JPH1138006A - Inspection method and inspection kit - Google Patents

Abstract

PURPOSE: To increase the detection sensitivity of a specimen and shorten the time required for detection by adding gelatin at the specific position of a chromatographic carrier when a sample is added, or arranging gelatin in advance. CONSTITUTION: A marker region 3 is arranged on a chromatographic carrier 2 so that a tracer can be deployed by an aqueous solvent. An auxiliary material filtering a suspended material in a sample liquid to smooth an inspection is arranged in the upstream area 5 of the marker region 3. The sample liquid is added in the upstream area 5 of the marker region 3 or in the marker region 3, and a specimen in the liquid is marked by the tracer. The tracer and specimen are chromatographically deployed in the downstream direction and reach a coupling region 4. Gelatin is arranged in a region 7 between the marker region 3 and the coupling region 4, and a marker color former is arranged. The tracer coupled with the specimen is captured in the bonding region 4 for coloration. The tracer uncoupled with the specimen and a soluble material other than the specimen flow out in the downstream area 6 of the coupling region 4.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a test method and a test kit, and more particularly, to a test method using a chromatography strip and a test kit used for the test method.

[0002]

2. Description of the Related Art As a quick and simple test method, a method using a chromatography strip is known. Chromatographic strips are disclosed in JP-A-60-1922.
6, JP-A-1-63865 and JP-A-3-176669
As described in, for example, a tracer composed of a capture substance 1 and a marker according to the present invention, which specifically binds to an analyte, is arranged so as to be able to be developed by chromatography, or is added at the time of adding a sample, and is added downstream. And a chromatography carrier on which the capture substance 2 of the present invention that specifically binds to the analyte is fixed. When the sample solution is added to the region of the chromatography carrier where the tracer is arranged, or when the tracer and the sample solution are added together, the analyte binds to the tracer, and the tracer and analyte to which the analyte is bound are bound. Both unbound tracers are deployed along the long side of the chromatography strip, ie, downstream. When the tracer reaches the area where capture substance 2 is immobilized,
In this case, only the tracer to which the analyte is bound binds to the trapping substance 2 and is accumulated there, and the tracer to which the analyte is not bound flows downstream. Thus, the presence or absence and the amount of the analyte in the sample aqueous solution are determined by the capture substance 2
Can be known from the color development or the like at the portion where is fixed.

In connection with the present invention, Japanese Patent Laid-Open No.
6659 describes that the whole or a part of the labeling region in the present invention is wrapped with gelatin.

[0004]

The inspection method using such a chromatography strip can perform inspection in a short time and its operation is simple, but it is necessary to improve the detection sensitivity of an object to be detected. was there. That is, the problem to be solved by the present invention is to provide a test method using a chromatography strip with improved detection sensitivity and the like, and a test kit used for the test method with improved detection sensitivity and the like. To provide.

[0005]

Means for Solving the Problems The present inventors significantly increase the detection sensitivity by adding gelatin to a specific position of a chromatographic carrier at the time of adding a sample or previously disposing the gelatin at a specific position of a chromatographic carrier. It has been found that various points of the conventional test method and test kit can be improved, such as that the detection time can be shortened and the prozone phenomenon in the sandwich method is reduced.

That is, according to one aspect of the present invention, the capture substance 2 which is directly bound to the analyte or indirectly binds via the capture substance 3 which binds to both the analyte and the capture substance 2 is fixed or A capture substance 1 that specifically binds to the analyte or a capture substance 1 that specifically binds to the capture substance 2 in competition with the analyte is fixed downstream of a region where the capture substance 2 is immobilized. A chromatography carrier having at least a binding region is used, or a chromatographic strip to be added at the time of addition of a sample, which is previously disposed upstream of the binding region so that a tracer comprising a capture substance 1 and a marker can be developed by chromatography. In the test method, gelatin is added at the time of sample addition to the upstream of the binding region, or gelatin is added to the tray upstream of the binding region. Chromatography is inspection method characterized by pre-arranged in the region except for the region (hereinafter referred to as indicator area) which is arranged in advance.

Further, another aspect of the present invention is that the gelatin is previously arranged in the above-mentioned chromatographic strip and gelatin, or in a region upstream of the binding region of the above-mentioned chromatographic carrier and excluding the labeling region. This is a test kit having at least a gelatin arrangement chromatography strip.

In the present invention, the analyte to be detected in the presence or absence or the amount of the analyte in the sample may be any as long as a compound that specifically binds to the analyte is present. For example, a compound into which biotin is introduced (avidin specifically binds), a compound having avidin,
Complementary DNA, a compound or hapten having antigenicity, a compound that specifically binds to an antibody of a specific antigen, an antibody,
Antibody fragments that specifically bind to antigens, lectins that specifically bind to sugar ligands, sugar ligands that specifically bind to lectins, other ligands that specifically bind to receptors, and receptors that specifically bind to ligands, etc. can give. Of course, the higher the reactivity or affinity of these binding reactions, the higher the detection sensitivity. Specifically, microorganisms; viruses; specific antigens such as pathogenic microorganisms and viruses; antigens such as tumor marker antigens such as prostate cancer; antibodies such as antibodies and autoantibodies generated by microorganisms and viral infections; Stool hemoglobin or an antigen specific to hemoglobin; cells presenting a specific antigen, cells having a specific sugar ligand, cells having a specific receptor, etc .; thyroid hormone, parathyroid hormone, gonad stimulation Hormones such as hormones, cytokines, bioactive proteins or peptides such as monokines; enzymes, proteoglycans, gamma seminoproteins,
Proteins or glycoproteins such as amyloid precursors; low molecular weight chemicals or drugs in blood; signaling regulators such as agonists and antagonists; thrombolysis / coagulation regulators; gene expression regulators; cell adhesion molecules; Lectin; nucleic acids such as RNA and DNA are exemplified. In particular, feces, urine, etc. are desirable as non-invasive specimens.

The capture substance 1 is composed of a compound that specifically captures and binds to the analyte or competes with and specifically binds (captures) the capture substance 2 described below with the analyte. That is,
For example, when the analyte is an antigen, an antibody or an antibody fragment that specifically binds to the antigen (hereinafter referred to as “antibody”)
Also includes an antibody fragment that specifically binds to the antigen. ) Or a compound that specifically binds to an antigen or an antibody (hereinafter, “antigen” includes a compound that specifically binds to an antibody). When the analyte is an antibody, it is an antigen or an antibody. When the analyte is a ligand (or a compound having a ligand),
The capture substance 1 is a receptor (or a compound having a receptor) or a ligand, and when it is a receptor, it is a ligand or a receptor. Capture substance 1
In some cases, it specifically binds to the capture substance 3 via the target substance, and as a result, binds to the capture substance 2 via the capture substance 3. The binding between the analyte and the capture substance 1 may be reversible or irreversible, but if the binding strength is too weak, the detection sensitivity is reduced.

[0010] A marker is added to the capture substance 1 to form a tracer. As a method of adding a marker to the capturing substance 1, the capturing substance 1 may be adsorbed or included in the marker, or may be bonded by a hydrogen bond or a covalent bond. In short, it suffices that the capture substance 1 is added to such an extent that the capture substance 1 is not easily detached from the marker and is added so as to be recognized by the target substance or the capture substance 2.

The marker emits a signal, that is, a color indicating the presence thereof, when the capture substance 1 binds to an object to be detected, a capture substance 2 described below, or a capture substance 4 described below. The marker is preferably composed of fine particles that are insoluble in water and have poor water repellency. Examples of desirable markers include metal colloids such as colloidal gold, non-metallic colloids such as selenium colloid; organic colloids; dye particles; colored organic polymers such as colored polystyrene; and oil-based fine particles such as liposomes containing a coloring agent. . When the colloid or the like is not coloring, a coloring agent is added. Examples of the coloring agent include a fluorescent material and a coloring material in the ultraviolet region in addition to an ordinary coloring agent in the visible region. In addition, a substance that develops a color including fluorescence by an enzyme or the like disposed in a binding region or the like is also included in the color former of the present invention.

The tracer, which is either itself or bound to the analyte, develops and moves in the chromatographic carrier when the sample solution is added to the chromatographic strip. The tracer is used as a solution (or suspension) at the time of use, but can be stored in a powder form by freeze-drying or the like.

There are two methods for arranging the tracer such that the tracer can be developed on a chromatography carrier with an aqueous solvent. The first is a method in which a chromatographic carrier section which has been impregnated with a tracer aqueous solution and dried is placed on a base material or a chromatographic carrier at an appropriate distance from the binding region, which is upstream of the binding region described later. In this case, the place where the tracer is placed is called a sign area. The second is a method in which a tracer is added onto a chromatography carrier when a sample is added. Thus, in this case, the chromatography strip has no labeled area.

The capture substance 2 binds a tracer having an object to be detected through the object to be detected, or binds a tracer having the capture substance 1 through the capture substance 1. In the case of binding via the latter capture substance 1, it competes with the analyte and binds to the capture substance 2. In any case, the capture substance 2 specifically binds to the analyte. Furthermore, it may be configured such that it specifically binds to the capture substance 3 that specifically binds to the analyte, thereby indirectly binding to the analyte. More specifically, when the analyte is an antigen, the capture substance 2 is an antibody (here, “antibody” also includes an antibody fragment that specifically binds to the antigen as defined above). . When the capture substance 2 is an antibody and the capture substance 1 is also an antibody, it is desirable that the capture substance 2 recognizes a different epitope of the analyte antigen. Similarly, when the analyte is an antibody, it is an antigen ("antigen" also includes a compound that specifically binds to the antibody as defined above), and when it is a receptor, it is a ligand;
When it is a ligand, it is a receptor. When the capturing substance 2 specifically binds to the capturing substance 3, avidin, anti-biotin antibody, lectin, and the like are examples of the capturing substance 2.

In order to immobilize the capture substance 2 on the chromatography carrier, an appropriate functional group of the chromatography support and a functional group of the capture substance 2 may be covalently bonded, or the capture substance 2 may be bound to the chromatography support. May be firmly adsorbed. Further, the capture substance 2 may be bound or adsorbed to the fine particles, and the fine particles may be dispersed and fixed in the chromatography carrier. Here, the term “fixed” refers to being arranged so as not to be substantially diffused and developed by the aqueous solvent. The region where the capture substance 2 is fixed is called a binding region.

When the capture substance 1, capture substance 2, capture substance 3 or capture substance 4 is an antibody, a monoclonal antibody is used in many cases. It can be used after appropriate means such as selection of antigen or animal species.

The capture substance 3 is composed of a part that specifically binds to the analyte and a part that specifically binds to the capture substance 2. The portion that specifically binds to the analyte is an antigen when the analyte is an antibody, an antibody when the antigen is an antigen, and a receptor and a receptor when a ligand is a ligand, as described in detail in Capture Substance 1. Is a ligand. The portion that specifically binds to the capture substance 2 is, for example, biotin when the capture substance 2 is avidin, an anti-biotin antibody or avidin when it is biotin, or a sugar ligand when it is a lectin.

The capture substance 3 is pre-arranged on the chromatography carrier so that it can be developed and moved into the chromatography carrier when an aqueous solution of the sample is added to the chromatography strip in any region selected appropriately upstream of the binding region. Or added to the chromatographic support at the time of sample addition. The arrangement method was described in detail in the section of tracer arrangement.

The chromatography carrier may be any carrier as long as it can develop a solute in an aqueous solvent. For example, the nonwoven fabric is made of cellulose, nitrocellulose, cellulose acetate, polyacrylate, glass, agarose, polyurethane, or the like, or a combination of these and inorganic powder.

A chromatographic strip can be obtained by, for example, laminating an appropriate substrate to the chromatographic carrier as required.

As is well known, gelatin is obtained from vertebrates such as fish and mammals. The melting temperature of the gelatin gel is preferably 15 ° C. or higher, more preferably 25 ° C.
That is all.

In the present invention, the melting temperature of a gelatin gel is measured by the following method.

Gelatin is dissolved in a 10 mM phosphate buffer (pH 7.4) containing 0.9% sodium chloride to a concentration of 10 g / dl. 1 ml of this gelatin solution is put into a test tube having a length of 100 mm and a diameter of 12.5 mm, and the test tube is stoppered. The test tube is immersed sufficiently in a water bath at 0 ° C. to gelatinize the gelatin. The temperature of the water bath is slowly increased at a rate of about 2 minutes per degree Celsius, and the temperature at which the gelatin gel falls when the test tube is inverted is measured.

Gelatin is used at the time of adding a sample solution, or is used by being arranged on a chromatography carrier in advance. When used at the time of addition of the sample solution, it may be dissolved in the sample solution, or may be added as an aqueous gelatin solution separately from the sample solution. Usually, it is desirable to use the same aqueous solvent as when preparing the sample liquid as the aqueous solvent when preparing the aqueous gelatin solution. When gelatin is used at the time of adding a sample, the gelatin concentration of the gelatin solution is preferably in the range of 0.1 to 1.5% by weight. Even when gelatin is placed in advance, the amount of gelatin is the weight% of the above sample solution.
It is good to arrange in advance.

When gelatin is added at the time of adding a sample solution, it is added to any region upstream of the binding region. When the gelatin is previously arranged, it is arranged upstream of the binding region and in a region other than the labeling region. The optimum addition or arrangement site is appropriately selected in advance, but if it is arranged in the region 7, the best result may be obtained.
When obtaining a gelatin-located chromatographic strip in which gelatin has been pre-arranged on a chromatographic carrier, it is arranged upstream of the binding region and in a pre-selected region other than the labeling region so as to be developed with an aqueous solvent. I just need. That is, a chromatographic carrier section that has been impregnated with an aqueous gelatin solution and dried may be placed on a substrate or a chromatographic carrier.

When the capture substance 4 that specifically binds to the capture substance 1 is fixed downstream of the region of the chromatography carrier to which the capture substance 2 is bound to form a binding area, gelatin is immobilized on the capture substance 2. Area and trapping substance 4
Is preferably added or pre-arranged between the regions in which is fixed.

When gelatin is used by a method other than the method of the present invention, for example, when gelatin is previously arranged in a labeling region, problems such as an increase in detection time and a decrease in detection sensitivity often occur. On the other hand, for example, when gelatin is used at the time of adding a sample, such a problem does not occur even if it is added to the labeled region. This is presumably because when gelatin was previously placed in the labeling area, the gelatin had some effect on the labeling substance during storage of the chromatography strip.

The aqueous solvent of the sample solution may be water, but usually a pH buffer is used. PH of aqueous solvent
Is preferably in the range of 5 to 9.

The sample solution is added to the labeled region or upstream of the labeled region when there is a labeled region, or upstream of the binding region when there is no labeled region. The addition here means
There are cases where the sample aqueous solution is placed on the chromatography strip and cases where the uppermost end of the chromatography strip is immersed in the sample solution.

If the tracer and / or the capture substance 3 are not previously arranged on the chromatographic carrier, they are added when the sample is added. The addition method is usually added together with the sample.

There are two cases for the test kit of the present invention.

The first case is when a chromatographic strip in which gelatin is not arranged in advance and gelatin are combined at least. In this case, an aqueous solvent or the like may be further combined. In addition, gelatin
It may or may not be dissolved in an aqueous solvent.

The second is the case where it is constituted by a gelatin strip having a chromatographic arrangement. Also in this case, other components such as an aqueous solvent may be further combined. In any of the above cases, when the tracer and / or the capturing substance 3 are not previously arranged on the chromatography carrier, they are combined as a powder or aqueous solution (or suspension) such as freeze-dried.

Further description will be made with reference to the drawings.

FIG. 1 shows a chromatographic strip where the labeling area is located. The chromatography strip 1 is composed of a chromatography carrier 2 and a base material as required.

The chromatographic carrier 2 has a labeled area 3 in which the tracer is arranged so as to be developed with an aqueous solvent.

The upstream area 5 of the labeling area 3 is arranged as required, and when it is arranged, it often serves as a sample liquid addition site. Here, the suspension in the sample solution can be filtered, and an auxiliary substance for smoothly performing the test can be included. Examples of the auxiliary substance include a substance that makes the binding reaction between the analyte and the capture substance 1 more specific or more quantitative, a coloring reagent for an insoluble marker, and the like.

When a sample solution is added to the upstream region 5 or the labeling region 3 of the labeling region 3, the analyte in the sample is labeled with a tracer if it binds to the capture substance 1. Next, the tracer bound to the analyte and the tracer not bound to the analyte and the analyte were chromatographed and moved rightward, ie, downstream, in FIG. The connection region 4 is reached.

The labeling region 3 and the binding region 4 are not directly connected to each other and are arranged at a predetermined interval which is appropriately selected. In the area 7 for this interval,
In addition to the case where gelatin is placed, the labeling substance 3 may be arranged so that it can be developed by chromatography.
Further, a coloring agent for the marker may be arranged.

The coupling region 4 has the following two cases. That is, in the first case, the tracer that has captured the object is captured here and colored, or the tracer is captured and colored here in competition with the object. The second is a case where the capture substance 4 is fixed downstream of the area where the capture substance 2 is fixed (in the first case, this area is used as the binding area). In this case, they are appropriately arranged as necessary, but when they are arranged and used for color detection, they are referred to as a combined area 4. In the second case, the tracer that cannot compete with the substance to be detected and binds to the capture substance 2 and is not captured is captured by the capture substance 4 in the binding region and colored.

When the trapping substance 3 is disposed, the trapping substance 3 binds to the detection target captured by the tracer, and the complex of the tracer / detection target / capture substance 3 is formed via the capture substance 3. And binds to the capture substance 2 and stops at the binding region 4. Alternatively, the capture substance 3 binds to the capture substance 2 and specifically binds to the analyte in the binding region 4.

The downstream region 6 of the binding region 4 is used for allowing the tracer to which the target substance is not bound via the capture substance 1 to flow out when the target substance 1 binds to the target substance, or It is provided as necessary so that soluble substances other than the analyte in the sample liquid flow out. Further, in the downstream region 6, for example, a pH indicator or the like that develops a color when the aqueous solvent of the sample aqueous solution flows is arranged, thereby making it possible to determine the reliability of detection.

When the labeling area 3 is not provided on the chromatography strip, the test is performed in the same manner as described above, except that the tracer is added at the time of adding the sample.

[0044]

【Example】

Example 1 Preparation of tracer consisting of anti-human hemoglobin antibody labeled with selenium colloid: 91 mM sodium L-ascorbate and 32 mM selenium oxide at about 4 ° C.
And then stirred at about 42 ° C. for about 70 hours to prepare a selenium colloid.

The obtained selenium colloid was 550 nm
Was diluted with 10 mM bistris buffer (pH 7.0) so that the absorbance of the solution became 15. A mouse monoclonal anti-human hemoglobin antibody (0.02%) was added to the diluted solution, and the mixture was stirred at room temperature for 1 hour. Next, the obtained anti-human hemoglobin antibody labeled with a selenium colloid was washed with a 10 mM Tris-HCl buffer (pH 7.2) to obtain a tracer.

Preparation of Labeling Region: A tracer suspension was obtained by placing the tracer in 10 mM Tris buffer (pH 7.2) containing 1% casein so that the absorbance at a wavelength of 550 nm became 1.0. A glass fiber membrane (Lypore9524, manufactured by LYDALL, USA) was immersed in the suspension to sufficiently soak the suspension, and then the glass fiber membrane was dried to form a label region.

Preparation of binding region: 2 mg of a mouse monoclonal anti-human hemoglobin antibody having a different binding site to human hemoglobin is used as the anti-human hemoglobin antibody.
/ Ml in 30 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride. On the other hand, 0.4 × 4.
Nitrocellulose membrane with rectangular pores of 5 cm and 5 μm (Schleicher & Schuell, USA)
A 0.4 × 6.0 cm rectangular laminate having a thickness of 100 μm (PET100 PE LR, manufactured by Lintec Corporation)
007A) was affixed such that the top edges were aligned when the long sides were vertical. A solution obtained by linearly mixing the antibody was dropped approximately 1 cm from the lower end of the nitrocellulose membrane to which the laminate was attached, and then dried sufficiently to fix the anti-human hemoglobin antibody.

Preparation of Chromatographic Strip: Pad containing selenium colloid-labeled anti-human hemoglobin antibody cut into squares of 0.4 × 0.4 cm on the lower laminate of nitrocellulose with immobilized anti-human hemoglobin antibody Area) was applied slightly in contact with nitrocellulose. 0.4 × 1.3 cm hydrophobic non-woven fabric (Sontara 8801 manufactured by DuPont, USA) as a pre-filter on the laminate under the pad
Is attached so that it is slightly in contact with nitrocellulose,
This was used as a chromatography strip.

Assay: Contains 0% human bovine serum albumin (manufactured by Sigma, USA) and 0.1% bovine serum albumin (Seikagaku Corporation, Tokyo), 0.9% sodium chloride, 0.1% sodium azide. .1M Tris-HCl buffer (p
H7.6) was used as a sample. To this, gelatin derived from pig skin (manufactured by Sigma, USA) was added to a concentration of 0.7%. 25 μl of this sample solution was added onto the pre-filter of the chromatography strip. Seven minutes after the addition of the sample solution, the determination was made by reading the "redness" derived from the selenium colloid with the naked eye. The sensitivity was defined as the lowest hemoglobin concentration at which "redness" could be observed with the naked eye.

Table 1 shows the determination results. As is clear from Table 1, when gelatin was used, the sensitivity was increased about 80 times compared to when gelatin was not used.

[0051]

[Table 1]

Example 2 In Example 1, the determination was made from 1 minute after the addition of the sample solution to 1 minute.
The interval was one minute up to two minutes.

FIG. 2 shows the results. As shown in FIG. 2, when gelatin was used, the inspection time was reduced to about one third as compared with when no gelatin was used.

Example 3 In Example 1, the concentration of human hemoglobin in the sample solution was set to the value shown in FIG. The results are shown in FIG. As can be seen from FIG. 3, the measurable upper limit of the concentration of human hemoglobin when gelatin was used was about 10 times higher than the measurable upper limit when gelatin was not used. That is, it is understood that the use of gelatin reduces the prozone phenomenon.

[0055]

By using the test method or test kit of the present invention, the detection sensitivity of an object to be detected can be increased. Further, the time required for detection can be reduced. In addition, the so-called prozone phenomenon in the sandwich method is reduced.

[Brief description of the drawings]

FIG. 1 is an explanatory view of a chromatography strip.

FIG. 2 shows an inspection result by the inspection method of the present invention (Example 2).

FIG. 3 shows an inspection result by the inspection method of the present invention (Example 3).

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Chromatography strip 2 Chromatography carrier 3 Labeling area 4 Binding area 5 Upstream area of labeling area 6 Downstream area of binding area 7 Area between labeling area and binding area a Result when gelatin was used b When gelatin was not used result

Claims (10)

[Claims] 1. A capture substance 2 which is directly bound to an analyte or indirectly bound via a capture substance 3 which binds to both the analyte and the capture substance 2 is fixed or specific to the analyte. Or capture substance 4 that specifically binds to capture substance 1 that specifically binds to or to capture substance 2 in competition with the analyte
Is preliminarily arranged on a chromatography carrier having at least a binding region immobilized downstream of the region where the capture substance 2 is immobilized, such that a tracer comprising the capture substance 1 and a marker can be chromatographed upstream of the binding region. In a test method using a chromatographic strip to be added or added at the time of adding a sample;
A test method, wherein gelatin is added at the time of adding a sample to the upstream of the binding region or is preliminarily disposed in a region upstream of the binding region and excluding a region where a tracer is preliminarily disposed. 2. The inspection method according to claim 1, wherein the gelatin gel melts at 15 ° C. or higher. 3. The method according to claim 1, wherein the gelatin gel melts at 25 ° C. or higher. 4. The test method according to claim 1, wherein the sample is feces or urine. 5. In a case where a region in which the capturing substance 4 that specifically binds to the capturing substance 1 is fixed downstream of the area in which the capturing substance 2 is fixed is used as the binding area, gelatin is immobilized on the capturing substance 2. 2. The inspection method according to claim 1, comprising adding or previously disposing between the region where the trapping substance 4 is immobilized and the region where the trapping substance 4 is immobilized. 6. A pre-placed chromatographic strip and gelatin according to claim 1 or gelatin in a region upstream of the binding region according to claim 1 but excluding a region where a tracer is pre-positioned. A test kit having at least a gelatin arrangement chromatography strip. 7. The test kit according to claim 7, wherein the gelatin gel melts at 25 ° C. or higher. 8. The test kit according to claim 7, wherein the gelatin gel melts at 15 ° C. or higher. 9. The test kit according to claim 6, wherein the sample is stool or urine. 10. In a case where a region in which a capturing substance 4 that specifically binds to the capturing substance 1 is fixed downstream of a region where the capturing substance 2 is fixed is used as a binding area, gelatin is immobilized on the capturing substance 2. Area and trapping substance 4
The test kit according to claim 1, wherein the test kit is arranged in advance between regions where the is fixed.