JPH06289024A - Method for confirming reaction completion of diagnostic and sheet for chromatography used for it - Google Patents

Abstract

PURPOSE:To determine the presence of an antibody easily by providing a color developing agent which develops color with water on the reaction layer of a method for detecting an antigen where latex is a labeled substance for antigen/antibody reaction. CONSTITUTION:An organic synthetic polymer with less impurity such as polypyrrole and polyphenylene is used for latex. Antigen/antibody reaction is made on sheets such as the film of nitrocellulose use in filter form and the waxed paper of chromatography. An antibody performing the antigen/antibody reaction is immobilized on the sheets and a color developing agent is spread or impregnated. For example, bromocresol green, 2,5-dinitrophenol, etc., which develop color or are discolored when they are wetted by water contained in urine or serum are used as color developing agents.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for confirming the completion of a reaction of a diagnostic agent in a labeled immunoassay and a chromatographic sheet used for the method.

[0002]

2. Description of the Related Art Although a specific protein has been conventionally detected for diagnosis of various diseases, a labeled immunoassay applying an immunological antigen-antibody reaction is widely used as a method for detecting the specific protein. There is.

In this case, enzymes and colloidal metal particles are used as labels. However, although a test device that uses an enzyme or colloidal metal particles is simple, it is difficult to confirm that the reaction has ended because the change on the film does not occur if it is negative. There is a method of previously attaching the antigen to another position, but this method has a problem that the reaction may not occur due to deterioration depending on the antigen.

Further, in the case of a chromatographic test device using colloidal metal particles, it is difficult to determine the end of the reaction (end of development). Therefore, in order to confirm this, for example, above the determination surface, that is, the developing solution. Although the antigen was attached to the upper end of the rising part, there were some antigens that did not react due to deterioration, and thus the completion of expansion could not be confirmed, which was a problem.

In view of the above circumstances, as a method for easily and surely confirming the completion of the reaction of the diagnostic agent, there is a method disclosed in Japanese Patent Laid-Open No. 353764. This is to confirm the end of the reaction by wetting the color-developing agent that develops when it gets wet with water on the reaction layer to develop the color when detecting the antigen using the enzyme or colloidal metal particles. .

[0006]

[Patent Document 1] Japanese Patent Application Laid-Open No. 4-35376
Even with the method disclosed in 4, there is a problem that it is difficult to determine the presence or absence of an antibody because the antibody attached to the metal colloid is unstable to acid. The present invention solves this problem.

[0007]

The present invention provides a method for detecting an antigen using a latex as a labeled antibody in an antigen-antibody reaction,
The gist is a method for confirming the reaction completion of a diagnostic agent, characterized in that a color former that develops color with water is present on the reaction layer.

Further, according to the present invention, in the method for detecting an antigen by an antigen-antibody reaction, a coloring agent that develops color with water is provided above the judgment surface of the spreading layer, and a latex is immobilized as a labeled antibody in the middle portion. The main feature is the characteristic chromatograph sheet.

In the present invention, the latex is preferably an organic synthetic polymer containing few impurities. Examples thereof include polypyrrole, polyphenylene, polyaniline, polythiophene, polynaphthalene, polythiophenol, polyacetylene, polyvinyl chloride, polystyrene, polyvinyltoluene, polyacrylamide, and polyN-vinylpyrrolidone.

In the present invention, the antigen-antibody reaction is performed on the sheet. Examples of the sheet include a nitrocellulose film used in a filter format and a chromatograph filter paper. An antibody that undergoes an antigen-antibody reaction is immobilized on such a sheet to provide a reaction layer, and in the present invention, a color former is further applied or impregnated. The arrangement of the antibody and the color-developing agent on the sheet is not bad because they are mixed with each other, but it is preferable that these are partitioned.

As the color-developing agent used in the present invention, a color-developing agent or a color-changing agent is used when it is wet with water in urine or serum. When a chromatographic sheet is coated or impregnated with a color former, the position is above the application position of the antibody, so a color former that reacts with the antibody may be used, but generally one that does not react with the antibody of the test tool is selected. Generally, the test sample liquid used for diagnosis is blood or urine, and the pH of blood is usually 7.2 to 7.4 and the pH of urine is 4.6 to 8.0. A color former that surely develops a color at 6 to 8.0 is suitable.

Examples of such a color-forming agent include bromocresol green (yellow at an acidity of pH 3.8 or less, and a bright blue color when the pH exceeds 5.4) or 2,5 dinitrophenol (pH 5.8). When the pH exceeds 3.0, it becomes yellow), and bromophenol blue (light yellow at pH 3.0 or less, when it exceeds pH 4.6, it becomes reddish purple or blue, and the discoloration becomes faster as the pH increases).

In the present invention, the chromatographic sheet means a sheet used for filter paper chromatography or thin layer chromatography, and the sheet may be filter paper or a thin film attached to a glass plate. Good.

[0014]

When the test sample solution is dropped on the reaction layer, if the test sample solution contains an antigen, the antigen reacts with the latex-adhesive antibody and the antibody on the reaction layer to form the latex-adhesive antibody-antigen- The antibody complex is generated and color is developed, whereas when the test sample solution does not contain the antigen, the complex is not produced and thus color is not developed. On the other hand, at the same time, since the water in the sample liquid to be tested causes the color-developing agent to develop color, the completion of the reaction can be confirmed reliably and easily regardless of whether it is negative or positive.

Further, when the origin of the development layer of the chromatograph is immersed in the test sample solution, when the test sample solution contains an antigen, the antigen in the test sample solution and the latex adhered to the development layer are attached. When the antibody migrates in the direction of the antibody immobilized on the spreading layer and reacts with them to form a latex-adhesive antibody-antigen-antibody complex and develops color, whereas there is no antigen in the test sample solution In the above, since the above complex is not formed, no color is developed. On the other hand, the water in the test sample liquid moves to the upper part of the spreading layer and wets the color former that has been applied in advance to develop the color, so that the end of the reaction can be confirmed reliably and easily regardless of whether it is negative or positive. .

Although the present invention detects an antigen by an antigen-antibody reaction as described above, the use of latex as a labeled antibody is a feature of the present invention.

In the present invention, since latex is an organic synthetic polymer, it is stable against acid and is not affected by ascorbic acid or phosphoric acid in the test sample solution. It is possible to perform highly accurate detection because the binding between antibodies is not broken.

[0018]

EXAMPLES The present invention will now be specifically described with reference to Examples. [Example 1] A test sample liquid containing a large amount of ascorbic acid and prepared with a concentration of hCG as an antigen in the range of 0 to 1000 IU / l and containing a blue latex-attached anti-hCG antibody was added to 5 types of urine. Got ready.

From the center of the filter paper to one half,
The thing which apply | coated the anti-hCG antibody was prepared. And as a reaction completion confirmation part, the solution saturated with anhydrous cobalt chloride in isopropanol, the other part of the filter paper, that is,
A test tool was prepared by sufficiently spraying one side on which no antibody was applied by spraying and then drying.

As a result of dropping the above-mentioned five kinds of test sample liquids on this test tool, as shown in Table 1, spots colored in pink were observed in the reaction completion confirmation portion, and hCG which is an antigen was present. In this case, a blue spot was observed in the vicinity of the center of the filter paper where the test sample liquid was dropped, confirming that the spot was positive.

[0021]

[Table 1]

As a comparative example, a test sample in which 5 kinds of urine containing a large amount of ascorbic acid and prepared with an antigen concentration of hCG in the range of 0 to 1000 IU / l were made to contain a gold colloid-attached anti-hCG antibody The liquid was prepared. As a result of dropping 5 kinds of test sample liquids containing the anti-hCG antibody attached to gold colloid on the same test tool as in Example 1, as shown in Table 2, spots colored in pink were observed in the reaction completion confirmation part. Was
When hCG which is an antigen is present, a blue spot is observed near the center of the test paper onto which the test sample solution has been dropped.
In some cases, it could be confirmed to be positive, but in other cases, no spots were found and it was negative.

[0023]

[Table 2]

Example 2 Five kinds of urine containing a large amount of ascorbic acid and having an antigen concentration of hCG in the range of 0 to 1000 IU / l were used as test sample solutions.

A blue-latex-adhering anti-hCG antibody was applied to the lower part of the chromatographic filter paper, and a hCG antibody was applied to the center of this filter paper.

50 g of calcium chloride as a hygroscopic agent and 0.1 g of crystal violet, a basic dye, were dissolved in 100 cc of methanol, and this solution was applied on the filter paper and dried to prepare a test tool.

When the lower end of this test device was dipped in the above-mentioned five types of test sample solutions, as shown in Table 3, the reaction completion confirmation part on the upper part was colored green, and hCG which is an antigen was observed. In the presence of, a bright blue color is produced in the center of the test paper,
It was confirmed to be positive.

[0028]

[Table 3]

As a comparative example, the colloidal gold-attached antibody was placed at the bottom of the chromatographic filter paper and hCG was placed at the center of the filter paper.
The thing which apply | coated the antibody was prepared. In the same manner as in Example 2,
When the lower end of this test device was dipped in the above-mentioned 5 types of test sample liquids, as shown in Table 4, green color was observed in the reaction completion confirmation part on the upper part, and when hCG which is an antigen was present. ,
A very faint reddish purple coloring was seen in the center of the test paper, but it was difficult to distinguish between positive and negative judgments.

[0030]

[Table 4]

[0031]

As is apparent from what has been described above, by adopting the method of the present invention, it is possible to easily confirm the end of the reaction of the diagnostic agent in a short time, so that the examination time can be shortened. it can. Further, since the arrival or non-arrival of the test sample solution to the reaction layer can be confirmed by the presence or absence of color development, it is easy to determine positive and negative.

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[Procedure amendment]

[Submission date] May 21, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 1

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 2

[Correction method] Change

[Correction content]

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction content]

[0007]

The present invention is a method for detecting an antigen which utilizes latex as a label in an antigen-antibody reaction, characterized in that a color-developing agent that develops color with water is present on the reaction layer. The gist is how to confirm the end of the reaction of the diagnostic agent.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

Further, in the method for detecting an antigen by an antigen-antibody reaction, the present invention has a color former which develops color with water above the judgment surface of the spreading layer, and a label in which latex is used as a marker in the middle portion. The gist is a chromatographic sheet characterized by immobilizing an antibody.

Claims (2)

[Claims] 1. A method of detecting an antigen using a latex antibody as a labeled antibody in an antigen-antibody reaction, wherein the reaction layer contains a color former that develops color with water, and a method for confirming the end of the reaction of a diagnostic agent. 2. A method for detecting an antigen by an antigen-antibody reaction, characterized in that a color-developing agent that develops with water is provided above the determination surface of the spreading layer, and latex is immobilized in the middle as a labeled antibody. Graffiti sheet.