12 so that the gap between the magnetic pole piece 5 and the inspection container 1 can be adjusted.

The laser incident optical axis 7 is 30 degrees to the water surface of the inspection container 1,
The scattered light detection axis 8 is set at an angle of 45 degrees with respect to the water surface. The slit 9 and the condenser lens 10 are used to guide only the scattered light from the specimen concentrated under the magnetic pole piece 5 to the photomultiplier tube 11. These slit 9, lens 10, and photomultiplier tube 11 constitute a light receiving system for receiving scattered light of laser light. The scattered light detection axis 8 is set so as to avoid the reflected light from the water surface of the laser irradiation beam that interferes, and the laser incident light axis 7 is preferably at a lower angle than the scattered light detection axis 8. The electronic circuit section 100 processes the output of the photomultiplier tube 11.

FIG. 2 is a diagram for explaining the operation principle of the device of the present invention, in which 15 is a DC power supply, 16 is an intermittent pulse power supply, and 20 is a magnetic substance labeled specimen. In (a), the adjusted sample is the sample container 2
Immediately after being placed in the
15 is connected to 15 and is excited by direct current, (c) shows electromagnet 3
Is connected to the intermittent pulse power supply 16 and is excited,
(D) schematically shows the dispersed state of the magnetic substance-labeled specimen in the non-excited state.

In step (a), the electromagnet 3 is non-excited, so that the sample is uniformly distributed in the container. In the step (b), since the magnetic field of the electromagnet 3 is concentrated on the magnetic pole piece 5 placed directly above the inspection container 1, the magnetic substance labeled sample 20 is concentrated on the water surface immediately below the magnetic pole piece 5. Therefore, the sharper the tip of the pole piece 5, the more localized the concentration. The pole piece 5 is used in the steps (c) and (d).
Is a step of exciting the concentrated sample immediately below the pulsed pulse, and detecting the laser scattered light from the sample. When the intermittent pulse power supply 16 is de-excited, the concentrated sample diffuses in the solution due to Brownian motion. Therefore, when the electromagnet 3 is intermittently excited, the sample repeats concentration and diffusion in synchronization with the excitation period. In order to cause rapid diffusion of the sample in the non-excited state, the electromagnet 3
It is essential that the magnetic core 4 and the magnetic pole pieces 5 have no residual magnetization. Gravity acts mainly on the diffusion of the sample, so that it mainly goes downward. Therefore, as described above, it is better to set the laser incident optical axis 7 at a low angle and irradiate only the vicinity of the water surface of the sample container 2 from the sample. The change in scattered light intensity becomes large.

In this device, the magnetic substance-labeled specimen after concentration is periodically driven as described above. The scattered light from the sample is received by the photomultiplier tube 11. The output of the photomultiplier tube 11 is supplied to the electronic circuit unit 100, and the electronic circuit unit 100 selectively detects only the scattered light synchronized with the intermittent pulse, and repeatedly adds and averages the scattered light signals. . For the detection of scattered light from the specimen, the influence of disturbance or background can be removed very effectively by adopting a method of repeatedly accumulating only the fluctuation component synchronized with the intermittent pulse frequency and performing the averaging process. The range of intermittent pulse frequency is 0.05Hz to 20Hz. This is because the measurement takes a long time below 0.05 Hz and the sample does not follow above 20 Hz. Also,
The pulse has a peak value smaller than the DC exciting current value,
There is preferably no offset. Thus, according to this apparatus, it is possible to detect a very small amount of the magnetic substance-labeled body on the picogram level.

Although the inspection container 1 is configured to be movable in the horizontal plane in the above embodiment, the electromagnet and the magnetic pole piece may be configured to be moved in the horizontal plane with respect to the inspection container instead of this configuration. .

〔The invention's effect〕

The laser magnetic immunoassay device according to the present invention has a configuration that uses an electromagnet and a magnetic pole piece that faces the electromagnet, and thus has a simple configuration and can locally generate a high gradient magnetic field.
Therefore, the adjusted sample after the antigen-antibody reaction can be locally concentrated in an extremely short time.

Further, since the specimen concentrated just under the magnetic pole piece moves up and down by the intermittent pulsed magnetic field, the intensity of scattered light from the specimen can be increased by making the laser enter the specimen at a low angle.

When measuring laser scattered light, in addition to the scattered light detection method synchronized with the intermittent pulse period, the scattered light signals from the sample are repeatedly added and averaged to improve measurement sensitivity and measurement reproducibility. It can be significantly improved. When the scattered light synchronized with the intermittent pulse is selectively measured, the influence of the external environment, the scattering of the background, and the like can be extremely effectively performed, so that the detection sensitivity can be further improved.

With these characteristic configurations of the present invention, it is possible to break through the limit of the method using AFP while using the same laser scattered light measurement. Further, such a feature not only contributes to the improvement of detection sensitivity, but also makes the automation of measurement extremely easy.

The magnetic ultrafine particles used as the label have no problem in terms of radiation or toxicity, and those that are stable with respect to the sample can be easily obtained. The laser magnetic immunoassay device according to the present invention can be applied not only to the antigen-antibody reaction but also to the measurement of various hormones such as peptide hormones to which the RIA method has been conventionally applied or various enzymes, vitamins, drugs and the like. It is possible.

Therefore, it becomes possible to perform a wide range of precise measurements in a general environment, which could not be performed conventionally in limited facilities without using the RIA method. In general situations such as mass screening, if accurate measurements such as screening tests for various viruses and cancers can be widely performed, early diagnosis of cancer or viral diseases can be made, and effective early treatment can be accurately performed. Can be implemented. Thus, the effect of the present invention in the medical and medical fields is immeasurable.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a laser magnetic immunoassay device for explaining an embodiment of the present invention, and FIGS. 2 (a) to (d) are diagrams for explaining the operation principle of the device of the present invention. FIG. 7A is a diagram showing a state immediately after the adjusted specimen is put into the specimen container 2, and FIG. 7B is a diagram showing a state in which the electromagnet 3 is connected to the DC power supply 15 and is DC-excited. FIG. 7C is a diagram showing a state where the electromagnet is connected to the intermittent pulse power supply 16 and is excited, and FIG. 7D is a diagram schematically showing a dispersed state of the magnetic substance labeled sample in the non-excited state. 1 ... inspection container, 3 ... electromagnet, 4 ... magnetic core, 5 ... magnetic pole piece, 6 ... laser light source, 9 ... slit (light receiving system),
10 …… Condensing lens (light receiving system), 11 …… Photomultiplier tube (light receiving system), 15 …… DC power supply, 16 …… Intermittent pulse power supply, 20…
… Magnetic substance labeled specimen, 100 …… Electronic circuit.

Claims (4)

(57) [Claims] 1. A test container having an opening above which houses a sample labeled with magnetic ultrafine particles, a laser light source that guides laser light to the liquid level of the sample in the test container, and scattering of laser light by the sample. A light-receiving system that receives light, an electronic circuit unit that processes the output of the light-receiving system, a concentration mechanism that concentrates the magnetic substance-labeled sample just below the liquid surface of the sample, and a magnetic substance-labeled sample after concentration periodically. A driving mechanism for driving the concentrating mechanism and the driving mechanism, wherein the concentrating mechanism and the driving mechanism are provided with an electromagnet, a magnetic pole piece disposed so as to sandwich the inspection container so as to face the magnetic core of the electromagnet, and a direct current for exciting the electromagnet. It is composed of a power source and an intermittent pulse power source, and the electronic circuit section is configured to selectively detect only scattered light synchronized with the intermittent pulse and repeatedly add and average the scattered light signals. Characterized by The magnetic immunoassay apparatus. 2. The magnetic core of the electromagnet and the magnetic pole piece are made of a high-permeability material having a small residual magnetization, and the magnetic pole piece having a needle-like tip portion is installed directly above the liquid surface of the sample, and the laser light source is provided. 2. The laser magnetic immunoassay device according to claim 1, wherein the laser magnetic immunoassay device is attached so as to irradiate a liquid surface in the inspection container just below the magnetic pole piece. 3. The DC power supply and the intermittent pulse power supply have a cycle of 0.05 when a large DC is continuously output for a predetermined time.
The laser magnetic immunoassay device according to claim 1, wherein the laser magnetic immunoassay device is controlled so as to output a pulse having an intermittent pulse within a range of Hz to 20 Hz and having no DC offset. 4. The laser magnetic immunoassay device according to claim 1, wherein either the inspection container or the electromagnet and the magnetic pole piece move in a horizontal plane.