JPH07294527A - Test tool capable of easily confirming completion of reaction - Google Patents

Abstract

PURPOSE:To provide a test tool capable of easily and certainly confirming the completion of the reaction of a diagnostic drug. CONSTITUTION:In the test tool utilizing metal colloid or enzyme, a reaction layer is provided on a plate composed of filter paper and a color forming agent forming a color upon the wetting with water is applied to the reaction layer. When an antigen is present in a sample soln. to be examined, the antigen is reacted, for example, with a metal colloidal particle bonded antibody and the antibody in the reaction layer when the sample soln. to be examined is dripped on the reaction layer to form a metal colloidal particle-bonded antibody-antigen- antibody composite to form a color and, when there is no antigen in the sample soln. to be examined, no color is formed because there is no composite. Since water in the sample soln. to be examined allows the color forming agent to form a color at the same time, the completion of reaction can be certainly and easily confirmed regardless of negative.positive.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a diagnostic test tool using a metal colloid or an enzyme, which can easily confirm the completion of the reaction.

[0002]

2. Description of the Related Art Conventionally, detection of a specific protein has been carried out for the diagnosis of various diseases, but the detection method applying an immunological antigen-antibody reaction is most widely used as the detection method of the specific protein. . This utilizes the property that an antibody specifically recognizes and binds to an antigen substance, and an antibody that recognizes a specific protein serving as an antigen is prepared, and using this, the specific protein and the antigen in a test sample are used. An antibody reaction is performed, and the result is detected using a secondary phenomenon such as an agglutination reaction or a label. Such detection methods are roughly classified into qualitative detection methods and quantitative detection methods according to the degree of detection. The qualitative detection method is a method for detecting the presence or absence of a certain amount of protein or more by a simple method such as coloring, and is generally applied to clinical diagnostic agents and the like.

The labeled immunoassay method is intended to directly measure the antigen or antibody by labeling the antigen or antibody with an enzyme or a highly sensitive radioisotope and the like, and is originally quantified based on the label. Although it is a so-called quantitative detection method, it is also applied as a simple qualitative detection method because of its extremely high sensitivity. As a qualitative detection method to which such a labeled immunoassay method is applied, there is a detection method to which an enzyme immunoassay method using an enzyme as a label is applied, and as a method to which a direct detection method of the labeled immunoassay method is applied, a detection substance There is a detection method using colloidal metal particles (metal colloid particles).

There are various measuring methods in the enzyme immunoassay method, and the sandwich method is the most practical method. This consists of preparing two types of antibodies specific to the antigen, one of which is bound to the solid phase, the other of which is bound to the enzyme that serves as a label to obtain a complex, and first, the solid phase to which the antibody is bound is covered. After adding a test sample solution and washing well to remove unreacted substances, the enzyme-antibody complex is reacted to form a sandwich-like bound substance which is an antibody-antigen protein complex on the solid phase surface. After completely removing the excess complex, the enzyme activity of the complex bound on the solid phase surface is measured.
As an application of this as a simple qualitative detection method, a test sample solution containing an enzyme-antibody complex is added to a solid phase to which an antibody is bound and reacted, and an antibody-antigen protein complex is formed on the solid phase surface. A test sample solution (enzyme-antibody complex is released from the filtration membrane by the enzyme-antibody complex-containing filter membrane that contains the enzyme-antibody complex in the solid phase to which the body is formed or the antibody is bound (Included in the liquid) and reacted to form an antibody-antigen protein complex on the surface of the solid phase, ortho-phenylenediamine, etc. to prove that the antibody-antigen protein complex has been formed. The color developing reagent of is used.

The detection method using metal colloid particles as a detection substance is to use metal colloid particles instead of the enzyme in the enzyme immunoassay method. The metal colloid particles have a unique color tone due to their respective physical properties, which allows the result of the antigen-antibody reaction to be directly and visually displayed. Metal colloid particles are a colloidal dispersion of a metal, a metal compound, a polymer nucleus coated with a metal or a metal compound,
The particle size is 10-100 nm. Examples of the metal colloidal particles include metals such as platinum, gold, silver and copper, silver iodide, silver bromide, copper hydrate oxide, iron oxide, iron hydroxide or hydrate oxide, aluminum hydroxide. Or hydrated oxide,
Chromium hydroxide or hydrated oxide, vanadium oxide,
Examples thereof include metal compounds such as arsenic sulfide, magnesium hydroxide, lead sulfide, mercury sulfide, barium sulfate and titanium dioxide.

As a typical example of the metal colloid particles, there are colloidal gold particles (hereinafter referred to as gold colloid). This is a colloidal form of tetrachloroauric acid under certain specific conditions, which changes from pink to magenta according to the particle size of the gold colloid. When this gold colloid and protein are placed under certain conditions, they bind by non-covalent and electrostatic adsorption to form a stable conjugate. Since the metal colloid particles are bonds that do not require chemical modification, they are characterized in that protein denaturation does not occur and physiological activity does not decrease.

[0007]

However, although a test device using metal colloid particles or an enzyme is simple, it is difficult to confirm that the reaction has ended because the change on the film does not occur if the test device is negative. As a general method, there is a method of previously attaching an antigen to a position different from the judgment surface, but this method has a problem that the reaction may not occur due to deterioration depending on the antigen. The present invention has been made in view of the above circumstances, and an object thereof is to provide a diagnostic test device utilizing a metal colloid or an enzyme, which can easily confirm the completion of the reaction. To do.

[0008]

In order to solve the above problems, the present invention provides a diagnostic test device for detecting an antigen by using a metal colloid or an enzyme, wherein a reaction layer is formed on a plate formed of filter paper. The test tool is provided and the reaction layer is coated with a color-developing agent that develops color when wet with water. In the present invention, "applying a color former" includes impregnating a color former. Further, "coloring" includes discoloration.

[0009]

According to the above construction, when the test sample solution is dropped on the reaction layer and the test sample solution contains an antigen, the antigen is, for example, an antibody attached to the metal colloid particles and an antibody on the reaction layer. Upon reaction, a metal colloid particle-adhered antibody-antigen-antibody complex is produced and develops color. When no antigen is present in the test sample solution, the complex is not produced and does not develop color.
On the other hand, at the same time, since the water in the test sample liquid 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.

[0010]

EXAMPLES Next, examples of the present invention will be described. Figure 1
FIG. 2 is an explanatory view showing a reaction mechanism when the test tool using the metal colloid particles of the present invention is used, FIG. 1 is a case where the test sample liquid contains the metal colloid particles, and FIG. The case of using the law is shown. As can be seen from the reaction mechanism of FIGS. 3 to 4, in the conventional test device, the end of the reaction cannot be confirmed in the case of a negative reaction, whereas in the test device of the present invention, the color development of the color-developing agent causes a negative reaction. It can be easily confirmed that the test sample solution has reached the reaction layer, that is, that the reaction has ended.

The color-developing agent used in the present invention is selected from those which develop color when wet with water, but do not react with the antibody of the test device. Generally, the test sample solution used for diagnosis is blood. Although the pH of these samples is slightly different depending on sex, age and health condition, the pH of blood is usually 7.2-7.4 and the pH of urine is 4.6-.
Since it is 8.0, a color forming agent that surely develops or changes color at pH 4.6 to 8.0 is preferably used. Examples of such a color former include bromocresol green (pH 3.8
Yellow under the following acidic conditions, bright blue when pH exceeds 5.4), 2.5 dinitrophenol (yellow when pH exceeds 5.8), bromophenol blue (pH 3.0)
Below, it becomes pale yellow, and when it exceeds pH 4.6, it becomes reddish purple to blue. The higher the pH, the faster the color change).

[Example 1] Bromocresol green was dissolved in ethanol (95%), and then diluted with water 3 to 4 times to prepare a color former solution, which was applied to a test paper (filter paper to which the antigen was applied. It was applied to the surface of the test piece and dried to prepare a test tool. When a test sample solution containing a colloidal gold-adhering antibody (including a urine component) was dropped onto the test device, a vivid blue color was developed, and a purple spot was observed near the center of the test paper. The blue color confirms that the test sample solution has reached the reaction layer, and the violet spot confirms positive.

[0013]

As is apparent from what has been described above, by adopting the test device of the present invention, it is possible to easily confirm the end of the reaction in a short time, thus saving time. Further, since the arrival or non-arrival of the test sample liquid to the reaction layer can be confirmed by the presence or absence of color development, the number of erroneous judgments such as diagnosing positive as negative is reduced.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing a reaction mechanism when a test device using metal colloid particles of the present invention is used, showing a case where a test sample liquid contains metal colloid particles.

FIG. 2 is an explanatory diagram showing a reaction mechanism when a test device using the metal colloid particles of the present invention is used, and shows a case where a membrane filtration method is used.

FIG. 3 is an explanatory diagram showing a reaction mechanism when a conventional test device corresponding to FIG. 1 is used.

FIG. 4 is an explanatory view showing a reaction mechanism when a conventional test device corresponding to FIG. 2 is used.

Claims (2)

[Claims] 1. A reaction layer is provided on a plate made of filter paper, and the reaction layer is easily coated with a color-developing agent that develops a color when wet with water. Confirmable test tool. 2. The color former is bromocresol green, 2.
5. The test device according to claim 1, which is at least one selected from the group consisting of dinitrophenol and bromophenol blue.