JPH0390861A - Immunoassay and implement for immunoassay - Google Patents

Abstract

PURPOSE:To detect the specific material in a specimen in a short period of time by adding the specimen into a liquid dispersion dispersed into a coloring liquid of the color different from the color of fine particles. CONSTITUTION:The fine particles 1 are dispersed into the previously obtd. coloring liquid 3. The specimen is then added into the dispersion. Desired antigens 4 and antibodies 2 are bonded by immune reaction and are further flocculated, by which the flocculated lump (conjugate) 5 including the chromatic fine particles 1 is formed if the desired antigens 4 exist in the specimen. The flocculated lump 5 precipitates upon growing and depositer in the lower past of the dispersion. The chromatic fine particles 1 from the dispersion are the mixed colors of the fine particles 1 and the coloring liquid 3, for example, purple, but the fine particles 1 are separated by the precipitation of the flocculated lump 5 and, therefore, the color of the dispersion changes to the original color of the coloring liquid 3, for example, blue. The presence of the desired antigens in the specimen is known by this change in color. The specific matter in the specimen is easily and rapidly detected in this way.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to an immunoassay method for detecting a specific substance in a specimen and an immunoassay tool for carrying out the method.

<Prior Art> Immunoassays detect the presence of specific substances (antigens, antibodies, etc.) in specimens such as blood and urine, and are used to determine pregnancy and diagnose various diseases.

One such immunoassay method is one that is performed using the following procedure.

When detecting a specific antigen in a sample (hereinafter referred to as the target antigen), white polystyrene microparticles (diameter 0.2 to 20p) immobilized with an antibody that can specifically bind to the target antigen are used.
) is added to the solution in which the sample is dispersed. As a result, the target antigen and white fine particles combine to form an aggregate.

Such an operation is performed, for example, on a glass plate, and after mixing the dispersion of white fine particles and the sample, when the aggregate grows to a diameter of 1 mm, its presence is visually determined, and the presence of the aggregate in the sample is determined. It can be seen that the target antigen is present in the target antigen.

However, this method requires skill because the presence of aggregates is determined visually, and the size of the aggregates (
There is a drawback that it takes time to detect the target antigen, as it is necessary to wait until the target antigen grows to a diameter of approximately 1 mm.

<Problems to be Solved by the Invention> The present invention has been made in view of the above-mentioned shortcomings of the prior art. Our goal is to provide measuring tools.

Means to solve problems〉 Such an object is achieved by the following invention.

That is, the present invention, when detecting a specific substance in a specimen,
A sample is added to a dispersion liquid in which colored microparticles immobilized with a ligand capable of specifically binding to the specific substance are dispersed in a colored liquid of a different color from the microparticles. This is an immunoassay method characterized by separating fine particles from the dispersion liquid and detecting the specific substance based on a change in color of the dispersion liquid caused by this separation.

Preferably, the separation of the fine particles is performed by an immunoassay method by precipitating a combination of the fine particles and a specific substance.

Further, the separation of the fine particles is preferably performed by an immunoassay method in which the bound substance is separated and removed using a filter having a pore size smaller than the diameter of the bound substance between the fine particles and the specific substance.

Further, the separation of the fine particles is performed by using a support on which a ligand different from the above-mentioned ligand is immobilized, binding a specific substance to the ligand, and adsorbing the bound substance of the fine particles and the specific substance to the support. Preferably, the method is an immunoassay.

Further, the separation of the colored fine particles uses a granular support with a high specific gravity on which a ligand different from the ligand is immobilized,
Preferably, the method is an immunoassay method in which a specific substance is bound to the ligand to obtain a conjugate of fine particles, a specific substance, and a granular support, and this conjugate is precipitated.

In addition, in the separation of the colored fine particles, a granular support on which a ligand different from the above-mentioned Gant is immobilized is used, and a specific substance is bound to the ligand to obtain a combination of the fine particles, the specific substance, and the granular support. Preferably, the method is an immunoassay method in which the bound substance is separated and removed using a filter having a pore size smaller than the diameter of the particulate support.

The present invention also relates to an immunoassay device used to carry out any of the above immunoassay methods, which comprises colored microparticles immobilized with a ligand capable of specifically binding to a specific substance in a sample. This is an immunoassay device characterized by containing the fine particles and a colored liquid or coloring agent of a different color.

<Configuration of the Invention> Hereinafter, the immunoassay method and immunoassay device of the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1, FIG. 2, and FIG. 3 are explanatory diagrams each schematically showing an example of the procedure of the immunoassay method of the present invention.

As shown in Figure 1, colored (e.g. red) microparticles 1 are immobilized with an antibody (ligand) 2 that specifically binds to the target antigen 4 in a sample such as blood, serum, or urine. The fine particles 1 are dispersed in a colored liquid 3 of a different color (for example, blue). In this state, this dispersion (also referred to as a solution in the case of colloidal particles) has a mixed color (for example, purple) of the fine particles 1 and the colored liquid 3.

For the fine particles 1, latex (especially polystyrene latex), colloidal particles, etc. are preferably used, and the particle size is as follows:
It is preferably about 0.02 to 50.

The colored liquid 3 may be any type of liquid, such as one colored with a coloring agent such as various pigments (for example, indigo blue), or colored by dispersing particles similar to the fine particles 1 (different in color). It can be anything.

The color combination of the fine particles 1 and the colored liquid 3 may be a combination of chromatic colors or a chromatic color and an achromatic color, such as red and blue, blue and yellow, red and white, etc. Among these, combinations of chromatic colors are particularly preferred, and combinations of colors that are not similar are preferred. Also,
It is also preferable that at least one of them is fluorescent in order to improve visibility.

As antibody 2, it is preferable to use a polyclonal antibody.

In addition, in the present invention, a method of dispersing the fine particles 1 in a colored liquid 3 obtained in advance, and a method of dispersing the colored liquid 3 in a liquid in which the fine particles 1 are dispersed are described.
Alternatively, a method of adding a coloring agent may be used.

Next, the specimen is added to the dispersion liquid. Target antigen 4 in sample
When present, the target antigen 4 and the antibody 2 are bound by the immune reaction, and further aggregated to form an aggregate (combined substance) 5 containing the colored fine particles i.

When the aggregates 5 grow (for example, have a diameter of Iota or more), they precipitate and are deposited below the dispersion liquid. This results in
Agglomerates 5 containing colored fine particles 1 are separated from the dispersion. The color of the dispersion is a mixture of the fine particles 1 and the colored liquid 3 (for example, purple), but due to the precipitation of the aggregates 5, the fine particles 1
is separated, so the color of the dispersion changes to the color of the original colored liquid (for example, blue). The presence of the target antigen in the sample can be determined by this color change.

In this way, since the target antigen is detected by a change in the color of the dispersion, anyone can easily and reliably determine the target antigen.

Note that the change in color of the dispersion liquid can be easily confirmed visually, but the change in color may be measured optically (for example, by measuring absorbance).

Particularly when quantitatively analyzing a target antigen in a specimen, optical measurement is preferable in terms of accuracy.

On the other hand, if it is sufficient to determine whether a certain amount of the target antigen is present in the sample (for example, in the example described later, 100 mIU/m or more), it is easy to visually determine the change in color. It is also preferable because it is quick.

Furthermore, in the method shown in FIG. 1, the aggregates 5 may be separated and removed using a filter (not shown). That is, the pore size is smaller than the diameter of the aggregate 5 (for example, 0.45~
A filter (for example, a membrane filter, a nylon filter) having a diameter of about 80 mm is prepared, and the dispersion liquid is passed through this filter to separate and remove the aggregates 5 in the dispersion liquid. The filtrate that passes through the filter assumes the color of the original colored liquid. This shows that the target antigen 4 is present in the sample.

Here, the colorant passes through the filter and is not filtered out.

The method of separating the flocs 5 by precipitation requires time for the flocs 5 to grow to a certain size (diameter 1011s or more) and time for the flocs 5 to settle.
In the method of separating vines using a filter, the size of the aggregates 5 that are separated by filtration tends to be relatively small (about 0.5 to 150 u), and because they are forcibly separated and removed by the filter, the size of the aggregates 5 is relatively small (about 0.5 to 150 u). The target antigen can be detected in a short time.

Note that if the target antigen is not present in the sample and no aggregates 5 are formed, the aggregates are not precipitated or removed by a filter, and the fine particles 1 remain in the dispersion, so the color of the dispersion is different. It does not change.

Note that the method for separating the aggregates 5 is not limited to the above-mentioned method, and may be performed, for example, by centrifugation, separation by adsorption to a support 9 or 11, which will be described later, or the like.

Figure 2 shows an example in which the method of separating colored fine particles is different.
Microparticles (for example, pink) 6 having immobilized antibodies 7 that specifically bind to the target antigen 8 are dispersed in the dispersion (for example, purple), and a specimen is added to this dispersion (for example, purple). When the target antigen 8 is present in the sample, the target antigen 8 binds to the antibody 7 at the recognition site 8a due to an immune reaction.

The fine particles 6 have a relatively small particle size compared to the fine particles 1,
For example, it is about 5 to 200 nm. Specifically, colloidal particles such as gold colloid (particle size 20 to 200 nm) and silver colloid, gelatin particles, and the like are preferably used.

Although it is preferable to use a monoclonal antibody as the antibody 7, it is also possible to use a polyclonal antibody.

On the other hand, a support 9 immobilized with an antibody 10 that binds to a recognition site 8b different from the recognition site 8a of the target antigen 8 is prepared, and the support 9 is brought into contact with the dispersion.

As the support 9, for example, filters such as membrane filters, nylon filters, filter paper, tubes, microplates, plate-shaped or rod-shaped plastic supports, etc. can be used. When using filters, the dispersion liquid can be Contact by filtration.

Further, as the support body 9 other than the above, one made of a porous material can be mentioned. When using this support, the support is immersed in the dispersion liquid to adsorb it.

A support made of such a porous material is preferable because it has a large contact area with the dispersion liquid and has high sensitivity.

Through such contact with the support 9, the target antigen 8 binds to the antibody 10 at the recognition site 8b, and the bound product of the fine particles 6 and the target antigen 8 is adsorbed to the support 9.

The remaining liquid (the filtrate that has passed through the membrane filter, etc.) has the color of the original colored liquid, which indicates that the target antigen 8 is present in the sample.

As for the antibody 10, it is also preferable to use a monoclonal antibody, but it is also possible to use a polyclonal antibody. In addition, antibodies homologous to antibody 7 and antibody 10,
In particular, polyclonal antibodies can also be used.

FIG. 3 shows an example in which the method of separating colored fine particles is further different. As shown in the figure, the target antigen 8 is bound to the antibody 7 of the microparticle 6 in the same manner as in FIG. 2 above.

On the other hand, a granular support 11 on which the same antibody 10 as described above is immobilized is prepared, and this support 11 is added to the dispersion liquid.
As a result, the target antigen 8 binds to the antibody 10 at the recognition site 8b, and a composite (sandwich) 12 of the microparticle 6, target antigen 8, and particulate support 11 is formed.

Latex, particularly polystyrene latex, is suitably used for the granular support 11, and the particle size thereof is 0.2 to 15
Preferably, it is about P.

It is also possible to use beads having a particle size of about 15-2 mm, especially polystyrene beads and polypropylene beads.

The combined material 12 containing such particulate support 11 is separated and removed by a filter (not shown). That is, the pore size is smaller than the diameter of the granular support 11 (for example, 0.15~
8-) is prepared, and the dispersion is passed through this filter to separate and remove the bound substance 12 in the dispersion. The filtrate that passes through the filter assumes the color of the original colored liquid. This shows that the target antigen 8 is present in the sample.

It should be noted that the above filters, especially those with relatively small pore sizes (
For example, when using a filter of 0.15 to 1-), it is preferable to pressurize the dispersion liquid to pass through the filter to increase the filtration rate.

Alternatively, the bound substance 12 can be separated by precipitation without using such a filter.

In this case, the granular support 11 has a high specific gravity (preferably,
1.5 to 2.5) is used. in particular,
Examples include high-density latex. Further, the particle size of the high specific gravity granular support is preferably about 0.5 to 15 μm.

By using such a granular support 11, the bound substance 12 is precipitated and separated from the dispersion liquid, and the color of the dispersion liquid changes to the color of the original colored liquid as described above, and the presence of the target antigen 8 is detected. I understand.

In this case, since the granular support 11 has a high specific gravity, the precipitation rate is high, contributing to shortening the detection time.

Note that the method for separating the bound substance 12 is not limited to the above-mentioned method.
For example, centrifugation, filtration, etc. may be used.

The method shown in FIGS. 2 and 3 does not require time for the formation or growth of the aggregate 5 described above, so the detection time for the target antigen is short, especially when using the support 9 or the support 11 and filter. If used, detection time is significantly reduced.

In addition, in the present invention, unlike the illustration, the granular support 11 may be added to the dispersion liquid or the colored liquid before adding the specimen to the dispersion liquid.

The immunoassay method as described above can be carried out using the following immunoassay tool.

The immunoassay device includes the colored fine particles 1 or 6 and the colored liquid 3 or a coloring agent for obtaining the colored liquid 3, and these are combined as a kit.

Each component of the immunoassay device is determined depending on the immunoassay method, and the following are exemplified. In addition, ■〜■
is used in the method shown in FIG. 1, (2) and (2) are used in the method shown in FIG.

■・Fine particles 1 (with antibody 2) ・Coloring liquid 3 (or coloring agent) ■・Fine particles 1 ・Antibody 2 ・Coloring liquid 3 (or coloring agent) ■・Fine particles 1 (with antibody 2) ・Coloring liquid 3 (or coloring agent) ・Filter ■・Fine particles 6 (with antibody 7) ・Coloring liquid (or colorant) ・Support 9 (with antibody 10) ■・Fine particles 6 ・Antibody 7 ・Coloring liquid (or colorant) ・Support 9 ・Antibody 10 ■・Fine particles 6 (with antibody 7) ・Coloring liquid (or coloring agent) ・Particulate support 11 (with antibody 10) ■・Fine particles 6 ・Antibody 7 ・Coloring liquid (or colorant) ・Particulate support 11 ・Antibody 10 ■・Fine particles 6 (attached to antibody 7) ・Colored liquid (or coloring agent) ・Particulate support 11 (attached to antibody 7) ・Filter The above-mentioned microparticles 1.6 are usually stored in a state where they are dispersed in a liquid. However, it may also be stored in a dry state, especially in a freeze-dried state.

Note that the immunoassay device as described above may include other components.

As described above, regarding the immunoassay method and immunoassay tool of the present invention,
Although the present invention has been described with reference to several examples, the present invention is not limited to these, and it is possible to add predetermined operations to each side, or to make various other modifications and applications.

Further, in the above description, an antigen was mentioned as a specific substance (object to be measured) in a specimen, but the substance is not limited to this, and may be, for example, a hormone, various virus antibodies, or the like.

The immunoassay method and immunoassay tool of the present invention can be used for determining pregnancy, diagnosing various diseases, identifying bacteria, and the like.

<Example> Hereinafter, specific examples of the present invention will be described.

(Example 1 of the present invention) Red polystyrene latex with a particle size of 0.5-
It was dispersed in M. glycine buffer (pH 9,6) to a concentration of 2%. To this was added 1 mg/mff1 anti-hCG polyclonal antibody (rabbit), and this antibody was immobilized at 4°C. This was centrifuged and collected.

Furthermore, after post-coating with bovine serum albumin, bovine serum albumin was solidified in the same manner and the particle size was 0.5.
An equal amount of blue polystyrene latex of - was added to prepare a purple dispersion.

Pour 40 ui of this dispersion into the wells of a U-shaped microplate.
40 IIt of the sample containing the target antigen shown in Table 1 was added to each well and allowed to react for 300 minutes. Aggregates were precipitated at the bottom of the well, and Table 1 shows the change in color of the dispersion at this time.

Table (Example 2 of the present invention) Red polystyrene latex with a particle size of 0.5-
It was dispersed in M. glycine buffer (pH 9,6) to a concentration of 2%. An ltag/aj anti-hCG polyclonal antibody (rabbit) was added to this, and this antibody was immobilized at 4°C. This was centrifuged and collected.

Furthermore, after post-coating with bovine serum albumin, bovine serum albumin was solidified in the same manner and the particle size was 0.5.
An equal amount of blue polystyrene latex from Step 4 was added to prepare a purple dispersion.

To 400 ul of this dispersion, 400 ul of a sample containing the target antigen shown in Table 2 was added, reacted for 2 minutes, and then filtered through a 3-pore size membrane filter. The color of the filtrate is shown in Table 2.

Table 2 (Example 3 of the present invention) Red smoke-colored colloidal gold particles with a particle size of 20 nm (A520 = 3
．． 5) was immobilized with lllIg7mi anti-hCG monoclonal antibody.

Furthermore, an equal amount of blue polystyrene latex having a particle size of 0.2 μm was added to prepare a purple dispersion.

On the other hand, a monoclonal antibody having a different recognition site from that of the antibody immobilized on the gold colloid particles was immobilized on a membrane filter support with a pore size of 1 μm by the CNBr method.

Specimen 4 containing the target antigen shown in Table 3 in dispersion liquid 400 aj
After adding 00 ul and reacting for 5 minutes, the mixture was filtered using the aforementioned antibody-immobilized membrane filter. Table 3 shows the color of the filtrate.
Shown below.

Table 3 (Example 4 of the present invention) Red smoke-colored colloidal gold particles with a particle size of 20 nm (A520 = 3
．． 5) was immobilized with IB/ml anti-hCG monoclonal antibody.

Furthermore, an equal amount of blue polystyrene latex having a particle size of 0.2711 was added to prepare a purple dispersion.

On the other hand, a monoclonal antibody having a different recognition site from that of the antibody immobilized on the gold colloid particles was immobilized on white polystyrene latex having a particle size of 8, and this was added to the dispersion liquid.

To 400 μj of this dispersion, 400 μm of the sample containing the target antigen shown in Table 4 was added and reacted for 5 minutes.
It was filtered with a μ membrane filter. The color of the filtrate is shown in Table 4.

Table 4 (Example 5 of the present invention) Red smoke-colored colloidal gold particles with a particle size of 2Or+m (A520=
3.5) was immobilized with IIIIg/rrrt anti-hCG monoclonal antibody.

Furthermore, an equal amount of blue polystyrene latex with a particle size of 0.2- was added to prepare a purple dispersion.

On the other hand, a gold coroinoclonal antibody was immobilized on a white high-density latex with a particle size of 514 m (manufactured by Japan Synthetic Rubber Co., Ltd., specific gravity 1.50), and this was added to the dispersion.

Dispense 400 of this dispersion into each well of a U-shaped microplate, and add 4 samples containing the target antigen shown in Table 5 to each well.
0 μf was added and reacted for 15 minutes. A precipitate was formed at the bottom of the well, and Table 5 shows the color change of the dispersion at this time.

Table 5 (Example 6 of the present invention) Red polystyrene latex with a particle size of 0.25-
It was dispersed in LM glycine buffer (pH 9,6) to a concentration of 2%. 1 mg/mt anti-hLH polyclonal antibody (rabbit) was centrifuged and collected.

Furthermore, after post-coating with bovine serum albumin, it was centrifuged and this solution was dispersed in a 0.4% indigo carmine solution (blue) to form a purple dispersion.

Transfer 40 μt of this dispersion liquid to the well of a U-shaped microplate.
40 uj of a sample containing the target antigen shown in Table 6 was added to each well and allowed to react for 30 minutes. Aggregates were precipitated at the bottom of the well, and Table 6 shows the color change of the dispersion at this time.

Table 6 <Effects of the Invention> As described above, according to the immunoassay method and immunoassay tool of the present invention, any person can easily and reliably detect a specific substance in a sample by changing the color of the dispersion liquid. Detection of specific substances can be performed quickly, and the detection time is short.

[Brief explanation of drawings]

FIG. 1, FIG. 2, and FIG. 3 are explanatory diagrams each schematically showing an example of the procedure of the immunoassay method of the present invention. Explanation of symbols 1.6... Microparticles 2.7.10... Antibody 3... Colored liquid 4.8... Target antigens 8a, 8b... Recognition site 5... Aggregate mass 9...・Support: Granular support 2: Conjugate applicant Almo Co., Ltd.

Claims (7)

[Claims] (1) When detecting a specific substance in a sample, the sample is placed in a dispersion liquid in which colored fine particles immobilized with a ligand that can specifically bind to the specific substance are dispersed in a colored liquid of a different color from the fine particles. an immunoassay method, characterized in that colored fine particles bound to the specific substance are separated from the dispersion through an immune reaction, and the specific substance is detected by a change in color of the dispersion caused by this separation. . (2) The immunoassay method according to claim 1, wherein the separation of the fine particles is performed by precipitating a combination of the fine particles and a specific substance. (3) The separation of the fine particles is carried out by separating and removing the bound substance using a filter having a pore size smaller than the diameter of the bound substance of the fine particles and the specific substance.
Immunoassay described in. (4) The separation of the fine particles is performed by using a support on which a ligand different from the ligand is immobilized, binding a specific substance to the ligand, and adsorbing the bound product of the fine particles and the specific substance to the support. The immunoassay method according to claim 1, which is carried out by. (5) The colored fine particles are separated using a high specific gravity granular support on which a ligand different from the ligand is immobilized,
2. The immunoassay method according to claim 1, which is carried out by binding a specific substance to the ligand to obtain a conjugate of the fine particles, the specific substance, and the granular support, and precipitating this conjugate. (6) The separation of the colored fine particles uses a granular support on which a ligand different from the above-mentioned ligand is immobilized, and binds a specific substance to the ligand to obtain a combination of the fine particles, the specific substance, and the granular support. 2. The immunoassay method according to claim 1, wherein the bound substance is separated and removed using a filter having a pore size smaller than the diameter of the particulate support. (7) An immunoassay tool used for carrying out the immunoassay method according to any one of claims 1 to 6, which is a colored immunoassay tool on which a ligand capable of specifically binding to a specific substance in a specimen is immobilized. 1. An immunoassay device comprising: microparticles; and a colored liquid or colorant having a color different from the microparticles.