JPH07111429B2 - Laser magnetic immunoassay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention belongs to an ultrasensitive assay method among immunoassay methods that utilize an antigen-antibody reaction, and a specific antibody or antigen is detected from a trace amount of a sample. The present invention relates to an immunoassay method capable of detecting

[Conventional technology]

Development of an immunoassay utilizing an antigen-antibody reaction as an early test method for new types of viral diseases such as AIDS and adult T-cell leukemia, and various cancers is currently underway on a global scale. This is to detect an antibody or the antigen itself by utilizing the property that an antibody formed when an antigen such as virus invades a living body specifically reacts with the antigen (antigen-antibody reaction). It is a thing. For this,
Radioimmunoassay (RIA), enzyme immunoassay, fluorescent immunoassay and the like have been put into practical use as a microimmunoassay method. These are isotopes, enzymes,
The presence or absence of an antibody or an antigen that specifically reacts with an antigen or antibody labeled with a fluorescent substance is detected. Of these, RIA is for quantifying the amount of the sample that contributed to the antigen-antibody reaction by measuring the radiation dose of the labeled isotope, and at present, it is the only method capable of measuring ultratrace amounts in the picogram range. is there. But,
Since RIA must handle radioactive substances, special equipment is required, and in terms of half-life and waste treatment,
There were restrictions such as when and where to use it. Further, in the method using an enzyme or a fluorescent substance, since the presence or absence of an antigen-antibody reaction is confirmed by using color development or luminescence, the measurement is semi-quantitative and the detection limit is about nanogram. Therefore, RIA
There has been a demand for an immunoassay method having the same detection sensitivity as that of the above and having no limitation in use.

As a method of using laser light to detect the presence 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 plastic based on the antigen-antibody reaction is attached. A method for investigating the change in mass caused by the aggregation of the two from the change in the scattering or transmission state of laser light has been published.
With 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.

[Object of Invention and Problems to Be Solved]

The present invention is intended to solve various restrictions such as half-life and waste treatment, and to realize a new immunoassay having picogram detection sensitivity comparable to that of RIA.

[Means for solving problems]

In the present invention, 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,
By knowing the presence or absence and the amount of the magnetic substance-labeled substance in the liquid phase, the sample can be specified and quantified. 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.

[Action]

Since the present invention uses magnetic ultrafine particles as a label for an antigen-antibody reaction, unreacted magnetic substance-labeled substances can be separated and removed from a sample by a magnetic field. In addition, after the liquid phase, the magnetic substance can be concentrated by a magnetic method to improve the detection sensitivity. In addition, by selectively measuring the change in scattered light or transmitted light that is synchronized with the magnetic field while driving the magnetic material in the alternating magnetic field, it is possible to effectively remove the influence of the external environment and the scattering of the background. Can be done. In the present invention, since magnetic ultrafine particles are used as a label, there is no limitation on the time of use or the place of use.

In the present invention, the pretreatment of reacting the sample with the magnetic substance-labeled substance is carried out by an indirect method of reacting a solid-phased known antigen or antibody with the antibody or antigen as the sample, and directly with the antibody or antigen as the sample. There is a direct method of immobilizing. Further, as a method of reacting the magnetic substance-labeled body, there are a method of positively reacting the sample and the magnetic substance-labeled body, and a method of inhibiting the reaction (competitive inhibition reaction detection method). Examples will be shown below.

〔Example〕

Example 1 FIG. 1 is a diagram for explaining a first example (indirect method) of the present invention, in which (a) to (d) show a sample adjusting step, and (e)
(F) shows the preparation process of the comparative control sample. 1 is a support made of agar, 2 is a virus antibody, 3 is a virus antigen, 4 is a virus antibody labeled with a magnetic substance, 5 is a rare earth magnet,
6 is a glass cell. (A) is a known viral antibody 2
Is immobilized on the support 1, (b) is a step of injecting an unknown viral antigen 3 in the blood of the patient into the immobilized antibody 2, and causing an antigen-antibody reaction, (c) is the viral antigen 3 The step (d) is a step of reacting the magnetic substance-labeled virus antibody 4 with a solution, and a step (d) of dissolving and removing the support 1 into a glass cell 6 and dispersing the sample in an aqueous solution. (E) and (f) show the steps of preparing the comparative control sample, and (e) shows the magnetic substance-labeled virus antibody 4 which did not react with the sample due to the absence of the virus antigen 3 by the rare earth magnet 5.
Step (f) is a step of separating and removing from the solid-phased antibody, and a step of liquidizing the comparative control sample. By performing the same step as (e) on (c), the unreacted excess magnetic substance-labeled virus 4 can be removed from the sample. In this example, the reason why the virus antibody was immobilized on the agar was:
This is for facilitating the operation of separating and removing the excess labeled substance with the rare earth magnet in the step (c). The removal step of (e) was performed using a magnet by utilizing the characteristics of the magnetic substance-labeled body, but it can also be removed by washing. The combined use of a magnet and cleaning is also effective. As the magnetic substance-labeled virus antibody 4, in the present example, a magnetite ultrafine particle coated with an antibody that specifically binds to the virus antigen 3 was used.
The reason why magnetite was selected as the magnetic ultrafine particles is that magnetite has a good affinity for viruses and specific antibodies and is suitable for labeling viruses and the like. Further, in order to efficiently and effectively perform separation by an external magnetic field, removal, and measurement of scattered light and transmitted light described below, the magnetite preferably has a single domain particle structure, and the particle diameter is appropriately about 50 nm. It was The magnetic ultrafine particles are not limited to magnetite, but a compound magnetic material such as γ-ferrite,
Of course, a metal magnetic material such as iron or cobalt may be used.

FIG. 2 is a view for explaining a method of measuring the prepared specimen and comparative control specimen by the laser light scattering / transmission method described above among the examples of the present invention, and 8 is an H of output 5 mW.
eNe laser, 9 incident light, 10 scattered light, 11 transmitted light, 12 Si photodiode, 13 lock-in amplifier, 14 electromagnet, 15 0.5 Hz for driving electromagnet 14.
Is a low frequency power source, 16 is a lens that collects scattered light, and 17 is a polarizing plate. The glass cell 6 containing the sample or the comparative control sample is mounted in the electromagnet 14, the magnetic label is induced / concentrated around the laser incident light beam 9, and the scattered light flux 10 from the liquid containing the sample by the laser 8 or The transmitted light beam 11 is detected by the photodiode 12. Since the movement of the magnetic substance-labeled virus antibody 4 in the liquid phase is controlled by the electromagnet 14, the intensities of the scattered light beam 10 and the transmitted light beam 11 are controlled by the low frequency power supply 15.
It will be tuned to the frequency of. Therefore, the scattered light flux tuned to the frequency of the low-frequency power source 15 by the lock-in amplifier 13
Alternatively, if only the transmitted light beam 11 is amplified, the intensity of scattered light or transmitted light from the liquid containing the sample can be measured without being affected by disturbance such as temperature fluctuation.
In the case of the present example, the scattered light intensity from the liquid containing the specimen was measured in synchronization with the cycle of the alternating magnetic field, but the component that is synchronized with the alternating magnetic field due to the absence of the magnetic substance labeled body in the comparative control sample. The intensity of scattered light is direct current, and the background level can be known by measuring through the lock-in amplifier 13. As a result of measurement while diluting a standard solution containing a known amount of the magnetic substance-labeled substance, it was revealed that the detection limit of picogram was almost the same as that of RIA.

It should be noted that the normal measurement may be performed by scattered light, but depending on the type and concentration of the sample, it may be possible to measure higher S / N by using transmitted light. The polarizing plate 17 is used for separating and measuring the polarized component from the sample. That is, as the incident laser light, one close to linearly polarized light is used, and crossed Nicols are set so as to extinguish when there is no magnetic substance labeled substance in the liquid phase, and when the magnetic substance labeled substance is guided into the optical path. The polarization state is changed so that outgoing light can be obtained. In the present embodiment, the magnetic label is 0.5 Hz.
The scattered light from the liquid containing the sample was measured in tune with the low frequency of 1. However, the low frequency power source is not limited to 0.5 Hz, and it is optimal according to the viscosity and magnetic field strength of the aqueous solution containing the sample. It is preferable to determine the frequency.

Even in the case where the concentration by the magnetic field and the application of the alternating magnetic field were not performed in this example, it was possible to detect micrograms due to the difference in the scattering intensity between the sample containing the magnetic label and the comparative control sample.

Example 2 FIG. 3 is a diagram for explaining a second example (indirect method) of the present invention, in which (a) to (d) show the sample adjusting step, and (e)
(F) shows the preparation process of the comparative control sample. 1 is a support made of gelatin, 2 is a viral antibody, 3 is a viral antigen, and 4'is a magnetic substance-labeled anti-immunoglobulin labeled with ultrafine particles of magnetite. Here, the anti-immunoglobulin is a specific antibody formed by introducing a viral antibody into another living body, and has the property of specifically reacting with the viral antibody by an antigen-antibody reaction. Reference numeral 5 is a rare earth magnet, and 6 is a glass cell. (A) a step of immobilizing a known viral antigen 3 on a support 1, (b) a step of reacting a viral antibody 2 and a viral antigen 3 in the blood of a sample patient, (c) a magnetic substance Labeled anti-immunoglobulin 4 '
And the step of reacting the virus antibody 2 with the antigen-antibody, (d) is the step of dissolving and removing the support 1, and then placing the sample in the glass cell 6,
This is a liquid phase process of dispersing in an aqueous solution. (E) is a step of separating and removing unreacted magnetic substance-labeled anti-immunoglobulin 4'from the solid-phased antigen by the rare earth magnet 5, and (f) is a liquid phase step of the comparative control sample.

After preparing the sample through these steps,
When the sample was quantified by the laser light scattering method of
It was possible to detect a picogram of a viral antibody in the same level as in Example 1.

Example 3 FIG. 4 is a diagram for explaining a third example (direct method) of the present invention, in which (a) to (d) show a sample adjusting step. Reference numeral 1 is a support made of gelatin, 4 is a magnetic substance-labeled virus antibody labeled with ultrafine iron particles, 3'is an influenza virus, 51 is an electromagnet, and 6 is a glass cell.
(A) is an unknown virus 3'in the blood of a patient as a sample
Is immobilized on a support 1, (b) is a step of reacting a known magnetic substance-labeled viral antibody 4 with a virus 3 ',
(C) is a step of separating / removing excess magnetic substance-labeled viral antibody 4 with an electromagnet 51, and (d) is a liquid in which the support 1 is dissolved and removed, and then the sample is placed in a glass cell 6 and dispersed in an aqueous solution. It is a phase formation process.

After preparing a magnetic substance-labeled virus antibody 4 labeled with various types of known influenza virus antibodies, and reacting this with an unknown influenza virus 3 ′ collected from a patient by antigen-antibody reaction, the sample is prepared through these steps. ,
By performing a sample test by the laser light scattering method of Example 1, the influenza virus can be identified. Since the detection method according to this method has high detection sensitivity, it was possible to identify the influenza virus at an early stage of virus infection as compared with the conventional method using an enzyme or a fluorophore.

Example 4 FIG. 5 is a diagram for explaining an example of the competitive inhibition reaction detection method according to the fourth example of the present invention, in which 1 is a support made of gelatin, 2 is a viral antibody, 3 is a viral antigen. , 21 is a magnetic substance-labeled virus antigen, and 5 is a rare earth magnet. In the present embodiment, (a) to (d) represent the sample adjusting step,
(E)-(f) have shown the preparation process of the comparative control sample. (A) is a step of immobilizing a known viral antibody 2,
(B) is a step of reacting the solid-phased antibody 2 with the patient's viral antigen 3 in an antigen-antibody reaction, (c) is a magnetic substance-labeled viral antigen 21 labeled with a magnetic substance in a separate step as the antigen of (b) above The step of reacting with the sample after the antibody reaction, (d) the step of collecting the unreacted magnetic substance-labeled virus antigen 21 in step (c) by the magnet 5, and (e) the magnetic substance-labeled virus in the comparative control sample. Step (f) is a step of reacting the antigen 21, and step (f) is a step of collecting the unreacted magnetic substance-labeled virus antigen 21 by the magnet 5.

After the sample and the comparative control sample were prepared by these steps, they were measured by the laser light scattering method of Example 1, and in the case of this example, the magnetic substance-labeled virus antigen 21 was a virus in the liquid phase containing the sample. Since the antigen 3 inhibits the reaction with the viral antibody 2, it is removed by the magnet without reaction. However, since virus antigen 3 does not exist in the liquid phase containing the comparative control sample, magnetic substance-labeled virus antigen 21
Will be detected by reacting with the viral antibody 2. As a result, the magnetic substance-labeled substance was not detected in the liquid phase containing the sample, and the magnetic substance-labeled substance was detected only in the liquid phase containing the comparative control sample. However, since the number of viral antigen 3 is extremely small in the sample from the patient in the early stage of virus infection, the step (b)
Since only a part of the immobilized antibody reacts with the viral antigen 3, the magnetic substance-labeled substance can be detected even in the liquid phase containing the sample, but the detected amount decreases as the amount of the viral antigen 3 increases. The amount of viral antigen 3 can be quantified by this reduced amount.

Embodiment 5 FIG. 6 is a fifth embodiment of the present invention. FIG. 3 is a diagram for explaining another example of the competitive inhibition reaction detection method, wherein 1 is a support made of gelatin, 3 is a viral antigen, 2 is a viral antibody,
4 is a magnetic substance-labeled virus antibody, and 5 is a rare earth magnet. In the present example, (a) to (d) show the adjustment step of the sample, and (e) to (f) show the adjustment step of the comparative control sample. (A) is a step of immobilizing a known viral antigen 3, (b) is a step of reacting the immobilized antigen 3 with a patient's viral antibody 2 and an antigen-antibody reaction, and (c) is a magnetic substance-labeled viral antibody 4 Reacting with the viral antibody 2, (d)
Is a step of collecting the unreacted magnetic substance-labeled viral antibody 4 by the magnet 5, (e) is a step of reacting the magnetic substance-labeled viral antibody 4 with a comparative control sample, and (f) is an unreacted magnetic substance In this step, the labeled virus antibody 4 is collected by the magnet 5.

The same results as in Example 4 were obtained in this example as well.

In the above examples, agar or gelatin is used as the support, but there is no essential difference between them, and it is empirically selected from the combination with the immobilized antigen or antibody. .

〔The invention's effect〕

As described above, the present invention uses magnetic ultrafine particles as a label for an antigen-antibody reaction, separates and removes unreacted magnetic ultrafine particles in a magnetic field, and then irradiates a liquid containing a sample with a laser beam in a magnetic field to perform labeling. It measures scattered light or transmitted light from the body, and there are no restrictions on the time of use, place, etc. compared to the RIA method. Further, the detection sensitivity can be improved by inducingly concentrating the magnetic substance-labeled substance to the laser irradiation portion from the outside by an electromagnet or the like. Further, it is possible to eliminate the influence of the external world by controlling the movement of the magnetic substance-labeled body from the outside by the same means.

Since this assay method is particularly suitable for automating the test of antigen-antibody reaction, it is particularly effective when used for screening tests for various viruses, cancers, etc., which are required for mass screening. In addition to the antigen-antibody reaction, it can also be applied 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. As described above, the method of the present invention can be used for early diagnosis and treatment of patients, and is very useful in the medical field.

[Brief description of drawings]

FIG. 1 is a first embodiment of the present invention, FIG. 2 is a view for explaining a method for measuring scattering / transmission of laser light in the embodiment of the present invention, and FIG. 3 is a second embodiment of the present invention. Embodiment, FIG. 4 is a third embodiment of the present invention, and FIG. 5 is a fourth embodiment of the present invention.
FIG. 6 is a diagram showing a fifth embodiment of the present invention. 1 ... Support, 2 ... Viral antibody, 3 ... Viral antigen, 3 '... Influenza virus, 4 ... Magnetic labeled virus antibody, 4' ... Magnetic labeled anti-immunoglobulin, 5 ... Rare earth magnet , 6 ... glass cell, 8 ... laser, 9 ... incident light, 10 ... scattered light flux, 11 ... transmitted light, 12 ... photodiode, 13 ... lock-in amplifier, 14 ... electromagnet, 15 ... … Low frequency power supply, 16 …… Lens,
17 …… polarizer, 21 …… magnetic substance labeled virus antigen, 51 ……
electromagnet

Continued Front Page (72) Hiroko Mizutani 6-11 Udagawacho, Shibuya-ku, Tokyo

Claims (6)

[Claims] 1. A step of labeling an antigen or an antibody with magnetic ultrafine particles to form a magnetic label, and allowing the magnetic label to react with a sample in an antigen-antibody reaction; From the step of separating and removing the magnetic substance-labeled body, the step of irradiating with laser light by dispersing the sample in the liquid after the step, and the step of measuring scattered light or transmitted light from the sample by the step A laser magnetic immunoassay characterized by: 2. A sample which is subjected to an antigen-antibody reaction with a magnetic substance-labeled body after the antigen-antibody reaction between the sample and a specific antibody or antigen of the sample. Laser magnetic immunoassay according to the item. 3. The laser magnetic immunoassay method according to claim 1 or 2, wherein the antibody labeled with the magnetic ultrafine particles is an anti-immunoglobulin. 4. The laser magnetic immunoassay method according to claim 1, 2 or 3, wherein the step of separating and removing the unreacted magnetic substance-labeled body is separation and removal by a magnet. 5. A sample dispersed in a liquid is placed in a magnetic field, and the magnetic field induces and concentrates the laser light irradiation portion, and the sample is characterized in that: Laser magnetic immunoassay according to the item. 6. A sample irradiated with laser light is placed in a periodically changing magnetic field, and scattered light or transmitted light synchronized with the cycle of the magnetic field is detected. The laser magnetic immunoassay method according to item 2, 3, 4, or 5.