JPH042963A - Method for measuring laser-magnetic immunity - Google Patents

Abstract

PURPOSE:To prevent coagulation of magnetic fine particles and enable stable and highly sensitive measurement by using a combination of stabilized radical compound and fine particles as magnetic fine particles. CONSTITUTION:A combination of stabilized radical compound and fine particles is used as magnetic fine particles. Compound having stable radicals in the air or in water solution is used as the stabilized radical compound which is one of components for constituting the magnetic fine particles. As fine particles being the other component, hydrophilic polymer particles whose particle diameter is approximately 5mum or less or non-magnetic fine particles such as DNA (deoxyribonucleic acid) is used. As shown in a graph indicating intensity of interference fringes obtained by radiating laser light, the interference fringe intensity of the combination increases in proportion to stabilized radical compound concentration while interference fringe is not formed only with the stabilized radical compound and the interference fringe intensity will not change even if the concentration is increased.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a laser magnetic immunoassay method that utilizes antigen-antibody reactions and can quantitatively detect a specific antibody or antigen from an extremely small amount of a sample.

[Conventional technology]

The laser magnetic immunoassay method developed by the present inventors has already been published in Japanese Patent Application No. 61-224567 and Japanese Patent Application No. 6i25.
This invention is disclosed in prior inventions such as No. 2427 and Japanese Patent Application No. 61-254164.

This laser magnetic immunoassay method uses magnetite (Fe304) microparticles or microparticles whose surfaces are coated with physiologically active substances such as sugars such as Textran and proteins such as protein A. A magnetic label is created by combining an antibody (or antigen) with another antibody. Then, this magnetically labeled material is subjected to an antigen-antibody reaction with the antigen (or antibody) of the specimen, such as influenza virus, to generate the antigen (or antibody) that is the specimen.
is magnetically labeled to create a magnetically labeled body (first step).

Next, as shown in FIG. 5, the liquid in which the magnetic label is dispersed is poured into a container 1, and the container 1 is placed in a gradient magnetic field generating device comprising an electromagnet 2 and a magnetic pole piece 3. When the electromagnet 2 is excited, the magnetic field at the liquid surface directly below the magnetic pole piece 3 is highest, so the magnetically labeled specimen is concentrated in this area. When the magnetic field in this area exceeds several kilograms, the liquid surface microscopically rises due to the balance between the vertically upward magnetic attraction force on the magnetically labeled specimen and the surface tension of the liquid. When laser light 5 is incident on this raised portion 4 from an oblique direction and the reflected light 6 from the liquid surface is received by the screen, interference fringes appear. By measuring the light intensity of this interference pattern, the amount of magnetically labeled specimen can be determined.

This laser magnetic immunoassay has the advantage of being able to detect trace amounts of viruses and the like with extremely high sensitivity (]X]]O-log/rnρ due to the magnetic concentration effect.

By the way, as the magnetic particles used in this laser magnetic immunoassay method, there are magnetite particles or magnetite particles coated with a physiologically active substance such as protein A as described above, or magnetite particles are monodispersed in particle size. There is a problem in that it is difficult to separate the particles, and magnetite fine particles coated with protein A inevitably cause aggregation of the particles. For this reason, the number of antigens that can be captured by antigen-antibody reaction is reduced, resulting in a relative decrease in antibody titer, which is disadvantageous in that it is difficult to improve sensitivity.

[Invention or problem to be solved]

Therefore, an object of the present invention is to develop a magnetic immunoassay using magnetic fine particles with good monodispersity without causing aggregation.
The objective is to provide the l+ constant method.

[Means to solve the problem]

This problem can be solved by using a combination of a stable Rancal compound and a fine particle as the magnetic fine particle.

The present invention will be explained in detail below.

As the stable Rancar compound which is one of the components constituting the magnetic fine particles of the present invention, a compound having a Rancar which is stable in air or in an aqueous solution is used.Specifically, N-oxyl 2. 2.6.6-Titramethylpipericine derivatives, 5-membered ring pyrroline derivatives shown in Table 2, and N-oxyl-4,4-dimethyloxazolinone derivatives shown in Table 3.

Note that rTEMPOJ in Table 1 represents 2,26.6-tetramethylpipericin-1-okyl, and f-PROXYLI in Table 2 represents 2,26.6-tetramethylpipericin-1-okyl. Represents 1-oxyl, r D in Table 3
OXYLJ represents 4.4dimethyloxazole/n-1-oxyl, and "A-]°゛," in these tables
The symbols such as "B-]" and "C-]" represent the structural formulas of the compounds corresponding to the symbols shown in FIGS. 2 to 4.

In addition, the other component of the magnetic fine particles, the fine particles, includes hydrophilic polymer particles with a particle size of 5 μm or less;
Non-magnetic microparticles such as silica beads, colloidal silica, DNA, and (deoxyribonucleic acid) are used. Examples of hydrophilic polymers include poly-2-hydroxymethacrylate-1, poly-2hydroquinoacrylate-1, polyglycinol acrylate-1, polyglycinol methacrylate, polyacrylamide 1-, and polymethacrylamide. And so on.

For the bonding of the stable rancal compound and the microparticles, any reaction may be used as long as it does not destroy the stable radicals of the stable radical compound, and various conventionally known organic reactions may be used, such as Another method is to add an aqueous solution containing a stable U/C compound to an aqueous suspension containing microparticles and stir the mixture.

The thus obtained conjugate consisting of a stable Rancal compound and microparticles can be used as magnetic microparticles as is or coated with a physiologically active substance such as a sugar such as textran or a protein such as protein A. . The reaction with antigen or antibody is similar to conventional laser magnetic immunoassay.

A combination of such a stable radical compound and microparticles is attracted to a magnetic pole of about 5 kG. Therefore, they are used in the same way as conventional magnetite particles as magnetic fine particles in laser magnetic immunoassay.

FIG. 1 is a graph showing the intensity of interference fringes obtained by dispersing this combined body in water, applying a magnetic field to this dispersion using the apparatus shown in FIG. 5, and irradiating it with laser light.
It can be seen that in the conjugate, the interference fringe intensity increases in proportion to the concentration of the stabilizing radical compound. On the other hand, if only the stabilizing radical compound is used, no interference fringes are formed, and even if the concentration thereof is increased, no change in the intensity of the interference fringes occurs. Of course, even small particles alone will not be attracted to the magnetic poles, and no interference fringes will be formed.

In the laser magnetic immunoassay method of the present invention, since DNA can be used as the microparticle, a combination of D'NA and a stabilized Rancar compound can be used as the magnetic microparticle. For this reason, a stable radical compound is chemically bonded to the hybridized DNA probe using the Southern blot technique.
In addition, this laser magnetic immunoassay method can be used to perform DNA probe measurements with higher sensitivity than conventional methods. It can be used in gene detection methods, viral nucleic acid assays, gene expression assays, mutation detection methods in nucleic acid base sequences, and the like. These methods are advantageous because there is no need to handle radioactive isotopes such as p-32, which are essential in the Sasan process.

A specific example will be shown below.

(Example 1) 4-Amino-22,6,6-titramethylpiperine as a stabilizing radical compound was added to an aqueous suspension of polyglycerol methacrylate fine particles having a particle size in the range of 05 to 3μ as microparticles. Add an aqueous solution of -1-oquine and stir at room temperature for 1 hour to covalently bond the glycidyl group of polyglyne/methacrylate and the amine 7 group of 4-amino-2,2,6,6-tetramethylpipericin-1-oxyl. A conjugate of both was obtained. The surface of this conjugate was coated with protein A, and an IgG antibody isolated from rabbit immune serum against influenza virus was bound to the surface of protein A to obtain a magnetically labeled product.

Next, influenza virus was bound to this magnetically labeled material through an antigen-antibody reaction to obtain a magnetically labeled specimen. When this sample in which the magnetically labeled specimen was suspended was measured using the measuring device shown in FIG. 5, interference fringes were formed by the laser beam due to the application of a 5 kG magnetic field.

As a result, it was found that conventional laser magnetic immunoassay is also possible by using a combination of a stabilized radical compound and a microparticle.

Furthermore, although the above conjugate was suspended in water for a long period of time, no aggregation of the conjugates was observed.

In addition, similar effects can be obtained by using modified polymers containing polyglycyl methacrylate as a main component or other hydrophilic polymers instead of polyglycyl methacrylate.
4-amino-2,2,6-rotetramethylpiperi/n-
Instead of 1-oxyl, the tetramethylpiperidine-1-oxyl compounds shown in Table 1, the tetramethylpiperidine-1-oxyl compounds shown in Table 2, and the methyloxazoline-1-oxyl compounds shown in Table 3 are used. A similar effect was obtained with other compounds.

(Example 2) Colloidal silica with hydroxyl or amine groups on the surface of the particles was used as microparticles, and 4-amino-
2,2,6,6-tetramethylpiperidine-1-oxyl was combined in the same manner as in Example 1 to obtain a conjugate. A γ-globulin antibody was bound to this conjugate to obtain a magnetic label. This magnetically labeled antibody and the antigen on the antigen-bonded polymer beads were bound by an antigen-antibody reaction, and the colloidal silica was bound to the antigen in a sandwich-like manner. After centrifuging the colloidal silica that did not bind to the antigen, the magnetically labeled specimen that bound to the antigen was measured in the same manner as in Example 1.
It was possible to detect even moles.

〔Effect of the invention〕

As explained above, in the laser magnetic immunoassay method of the present invention, since a combination of a stabilized radical compound and microparticles is used as the magnetic microparticles, aggregation of the magnetic microparticles does not occur. What is the effect of stably performing highly sensitive measurements?

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between the stable radical compound concentration and interference fringe intensity in the present invention, and Figures 2 to 4 all show chemical structural formulas showing specific examples of the stable radical compound in the present invention. 5 are schematic configuration diagrams for explaining the laser magnetic immunoassay method.・・Container, electromagnet, magnetic pole piece, raised part, laser beam, ・Reflected light. Table 0 (B-13) CH2NH-C-CH21 ○ (B (B-17) (B-18) CH2N 20 (B-19) (B-20) Figure 4(b) (C (C

Claims (1)

[Scope of Claims] A first step of causing an antigen-antibody reaction between a magnetic label obtained by adding magnetic fine particles to an antigen or antibody as a label and an antibody or antigen as a specimen, and a magnetic field obtained in this first step. A laser magnetic immunoassay comprising at least a second step of applying a magnetic field to a solution containing a magnetically labeled specimen, which is a complex of a magnetically labeled body and a specimen, to induce and concentrate the magnetically labeled body in a region irradiated with laser light. A laser magnetic immunoassay method, characterized in that the magnetic fine particles are a combination of a stable radical compound and a fine particle.