JPH01463A - Testing method, testing device and testing kit - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a test method, a test device, and a test kit suitable for detecting test components in samples such as biological fluids.

[Conventional technology]

In the field of clinical testing, analyzers that use conventional wet analysis methods have problems with equipment maintenance, reagent preparation and storage, and sloppy operation, but they are highly accurate.

It is superior in terms of high throughput and has been widely used, especially in the field of clinical chemistry testing. On the other hand, recently,
Dry analysis methods (dry chemistry) are being developed, and it is becoming possible to obtain test results in an emergency without having to operate an analyzer. For example, test strips used in urine tests are a type of dry chemistry, and there are also multilayer film dry chemistry products that have been developed in recent years.

Furthermore, a system in which wet analysis is performed using a disposable carrier and the water used in the reaction is retained in the carrier can also be broadly classified as a dry method. In recent years, these dry methods, which previously only produced results with low accuracy, have become able to improve the specificity of the reaction and produce highly accurate test results. In addition, a highly sensitive detection method for trace components that combines a dry method and an immunoassay method has also been attempted.

As a result of this process, the chemical reactions used in the dry method have often adopted reactions with high specificity, such as enzymatic reactions or immunological reactions. In addition, the development of analytical methods has been remarkable, with efforts being made to increase analytical accuracy and detection sensitivity by combining multiple reactions, and the range of future applications is extremely wide.

For example, a method for immunologically determining the presence or absence of pregnancy is disclosed in JP-A-61-182577. In this example,
hCG is detected using a combination of latex-supported β-hCG (β-chain subunit of human chorionic gonadotropin) antiserum having an active group. - Ho, Japanese Patent Publication No. 52-82
No. 892 proposes a method for determining pregnancy at home, which utilizes a wet reaction. In this example,
It uses the imperial reaction caused by a specific enzyme.

[Problem that the invention seeks to solve]

In the conventional technology, it has not been possible to check the progress of the reaction on the reaction carrier or to check the activity or deterioration of the reagent. The dry method described above has the advantage that even householders can use it due to its simplicity, but on the other hand, the reagent storage period is often long, and there are problems with the reagent storage environment. If the activity of the cell is degraded, it may cause an erroneous determination. It is important to be able to confirm that there is no deterioration of reagents during storage and that multiple chemical components are working effectively, etc. in order to improve the reliability of test results, but in the past, such confirmation was not possible. There was no easy way to do it.

An object of the present invention is to provide a test method, a test device, and a test kit that can easily check the deterioration of a reagent and also check the presence or absence of a test component.

[Means for solving problems]

In the testing method of the present invention, a liquid sample to be tested for the presence or absence of a test component is transferred to a first tube carrying a reactive substance that reacts with the test component.
and a second carrier carrying a substance of the same type as the test component, and a coloring agent that is involved in coloring when the test component is present is applied to the first and second supports. It is characterized by being brought into contact.

The test device of the present invention is characterized in that it includes a carrier carrying a substance reactive with a test component and a substance of the same type as the test component, and a liquid sample receiving container in which the carrier is placed.

The test kit of the present invention includes a first test piece carrying a reactive substance that reacts with a test component, a second test piece carrying a substance of the same type as the test component, and a test kit that includes The test piece is characterized by comprising a coloring agent that participates in coloring the first and second test pieces.

In a preferred embodiment of the present invention, a specific reaction such as an immune reaction or an enzyme reaction is used. When an immune reaction is used, the detection substance is an antigen or an antibody, and the substance of the same type is an antibody or an antigen of the same type. It is. When using an enzymatic reaction, the enzyme is supported as a homogeneous substance.

[Effect]

In the present invention, the reagents are active and effective.

If the test component is present in the sample, both the detection substance and the test component are colored.

If the reagent is active and the analyte is not present in the sample, only both types of substances will be colored. If the reagent has no activity and has deteriorated, neither will be colored. Therefore,
Easily check the effectiveness of reagents.

〔Example〕

For example, if a component to be measured is supported on a part of the reaction support in advance, the component to be measured in the sample and the supported component will show the same reactivity to the analytical reagent in one reaction support. The sample components can be analyzed while checking the state of the reagent and the progress of the reaction by the reaction caused by the supported components.

Also, for example, as a reaction in the case where a highly specific immune reaction is carried out on a reaction support.

After immobilizing the antibody against the antigen, which is the measurement component, on a reaction support and reacting with the sample component, the labeled second
A method can be considered in which the components are analyzed based on the amount of label on the reaction support by reacting with antibodies. For example, when the labeling substance is an enzyme, the progress of the enzymatic reaction can be confirmed by preparing in advance a portion of the reaction support that supports the enzyme. Not limited to immunoassay methods, when an enzyme is involved in a color reaction or the like, sample components can be similarly analyzed while confirming that the reaction is progressing.

In addition, even with enzymes other than those mentioned above, by supporting components involved in the reaction for analyzing sample components on a reaction support, it is possible to analyze sample components while checking the progress of the reaction. .

Until now, in the field of so-called dry chemistry, where components are analyzed using a reaction support, when determining results based on a color reaction, for example, when the expected color is not observed, the color reaction is included. It was not possible to distinguish whether there was a hindrance to the progress of the reaction for analysis, or whether one of the components was below the detection limit.

According to the present invention, this is achieved by supporting a substance involved in a reaction for detecting a test component in a liquid sample on a planar reaction support. In the second case, the supported substance is preliminarily supported on a reaction support at a site (location) different from the site where the test component in the sample, which is the original measurement component, reacts.

In other words, when determining the analysis results, the substances involved in the reaction for detecting the test component are separated so that the site for checking the progress of the reaction and the site for determining the analysis result of the test component can be determined separately. , supported on a reaction support.

If the reaction check site on the reaction support does not show any change, such as coloration, when reacted with the reagent, it is presumed that there was some kind of hindrance in the progress of the reaction. Therefore, even if no change such as coloration is observed in the analysis site of the sample component, it cannot be determined whether the sample component is not contained or whether the concentration is below the detection limit. However, e.g.
If the second reaction check site shows a change such as coloration, but no change is observed in the sample component analysis site, it is likely that the sample does not contain the test component, or Even if it exists, it can be determined that its concentration is below the detection limit of the analytical method.

The reaction support used in the present invention may be filter paper, which has been conventionally frequently used in the field of dry chemistry, or may be a nylon film, a polyester film, or the like. Moreover, various packing agents may be used as the reaction support, and these may be used by filling a column. Various fillers can be used, such as latex particles, bead-shaped agarose, and Sepharose.

The test kit based on the present invention comprises a reaction support carrying components involved in a reaction for analyzing a test component in a sample, an antibody solution, a coloring solution, and a buffer. Optimal conditions for the composition and concentration of each component are determined depending on the type of component to be analyzed.

The present invention will be explained in detail below.

Embodiment 1 In FIG. 1, the cylindrical case 10 is made of a material that is stable against reagents, such as glass or synthetic resin. A disk-shaped carrier 2o is fixed near the bottom of the case 10, and the carrier 20 includes a layer 21 made of a porous material and a cellulose membrane 22. The cellulose membrane 22 carries a substance 23 of the same type as the component to be measured in the sample in a localized manner, and a detection substance 2 that can react with a reagent and cause coloring.
4 is supported locally. These materials are localized to form a pattern of predetermined shape.

The case 10 is removably attached to a drainage liquid receiver 30 that receives the sample liquid and reagent liquid that have passed through the carrier. The original color of the support 20 is white, and the supported substance can be colored in various colors depending on the type of reaction.

Next, an experimental example using such an example will be described.

Human HCG 23 was bound to a part of a commercially available cellulose membrane 22, and anti-human HCG antibody 24 was bound to the other part. Next, the cellulose membrane 22 is attached to a water-absorbing porous material 2.
1 was adsorbed. After configuring as shown in Figure 1, this HC
100 μQ of a urine sample was added onto the reaction carrier 20 for G detection and left for 5 minutes. Next, 100 μQ of alkaline phosphatase-labeled anti-human HCG antibody was added, excess water was absorbed by the porous material, and the mixture was left at room temperature for 5 minutes.

Thereafter, 1 μQ of Tris acid buffer (pH 7, 8) was added, and water and excess labeled antibody solution were removed by allowing the porous material 21 to absorb water. Further, a solution of P-21-lophenyl phosphoric acid as a substrate was added, and enzymatic reaction was carried out for 5 minutes on the reaction support. It was determined whether or not the sample contained HCG based on the presence or absence of yellow coloring derived from the generated p-nitrophenol. Regardless of the presence or absence of HCG in the sample, the H
CG binding sites are colored yellow. This confirmed that the enzyme reaction proceeded without any problems.

FIG. 2 shows an example of the determination.

By devising the supporting locations (patterns) of the components that are pre-supported on the reaction carrier, in qualitative judgments, a negative judgment without the test component is marked with a minus mark, and a positive judgment with the test component is colored with a plus mark. It is also possible to do so. For example, using the example in Figure 2, if HCG is supported on site 23 and anti-HCG antibody is supported on site 24, when HCG is not contained in the sample (Figure 2A ) is colored negative, and when HCG is included (Figure 2B) it is colored positive.

However, it goes without saying that the parts on which each component is supported are not limited to this. Furthermore, if the reagent has deteriorated, the minus mark will not appear as a plus sign.

Table 1 Table 1 shows a comparison of test results between the method based on Example 1 and the conventional method. In Table 1, all normal pregnancy methods 10
The results of checking the effectiveness of the reagent over time using samples that were determined to be positive in 0 cases are shown. The numerical value of the numerator in the column to which the present invention was applied indicates the number of samples in which urine was positive, and the numerical value of the denominator is the number of samples in which the CG immobilization site was colored (i.e., the activity of the reagent was observed) of the carrier. Indicates the number.The numerator and denominator match.In the conventional method column, 1
The number of differences from 00 corresponds to the number of results that were incorrectly determined to be negative even though they were originally positive.

For example, in the case of the present invention, when using a reagent that has been prepared for 12 months after the reagent kit was prepared, there was no problem in the progress of the reaction in 80 out of 100 samples, and in addition, the sample component analysis section Coloration was observed. However, 10
For 20 out of 0 cases, HC, which is the 1 reaction check site,
No coloration was observed at the G-binding site, and it was determined that there was a problem in the progress of the 1-analysis reaction. With the conventional reagent, 75 out of 100 cases were HCG-positive, but the remaining 25 cases showed HCG-positive results even though their urine was HCG-positive.
It tested negative.

Example 2 Filter paper (TOYONa51) was cut out to IX2cM and used as a test piece base material. Approximately 1g (500 pieces) of base material in 20mQ
The suspension was suspended in 5M potassium phosphate buffer (pH 12.1), and 10 mQ of water was added. 1 while stirring with star 5-
A 0 mQ BrCN solution (0.1 g/mQ) was gradually added through the throat for 2 minutes, and the reaction was continued for an additional 6 minutes. The reaction was carried out at 4°C. Immediately after the reaction, a large amount of 0.005M was cooled on ice.
Na1 (washed with Co3 solution, 5 mg purified anti-AFP
It was allowed to react with the antibody at room temperature for 8 hours. Further, after reacting with 1M ethanolamine (pH 8,0) at 4°C overnight,
, 5M NaHCOa.

0.1M acetate buffer (pH 4,0), 0.015M P
Washed sequentially with BS (pH 7,2) and 1.5mM PB.
Freeze-dried in S (pH 7.2). In this way, a first test piece was obtained.

In a similar manner, AFP was bound to a piece of filter paper and freeze-dried to obtain a second test piece.

100 μQ of sample was added to anti-AFP antibody binding test strip and AFP.
Each was added to the bonded specimen and allowed to react at room temperature for 16 hours. After washing twice with 1 mQ of 0.9% NaCQ, 0.1 mQ of alkaline phosphatase-labeled antibody was added and reacted at room temperature for 24 hours.

Transfer each specimen to a separate glass test tube and
(After washing 3 times with 1 liter of 0.9% NaCQ, the substrate solution 1
mQ was added and reacted at 25°C for 60 minutes.

The case where both the AFT” binding test piece and the anti-AFP antibody binding test piece were colored was considered to be AFP positive. In this case,
AFP binding test strips have been used to determine the effectiveness of reagents.

This example method is provided to users as an AFP test kit. In other words, the test kit includes an anti-AFP antibody binding test piece, an AFP (antigen) binding test piece, and NaCQ for cleaning.
It is equipped with a solution, an alkaline phosphatase-labeled antibody reagent solution, a substrate reagent solution, and a test tube as a reaction container.

In Example 2 as well, when the reagent has activity but the concentration of the test component in the sample is below the predetermined concentration, the area containing the same substance as the test component becomes colored, and the reagent has activity and the concentration of the test component in the sample is colored. This method utilizes the phenomenon that when the concentration of the test component is equal to or higher than a predetermined concentration, both a location where a substance of the same type as the test component is located and a location where a substance reactive with the test component is located are colored.

In Example 2, each test piece was reacted in a separate reaction vessel, but two types of test pieces were joined together to substantially integrate them and reacted in one vessel as in Example 1. You can also do it. In this case, the test kit is prepared with a test piece in which two types are integrated.

〔Effect of the invention〕

According to the present invention, it is possible to check the progress of the reaction and the deterioration of the reagent, and at the same time detect the component to be tested, thereby improving the reliability of the test results.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view showing a schematic configuration of an embodiment of the present invention, and FIG. 2 is a diagram showing an example of a judgment pattern for a qualitative reaction. 10... Case, 20... Carrier, 23... HC
G-carrying part, 24... anti-HCG antibody-carrying part.
2. X', Fusa!・・・ Fraud

Claims (1)

[Claims] 1. A liquid sample to be tested for the presence or absence of a test component is carried on a first support carrying a reactive substance that reacts with the test component and a second support carrying a substance of the same type as the test component. A testing method characterized by contacting the first and second carriers with a coloring agent that participates in coloring when the test component is present. 2. A testing device comprising a carrier carrying a substance reactive with a test component and a substance of the same type as the test component, and a liquid sample receiving container in which the carrier is placed. 3. The test device according to claim 2, wherein the detection substance is an antigen or an antibody, and the homogeneous substance is a syngeneic antibody or antigen. 4. The test device according to claim 2, wherein the substance of the same kind is an enzyme. 5. A first test piece carrying a reactive substance that reacts with the test component, a second test piece carrying a substance of the same type as the test component, and when the test component is present, the first and A test kit comprising a coloring agent that participates in coloring a second test piece.