JPH07159398A - External diagnostic medicine - Google Patents

Abstract

PURPOSE:To provide a diagnostic tool having uniform coloration and the diagnostic tool, which can simply determine the quantity of a material to be detected. CONSTITUTION:This is a diagnostic tool, which can judge the presence or absence of the material to be detected in specimen liquid. In a base material 1, at least a specimen-liquid applying part 2, where specimen liquid is applied, the material to be detected and a positive judging part 4, on which a material that can be specifically bonded to the material to be detected is supported, are provided. The positive judging part 4 is constituted as a single region. The supporting amount of the material, which can be specifically bonded to the material to be detected, is proportional to the distance from the specimen- liquid applying part 2 and continuously increases in the single region. Or, the positive judging part 4 is constituted of a plurality of independent regions. The amount of the material, which can be specifically bonded to the material to be detected that is supported in each of a plurality of regions, respectively, increases stepwise in every region in proportion to the distance from the specimen-liquid applying part 2. This is the diagnostic tool performing these functions.

Description

Detailed Description of the Invention

[Background of the Invention]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a diagnostic tool for an in-vitro diagnostic agent using urine, blood and the like and a method for producing the same.

[0002]

2. Description of the Related Art In recent years, in vitro diagnostic agents using proteins, particularly antibodies, have been developed with the progress of trace substance detection systems using the antigen-antibody reaction. Among them, the deployable diagnostic tool using the sandwich method is capable of detecting trace components simply by dropping urine or the like, and is excellent as an OTC (over-the-counter drug). Therefore, some of them are already commercially available as pregnancy diagnostic agents.

The determination by a diagnostic agent is carried out by detecting a substance (for example, an antigen such as a protein) to be detected and a labeled substance (for example, an antigen such as a protein) which is capable of specifically binding to the substance at a specific portion on the diagnostic tool.
It is generally performed by binding with an antibody) and the presence or absence of coloration from the label, and it is important for accurate determination that the coloration is uniform and clear.

In addition, in the current diagnostic agents, the presence or absence of a specific substance is usually determined to make the diagnosis. However, for example, the amount of luteinizing hormone in urine peaks during ovulation, but its presence is confirmed before and after the ovulation. In such a case, not only the existence of it,
The usefulness of the diagnostic tool is considered to be high if a simple diagnostic tool that can easily measure and determine its amount can be realized.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a diagnostic tool having uniform and clear coloration. Another object of the present invention is to provide a diagnostic tool capable of easily quantifying a substance to be detected. The present inventors have completed the present invention by finding that the above object can be achieved by changing the amount of a substance capable of binding a substance to be detected supported on a substrate.

Therefore, the diagnostic tool according to the present invention is a diagnostic tool for determining the presence or absence of a substance to be detected in a sample liquid,
The base material is made of a porous material capable of developing a sample liquid, and the base material carries a sample liquid application site to which the sample liquid is applied and a positive substance carrying a substance capable of specifically binding to the substance to be detected. At least a determination portion, the carried amount of the substance capable of specifically binding to the substance to be detected, carried on the positive determination region, increases in proportion to the distance from the sample liquid application site. To do.

More specifically, in the diagnostic device according to the first aspect of the present invention, the positive determination site is constituted as a single region, and the amount of the substance capable of specifically binding to the substance to be detected carried by the specimen is It continuously increases in a single region in proportion to the distance from the liquid application site, and the diagnostic tool according to the second aspect of the present invention is characterized in that the positive determination site is independent from a plurality of regions. The amount of the substance that is configured and is capable of specifically binding to the substance to be detected carried in each of the plurality of regions is increased stepwise in each region in proportion to the distance from the sample liquid application site.

According to the first aspect of the present invention, there is provided a diagnostic tool having a uniform and clear coloration. Further, according to the second aspect of the present invention, there is provided a diagnostic tool that can easily quantify a substance to be detected.

[Detailed Description of the Invention] Sample liquids to which the diagnostic device according to the present invention is applied include those collected from animals including humans such as saliva, blood, urine, and stool, as well as those dissolved or suspended. A turbid aqueous solution is included. The diagnostic tool according to the present invention is used for determining whether or not a specific substance (substance to be detected) is present in these sample liquids, for example, to know the condition of animals including humans or for the diagnosis of diseases. It is something. Specifically, a pregnancy diagnostic agent using chorionic linear stimulating hormone (CG) as a substance to be detected, an ovulation diagnostic agent containing luteinizing hormone as a substance to be detected, and a fecal occult blood diagnostic agent containing hemoglobin as a substance to be detected, for example, It can be used as a virus infection diagnostic agent using a viral antibody such as HIV as a substance to be detected, and can also be used for detecting cancer tumor markers such as CEA and CRP and markers such as microalbumin. In addition, although the term “diagnostic agent” is sometimes used to mean the diagnostic tool itself, both are used in the same meaning in the present specification.

A preferred specific example of the diagnostic tool according to the first aspect of the present invention is shown in FIG. FIG. 1A is a perspective view of the diagnostic tool, and FIG. 1B is a partial cross-sectional view of the diagnostic tool. The diagnostic tool shown in the figure is provided with a sample liquid application site 2, a specific substance supply site 3, and a positive determination site 4 to which a sample liquid is applied in order from one end of a base material 1.

The base material 1 of the diagnostic tool according to the present invention uses a porous material as a base material on which the sample liquid can spread. Here, that the sample liquid can be developed means that the sample liquid can move through the porous material of the substrate by the capillary action. Preferable examples of the base material include glass fiber, cellulose, and filter paper, nylon non-woven fabric, polyester non-woven fabric, nitrocellulose, polyvinylidene fluoride, regenerated cellulose, cellulose acetate, charge-introduced nylon, and the like made of them. Further, it is also preferable to perform treatment with bovine serum albumin, collagen, casein or a synthetic polymer, if necessary, before or after the step of forming a positive determination site described later.

The sample liquid application site 2 is a site to which the sample liquid is applied, and after the sample liquid is applied, it spreads and moves in the porous material of the substrate 1 toward the specific substance supply site 3 by the capillary action. To start.

The specific substance supply part 3 is a part in which a substance capable of specifically binding to a substance to be detected (hereinafter, this substance may be referred to as “first substance”) is carried on the base material 1. . Specific examples of the first substance include an antibody against the antigen when the substance to be detected is an antigen, and an antigen when the substance to be detected is an antibody. This substance has the ability to specifically bind to the analyte. Therefore, when the substance to be detected exists in the sample liquid that has developed and moved from the sample liquid application site 2, the substance to be detected and the first substance bind to form a complex. Since the first substance needs to move with the development of the sample liquid, when the first substance is carried on the base material 1, the functional group existing in the first substance or the base material is
For example, it may be blocked with bovine serum albumin, collagen, casein, etc., and the first substance may be supported so as not to be strongly fixed.

The sample liquid that has passed through the specific substance supply site 3 then reaches the positive determination site 4. The positive determination part 4 is a substance that can specifically bind to the substance to be detected and is different from the substance carried in the specific substance supply part 3 described above (hereinafter, this substance will be referred to as “second Sometimes referred to as "material"). Specific examples of this substance include an antibody against an epitope of this antigen that is different from the epitope recognized by the first substance when the substance to be detected is an antigen, and an antibody as the substance to be detected when the substance to be detected is an antibody. Antibodies against.

When a complex of the substance to be detected and the first substance is present in the sample liquid, when the sample liquid reaches the positive determination site 4,
A binding reaction between the complex and the second substance occurs. The presence or absence of the substance to be detected can be determined by recognizing this binding using a labeling substance described later. That is, for example, when the first substance is labeled with colloidal gold, the complex of the substance to be detected and the first substance binds to the second substance of the positive determination region 4 and remains at the positive determination region and accumulates. . As a result, the coloration of the gold colloid is observed at the positive determination site 4. On the other hand, if the substance to be detected does not exist, the first substance labeled with the colloidal gold does not bind to the second substance, and therefore passes through the positive determination site 4,
No clear gold colloidal coloration is obtained in the positive determination region 4. As described above, the existence or non-existence of the substance to be detected in the sample liquid can be known, and thus the condition of animals including humans can be known or the disease can be diagnosed.

According to the first aspect of the present invention, the amount of the second substance carried in the positive determination part 4 increases in proportion to the distance of the sample liquid application site, that is, A on the sample liquid application site 2 side.
The amount of the second substance carried is gradually increased from the end to the B end.

The advantages of such a configuration are as follows.
When the complex of the substance to be detected and the first substance unfolds and moves to reach the positive determination site 4, the reaction between the second substance and the complex actively proceeds on the A end side of the positive determination site 4. Here, on the A end side, which is the stage where the sample liquid and the second substance first contact, if a large number of complexes are consumed by the reaction with the second substance, the B end side of the positive determination site 4 It is conceivable that the complex to be subjected to the reaction becomes insufficient and the color of the positive determination part 4 becomes uneven. Such non-uniform coloration often hinders accurate determination, but in the present invention, the amount of the second substance on the A end side is relatively smaller than that on the B end side. Because A
It can be effectively prevented that a large amount of the complex is consumed only at the end side. That is, according to the present invention, it is possible to secure a uniform reaction amount of the second substance and the complex in the positive determination part 4, and as a result, it is possible to observe the coloration due to the uniform labeling in the positive determination part 4, which is clear and accurate. It is possible to make a visual judgment.

Further, according to a preferred aspect of the present invention, when the positive determination part is formed in a circular shape, the amount of the substance capable of specifically binding to the substance to be detected is proportional to the distance from the sample liquid application part. Means that the amount of the substance capable of specifically binding to the substance to be detected increases from the peripheral portion of the circle toward the central portion in at least the semicircle of the circular positive determination portion on the specimen droplet portion side. It is also meant to include such a configuration. That is, when the positive determination site is circular, the peripheral portion of the semicircle on the detection liquid application site side is the position where the developed sample liquid comes into contact with the positive determination site for the first time, and the amount of the second substance carried is The present invention also includes a configuration in which the amount of the second substance at this position is sequentially increased in proportion to the distance from the sample liquid application site. With this configuration,
Strong coloration is observed in the first contact step between the above-mentioned sample liquid and the second substance, but the coloration becomes weaker thereafter,
As a result, uneven coloration can be effectively prevented. According to a further preferred aspect, it is preferable that the abundance amounts of the second substance in the peripheral portion of the semicircle are all the same.

The positive determination portion 4 is preferably formed by the following printing method using an intaglio, that is, a so-called gravure printing method. First, a composition in which the second substance is dissolved or dispersed in a suitable solvent is prepared. As the solvent, an aqueous solvent is preferable, but an organic solvent may be used alone or in combination with water as long as the physical properties of the second substance are not changed. Specific examples of preferable solvents include pure water, physiological saline, and phosphate buffer as the aqueous solvent, and hydrocarbons, alcohols, esters, ethers and the like as the organic solvent. Further, additives capable of improving various physical properties of this composition may be added so as to favorably perform printing on the concave plate, and examples thereof include a thickener, a surfactant, a preservative, and a stabilizer. According to a preferred embodiment of the present invention, the viscosity of this composition is preferably in the range of about 1 to 2000 cps,
More preferably, it is about 10 to 200 cps.

Next, this composition is filled in an intaglio plate of which the volume is successively increased in an arbitrary shape, and this is printed on a base material to form a positive judgment site. FIG. 2 schematically shows the situation. The intaglio plate 5 is a plate in which the recesses 6 are formed so that the volume thereof increases, that is, the depth of the recesses increases from the A end toward the B end. This recess 6
When the composition is filled in and the base 1 is imprinted so that the end A faces the direction of the sample liquid application site (FIG. 2 (a)), the composition permeates and is transferred to the substrate, and the sample liquid is applied. The positive determination part 4 is formed so that the amount of the loaded composition increases in proportion to the distance from the site, that is, the amount of the second substance carried increases in proportion to the distance from the sample droplet site. (Fig. 2
(B)).

This printing method is substantially the same as the so-called gravure printing, and thus the intaglio 5 can also be manufactured by a known method such as etching, helio, or laser engraving.

The shape and size of the concave portion 6 of the intaglio 5, that is, the amount of the second substance to be carried is arbitrary in consideration of the expected amount of the substance to be detected in the sample liquid, the reactivity with the second substance, and the like. According to a preferred embodiment of the present invention, the amount of the composition filled in the recess 6 is preferably 0.1.
~ 100 g / m ² or so, more preferably 0.5 to 50 g
/ M ² .

Unlike the above-mentioned first substance, it is necessary that the second substance at the positive determination site does not move as the sample liquid develops. Therefore, it is preferable that the positive determination portion is dried or heated after printing to fix the second substance on the substrate.

In the diagnostic device according to the present invention, it is preferable that the signal from the sign can be visually confirmed,
Furthermore, the label is preferably present on the first substance. Specific examples of preferred labels include enzyme labels such as alkaline phosphatase, β-galactosidase, and peroxidase; fluorescent labels such as fluorescein and rhodamine; dye labels such as red blood cells and colored latex; gold,
Examples thereof include metals such as silver and selenium, and inorganic particles.

The first embodiment of the present invention has been described above using the so-called sandwich method of antigen-antibody reaction. Therefore, two substances are required as substances that can specifically bind to the substance to be detected. However, it goes without saying that the present invention is also applicable to a system in which a substance to be detected is bound to only one substance and the presence or absence of the substance is detected by detecting the presence or absence of the binding. Examples of such a system include an enzyme and its substrate system, and specific examples of the enzyme include glucose oxidase, peroxidase, ascorbate oxidase, glutamate oxidase, alkaline fosterase, and amylase.

Another preferred example according to the second aspect of the present invention will be described with reference to FIG. FIG. 3A is a perspective view of the diagnostic tool, and FIG. 3B is a partial cross-sectional view of the diagnostic tool. The diagnostic tool shown in the figure has basically the same configuration as the diagnostic tool according to the first aspect of the present invention shown in FIG. 1 except for the difference in the configuration of the positive determination part 4 described below. . In the diagnostic tool shown in FIG. 3, the positive determination part 4 is composed of five independent regions 4a to 4e. The plurality of regions 4a to 4e are configured such that the amount of the second substance carried on each of the regions 4a to 4e increases step by step in proportion to the distance from the sample liquid application site. Interestingly, the diagnostic tool according to the second aspect of the present invention configured as described above has an advantage that the amount of the substance to be detected in the sample liquid can be easily determined. The reason is as follows. First, when the complex of the substance to be detected and the first substance unfolds and moves to reach the first region 4a of the positive determination part 4, the reaction between the complex and the second substance proceeds. If the amount of the complex present in the sample liquid is greater than or equal to the amount that gives a predetermined color intensity by the second substance present in 4a, the region 4a
The coloration of the predetermined intensity is observed. Since the complex in the sample liquid is consumed in the region 4a by the reaction with the second substance, the amount of the complex in the sample liquid decreases by a certain amount and the expansion is further continued. The sample liquid reaches the next region 4b due to the spreading movement. Since the region 4b carries a larger amount of the second substance than the region 4a, a coloration having the same intensity as that of the region 4a can be obtained with a relatively smaller amount of the complex than in the region 4a. If the amount of complex is in this region 4
In the area b as well, if the amount is more than the amount that gives the same intensity of color as that of 4a, the color of the same intensity of 4a is also observed in this region 4b. On the contrary, as a result of the amount of the substance to be detected in the sample liquid existing from the beginning being small and the complex being further consumed in the region 4a, the amount of the complex is more than the amount giving the predetermined coloring intensity in the region 4b. If the amount is small, no color is generated in this region 4b or a color weaker than that in the region 4a is observed. That is, since the amount of the second substance is gradually increased in these series of plural regions, it is configured so that the coloring of the same intensity is observed even with a smaller amount of the complex than in the case of the front region. There is. Therefore, a proportional relationship is established between the abundance of the substance to be detected in the sample liquid and the number of regions that give the same intensity of color in the region of this positive determination portion, and the amount of the substance to be detected in the sample can be quantified. It can be done.

In the embodiment of FIG. 3, the amount of the second substance carried is increased in each region in proportion to the distance from the sample liquid application site. That is,
This embodiment adopts a configuration that combines both the first aspect and the second aspect of the present invention. Such a configuration is preferable because uniform and clear coloration can be obtained in each of the regions 4a to 4e of the positive determination part. Furthermore, in the case where the regions 4a to 4e are circular, as described above, in at least the semicircle on the side of the sample droplet for the positive determination region of the circular shape, it is specific to the substance to be detected from the periphery of the circle toward the center. It is also preferable that the amount of the substance that can be chemically bound is increased. Therefore, for example, it becomes possible to quantitatively determine the amount of luteinizing hormone in urine. It is possible to determine the time of ovulation from the peak time.

The plurality of regions of the positive determination portion 4 are preferably manufactured by a so-called gravure printing method using an intaglio, as in the case of the positive determination portion 4 in the first aspect of the present invention described above. In this embodiment, an intaglio plate provided with a plurality of recesses is used, and each of the plurality of recesses is an intaglio plate whose volume gradually increases. Further, if desired, an intaglio plate is used in which the volume (that is, the depth of the plate) in the recess becomes deeper as the distance from the sample droplet site side increases. An intaglio plate preferably used in the first and second aspects of the present invention is shown in FIG.

[0029]

[Example] Example 1 3 mm × 2 as shown in FIG. 4 by Helio platemaking
An intaglio plate having a size of mm square, a line number of 60, and a plate depth sequentially from 30 μm to 150 μm in the long side direction (from A end to B end in the drawing) was prepared. Using this plate, an anti-hemoglobin antibody solution (manufactured by Denka Kogyo Co., Ltd.) was used as glass fiber paper GF.
/ DVA (made by Whatman) 100 mm x 200
Printed on mm substrate. After printing, it was dried at 50 ° C. for 2 minutes to immobilize the antibody and form a positive determination site. Base material 1
It was cut into a strip of 0 × 50 mm to prepare a fecal occult blood test diagnostic tool. The end on the A end side of this diagnostic tool is 10 μg /
A solution containing 0.5 ml of hemoglobin and an anti-human hemoglobin antibody-sensitized gold colloid (Meiji Seika Co., Ltd.)
ml) to develop the solution. In addition, B of the diagnostic tool
The end side was developed by contacting it with a cellulose pulp absorber. As a result, a uniform and clear coloration was observed over the entire area of positive determination.

Comparative Example A fecal occult blood diagnostic tool was manufactured in the same manner as in Example 1 except that an intaglio plate, which was intended to have a constant plate depth of 90 μm, was used. When the hemoglobin solution was developed with the diagnostic tool in the same manner as in Example 1, the color density was darker on the A end side and thinner on the B end side, and uniform and clear coloration could not be obtained.

Example 2 An intaglio plate having circle-shaped recesses having a diameter of 2 mm at intervals of 2 mm as shown in FIG. 5 was manufactured by Helio plate making. The five circle-shaped recesses are each 110 lines-75μ.
m, 100 lines-80 μm, 90 lines-95 μm, 75 lines-
It was 110 μm and 60 lines-150 μm. Printing was performed in the same manner as in Example 1 except that this intaglio plate was used. After printing, the base material was cut into a size of 10 × 100 mm to obtain a fecal occult blood test diagnostic tool having a positive determination site composed of five o-shaped regions 4a to 4e. The end on the region 4a side of this diagnostic tool was attached to a solution (0.5 ml) containing a hemoglobin concentration of 0.5 μg / ml or 10 μg / ml and an anti-human hemoglobin antibody-sensitized gold colloid (Meiji Seika Co., Ltd.). , The solution was developed. A cellulose pulp absorber was brought into contact with the B end side of the diagnostic tool for expansion. As a result, 4a and 4b were obtained in the case of a solution having a hemoglobin concentration of 0.5 μg / ml.
The same intensity of coloration was observed in the two regions, and in the case of the solution having a hemoglobin concentration of 10 μg / ml, the same intensity of coloration was observed in all five regions 4a to 4e.

[Brief description of drawings]

FIG. 1 is a diagram showing a preferred example of a diagnostic tool according to a first aspect of the present invention.

FIG. 2 is a diagram showing a preferred method of manufacturing a diagnostic tool according to the first aspect of the present invention.

FIG. 3 is a view showing a preferred example of a diagnostic tool according to the second aspect of the present invention.

FIG. 4 is a view showing an intaglio plate used in Example 1.

5 is a view showing an intaglio plate used in Example 2. FIG.

FIG. 6 is a view showing a preferable intaglio plate.

[Explanation of symbols]

 1 base material 2 specimen droplet site 3 specific substance supply site 4 positive determination site 5 intaglio

Claims (8)

[Claims] 1. A diagnostic tool for determining the presence or absence of a substance to be detected in a sample liquid, wherein the base material is a porous material capable of developing the sample liquid, and the sample liquid is applied to the base material. At least a sample liquid application site and a positive determination site carrying a substance capable of specifically binding to the substance to be detected, the positive determination site being carried, specifically binds to the substance to be detected A diagnostic tool, wherein an amount of a substance that can be carried increases in proportion to a distance from a sample liquid application site. 2. The positive determination site is constituted as a single region, and the amount of the substance capable of specifically binding to the substance to be detected is continuously within the single region in proportion to the distance from the sample liquid application site. The diagnostic tool according to claim 1, wherein the diagnostic tool increases in number. 3. The positive determination part is composed of a plurality of independent regions, and the amount of the substance capable of specifically binding to the substance to be detected carried in each of the plurality of regions is proportional to the distance from the sample liquid application site. The diagnostic tool according to claim 1, wherein the diagnostic tool gradually increases in each region. 4. The positive determination part has a circular shape, and the amount of the substance capable of specifically binding to the substance to be detected increases in proportion to the distance from the sample liquid application site, and the positive determination part has a circular shape. 4. The amount of a substance capable of specifically binding to a substance to be detected increases from at least the semicircle on the side of the sample droplet to the center of the circle at least in the semicircle of the sample droplet. Diagnostic tool. 5. The substance to be detected is an antigen and the substance carried on the positive determination site is an antibody, or the substance to be detected is an antibody and the substance carried on the positive determination site is an antigen for the analyte substance. The diagnostic tool according to claim 1, which is 6. A specific substance supply site carrying a substance capable of specifically binding to a substance to be detected, the substance being different from the substance capable of specifically binding to the substance to be detected carried on the positive determination site. The method further comprising: in the sample liquid application site, or between the sample liquid application site and the positive determination site.
Described diagnostic tool. 7. The substance to be detected is an antigen, the substance carried on a specific substance supply site is a labeled antibody against the antigen, and the substance carried on a positive determination site is a substance carried on a specific substance supply site. An antibody against an epitope of the antigen different from the recognizing epitope, or a substance to be detected is an antibody, a substance carried at a specific substance supply site is a labeled antigen for the antibody, and a substance carried at a positive determination site is The diagnostic tool according to claim 6, which is an antigen for a substance to be detected. 8. The method for producing a diagnostic device according to claim 1, wherein a composition in which a substance capable of specifically binding to a substance to be detected is dissolved or dispersed is prepared. A method comprising the steps of filling an intaglio plate having an arbitrary shape and increasing in volume in an arbitrary shape, and printing the intaglio plate on a substrate to form a positive determination site.