JPH0875734A - Criterion reagent - Google Patents

Abstract

PURPOSE: To decide whether an examination system is normally operated or examination is completed at a low cost by using a substance other than a biological substance changing in hue by pH upon the contact with a specimen soln. to be examined as a criterion reagent. CONSTITUTION: As the criterion reagent, a substance capable of being arranged on a porous material and changing in hue by pH, for example, chlorophenol red, thymol blue, thymol phthalein or neutral red is used. This criterion reagent is dissolved in a solvent to be applied to or dripped on a criterion region arranging position 4 and fixed to a nitrocellulose film 1 being a porous material. In the same way, a first antibody is dripped on a first antibody sensitizing carrier dripping position 2 and a second antibody is dripped on a second antibody dripping position 3 and both antibodies are dried to be fixed to the nitrocellulose film 1. When a specimen soln. is dripped on a specimen dripping position 5, the specimen soln. moves through the film 1 to reach the arranging position 4 and comes into contact with the criterion reagent to discolor the reagent. By this constitution, it can be decided whether an examination system is normally operated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a judgment reagent used in a simple clinical test system using a porous material such as cellulose, and particularly, to determine whether the test system is operating normally or not. The present invention relates to a reagent for determining whether or not it is finished.

[0002]

2. Description of the Related Art Conventionally, in the field of clinical examination, various systems have been developed which allow an individual to easily perform an examination. For example, Japanese Patent Application Laid-Open No. 2-136137 discloses a simple pregnancy diagnosis system using immunochromatography, which is not yet known.
No. 110522 proposes a system that can easily test fecal occult blood.

That is, in the pregnancy diagnosis system described in JP-A-2-136137, pregnancy is diagnosed by measuring the presence or absence of human chorionic gonadotropin (henceforth, hCG) in a sample (urine). Here, the colored carrier sensitized with the anti-hCG antibody moves in the porous material together with the sample solution. Then, at the termination determination site, regardless of the presence or absence of hCG in the sample, that is, regardless of whether it is positive or negative, the sample solution (urine) reaches the termination determination site, and thus a colored display is provided, The end of measurement is determined. In order to enable such a colored display, a substance that traps the colored carrier sensitized with the anti-hCG antibody is immovably immobilized at the termination determination site. Examples of the substance that traps the carrier include an antibody of a species that reacts with anti-hCG such as an anti-IgG antibody, or an antigen (hCG).

Further, in the above-mentioned simple fecal occult blood test system which has not been publicly known, the test system is constructed by utilizing immunochromatography. Here, the inspection apparatus body has an opening for inserting a defecation instrument and a determination window for externally determining the presence or absence of fecal occult blood. A buffer solution container is arranged in the main body so that a defecation instrument is inserted therein, and a filter for filtering the buffer solution in which the fecal sample is dissolved is arranged below the buffer solution container. Further, a spreading layer is provided so that the start end is located below the filter and the determination part faces the determination window. In this spreading layer, an anti-hemoglobin antibody (first antibody) sensitized carrier and an anti-human hemoglobin antibody (second antibody)
And are arranged. Then, by moving the buffer solution container or the stool collection tool inserted in the buffer solution container up and down, the buffer solution in which the stool sample is dissolved is flowed from at least a part of the lower surface of the buffer solution container to the spreading layer.

The spreading layer is made of a porous material such as nitrocellulose, and a carrier sensitized with the first antibody is fixed on one end side thereof. Further, a second antibody that recognizes an epitope different from the first antibody is arranged at a position separated from the portion where the first antibody-sensitized carrier is arranged by a predetermined distance. When the stool sample solution is developed from the side where the first antibody is arranged, the sample moves in the development layer and hemoglobin causes an antigen-antibody reaction with the first antibody. Hb
-When the first antibody-sensitized carrier complex moves and reaches the portion where the second antibody is arranged, the antigen-antibody reaction between Hb and the second antibody causes the Hb-first antibody-sensitized carrier complex to It is trapped in the second antibody-immobilized portion. Therefore, if the carrier has a color tone that has a clear contrast with the spreading layer, the presence of the carrier, that is, the presence of Hb can be easily confirmed visually.

Here, examples of the material forming the spreading layer include cellulosic materials such as nitrocellulose and cellulose acetate, nylon, fluorinated ethylene resin,
A porous material such as a membrane filter made of a synthetic polymer material such as polypropylene is used.

By the way, in the fecal occult blood apparatus, preferably, in addition to the determination part for determining positive or negative, a determination part for determining whether or not the sample solution is normally flown, that is, an end determination part is provided. Such an end determination part is Hb
Irrespective of the presence or absence of, the color tone is changed by the arrival of the sample solution, as in the case of the above-mentioned pregnancy diagnosis system, another type of antibody, that is, in the fecal occult blood test system, A method of immobilizing another different anti-Hb antibody or Hb as an antigen can be considered.

However, in the pregnancy diagnosis system and fecal occult blood test system as described above, apart from the determination part for determining positive or negative, the end determination part for determining the end of the test and the function confirmation of the test system are confirmed. Therefore, there is a problem in that the cost is very high if a bio-related substance such as an antibody or an antigen of another type as described above is used, though the device is a simple device.

[0009]

SUMMARY OF THE INVENTION The present invention has been made in order to reduce the cost of the above-described conventional simple inspection system. In the simple inspection system using the porous material, the end determination and the system operation confirmation are performed. It is an object of the present invention to provide an inexpensive determination reagent for performing.

[0010]

The present invention relates to a determination reagent used in a clinical test system using a porous material, which is provided when it is placed on the porous material and brought into contact with a sample solution to be tested. In addition, the determination reagent is characterized by comprising a substance whose color tone changes depending on pH, and does not use a bio-related substance as described above, so that the cost of the determination reagent can be reduced.

As the above-mentioned determination reagent, as long as it is a substance which is placed on a porous material and whose color tone changes with pH,
Not particularly limited, chlorophenol red, thymol blue, thymol phthalein, neutral red, phenol red, bromocresol green, bromocresol purple, bromocresol blue, bromophenol blue, methyl yellow, methyl orange, methyl red, Examples thereof include phenolphthalein and the like.

The amount of the determination reagent used is not particularly limited as long as it can sufficiently confirm the color tone change on the porous material. In addition, the inspection device in which the determination reagent of the present invention is used includes various inspection systems using a porous material and is not particularly limited. As such a test system, for example, a device using an immunochromatography method or an immunofilter method can be exemplified, and it can be used in the above-mentioned pregnancy diagnosis system, fecal occult blood test system, and the like.
As the porous material, in addition to celluloses such as nitrocellulose and cellulose acetate, an appropriate material such as a membrane filter made of synthetic polymer material such as nylon, fluorinated ethylene resin and polypropylene is used.

As a method of installing the judgment reagent in the porous material, the judgment reagent is mixed with an appropriate solvent and applied on the porous material, or the porous material is mixed with the judgment reagent in a solvent. It may be carried out by immersing in, and then drying. In addition, after the determination reagent is applied to the porous material, a hydrophilic polymer such as bovine serum albumin (BSA) or gelatin may cover the portion where the determination reagent is located.

[0014]

In the present invention, the determination reagent is composed of a substance which is placed on the porous material and whose color tone can be changed depending on the pH of the sample solution brought into contact therewith. Therefore,
It is possible to use an appropriate material that can change color depending on the above-mentioned pH, and it is possible to effectively reduce the cost of the determination reagent. That is, in the conventional simple test system, since the determination end site is constituted by using a biological substance such as an antibody or an antigen, there is a problem that the cost is high, whereas the determination reagent of the present invention is Since the substance is not a biological substance, the cost of the reagent required to complete the determination can be effectively reduced.

Therefore, according to the present invention, the cost of the simple pregnancy diagnosis system and the fecal occult blood test system can be reduced, and the spread of the simple test system can be promoted.

[0016]

EXAMPLES Hereinafter, non-limiting examples of the present invention will be described. This embodiment is an embodiment applied to a fecal occult blood test system.

First, the nitrocellulose membrane 1 having a planar shape of 10 × 50 mm and a thickness of 1 mm shown in FIG. 1 was prepared as a porous material. Next, as a first antibody, a phosphate buffer solution (pH 7.2: 20) of an anti-Hb monoclonal antibody (mouse MU-110: manufactured by Japan Biotest Institute).
0 μg / ml) 10 ml was prepared. Furthermore, polystyrene-based blue latex (average particle size 0.1 μm: solid content 5)
Wt%, Sekisui Chemical Co., Ltd.) 10 ml, 37 ° C
And stirred for 60 minutes. The resulting dispersion was 18.0 at 4 ° C.
Centrifuged at 00 rpm for 50 minutes. After removing the supernatant, a 1 wt% BSA (manufactured by Sigma) phosphate buffer solution (pH 7.2) was added to prepare a latex concentration of 1 wt%.

0.5 μl of the first antibody-sensitized carrier solution obtained as described above was added dropwise to the position of reference numeral 2 of the nitrocellulohys membrane 1 shown in FIG. Next, as a second antibody, an anti-human Hb monoclonal antibody (mouse MU-10
7: 5 mg of phosphate buffer solution (pH 7.2) of 1 mg / ml (manufactured by Japan Biotest Laboratories) was added dropwise to the position indicated by reference numeral 3 on the nitrocellulose membrane 1 and dried at room temperature for 12 hours. Further, after drying, it is immersed in a 1 wt% BSA phosphate buffer solution (opH7.2) at 37 ° C. for 30 minutes, and then again dried at room temperature for 12 hours to develop the first and second antibodies. It was immobilized on a layer of nitrocellulose membrane 1.

Preparation of judgment reagent 100% 1% by weight ethanol solution of phenolphthalein
10 ml of 0.1 mM NaOH aqueous solution (pH = 10) was added to ml to obtain a red solution. This red solution 100
μl was dropped on the position of the nitrocellose membrane 1 indicated by reference numeral 4.

Experimental Example Experiment Using Negative Specimen: Stool of a healthy person was sampled and dissolved in 10 ml of phosphate buffer (pH 7.2), and 1000 μl of this stool sample solution was added to the nitrocellulose membrane 1 with reference numeral 5.
It dripped at the position shown by. After about 5 minutes, it was possible to confirm that the color tone of the determination reagent had disappeared and the fecal sample solution had moved to the position indicated by reference numeral 4. At this time, no coloring was observed at the position indicated by reference numeral 3, and therefore it was confirmed that the result was negative.

Experiment using a positive sample: Feces of a healthy person were collected and dissolved in a phosphate buffer solution (pH 7.2) containing human Hb at a ratio of 50 μg / ml. 1000 μl of this solution was added to nitrocellulose membrane 1 with reference numeral 5
It dripped at the position shown by. After about 5 minutes the color tone of the determination reagent had disappeared, indicating that the solution had moved to the position indicated by reference number 4. At this time, coloring was observed at the position indicated by reference numeral 3, and therefore Hb was detected, and it was confirmed that it was positive.

[Brief description of drawings]

FIG. 1 is a schematic plan view for explaining a fecal occult blood test system prepared in an example.

[Explanation of Codes] 1 ... Nitrocellulose membrane as porous material 2 ... First antibody-sensitized carrier dropping position 3 ... Second antibody dropping position 4 ... Judgment reagent setting position 5 ... Sample dropping position

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yuji Kaneko 2-1 Hyakuyama, Shimamoto-cho, Mishima-gun, Osaka 2-1 Sekisui Chemical Co., Ltd.

Claims (1)

[Claims] 1. A determination reagent used in a clinical test system using a porous material, the color tone of which changes depending on pH when the sample solution is placed on the porous material and brought into contact with a sample solution to be tested. A determination reagent comprising a substance.