JPH11326325A - Immunity inspection body with determination efficiency - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply, quickly detect an indoor allergen, etc., collected in a solution by forming a detection part of a plurality of detection bands separated in parallel to a rear part of a chromatographic development. SOLUTION: When an immersion part 6 of an immunity inspection body is immersed in a liquid in which an antigen is dissolved, the solution is sucked by a base material 8, starting a chromatographic development. When the solution reaches a position 2 where a marker antibody is held, an antigen bonds with a marker antigen to a labelled antigen and moves further downstream. When reaching a position where an immobilized antibody is immobilized, namely, a detection part, the labelled antigen bonds with the immobilized antibody, and is caught there and at the same time colors the detection part. The marker antibody itself cannot bond with the immobilized antibody and therefore passes through the detection part to reach a test end confirmation part 5 set at the downstream. Since the test end confirmation part 5 is formed by immobilizing an antibody which can bond with the marker antibody, the marker antibody reaching the confirmation part is caught there and colors the test end confirmation part.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological test for detecting an allergen such as a test substance, particularly cedar or tick, by an antigen-antibody reaction. In particular, the present invention relates to a quantitatively imparted immunological test object incorporating a function capable of evaluating the range of abundance of a subject.

[0002]

2. Description of the Related Art Pain is caused by pollinosis caused by plant pollen such as cedar, cypress, or Aedes chinensis, and allergic diseases such as pediatric asthma and atopic dermatitis caused mainly by mites living in indoor dust. The number of recipients has increased significantly. Keeping patients away from allergies is the most effective way to improve the symptoms of allergic diseases, and it is necessary to understand the location and amount of allergens.

[0003] For this purpose, a mite collector for collecting house dust in a room attached to a vacuum cleaner and a system for measuring the amount of mite antigen from a filter for collecting mite have recently been put on the market. In these devices, a sample is first collected by a vacuum cleaner, then the filter that has collected the mites is immersed in an antigen solution to dissolve the antigen, and finally, the solution in which the antigen is dissolved is used for enzyme immunoassay (EIA) or immunochromatography. Inspection by law.

[0004] Among them, a preferable method for testing the presence of an antigen is an immunochromatography method used as a pregnancy test drug for OTC. That is, there are two types of antibodies against the allergen, one is fixed to the test substrate, the other is labeled with colloidal gold, colored latex, etc., and is held in a soluble state. These two antibodies can bind to different epitopes of the antigen to be tested. Further, an antibody against the labeled antibody is immobilized further downstream of the immobilized anti-allergen antibody. When a sample solution is provided and the sample solution contains an allergen, a labeled antibody and antigen complex (labeled antigen)
Is formed, and further bound to this antibody at the anti-allergen antibody fixing portion (that is, the antigen detecting portion) to form a sandwich complex, which is visually recognized.

However, the conventional immunochromatography method as described above has the following disadvantages. Since the detection unit is formed of a single zone, it is easy to determine the presence or absence of the antigen, but the amount of the antigen depends on the visual evaluation of the color density of the detection unit. Visual evaluation may give different judgments depending on the observer.

However, when it is desired to quantitatively determine the amount of an object to be detected as much as possible, such as indoor allergen test as a measure against asthma, etc., the above detection method cannot sufficiently cope with the problem. Especially in the case of indoor allergens, for example, when testing the amount of mites in the futon, urgent measures such as collecting mites with a vacuum cleaner etc. are required according to the test results, and a more quantitative detection method is required. Had been.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide an immunological test body which can easily and quickly detect indoor allergens and the like collected in a solution and can quantitatively measure the amount of the allergens visually. is there.

[0008]

The present invention is characterized in that one is fixed to a chromatographically developable base material, and the other is held by the base material in a form capable of binding to colored fine particles and dissolving ahead of the chromatographic spread. The present invention relates to an immunological test specimen comprising two types of antibody pairs, wherein the detection portion is formed by a plurality of detection zones separated along the back of the chromatograph. Specifically, the present invention is to capture the antigen by capturing the antigen after the antigen capturing ability of the immobilized antibody forming the upstream detection band among the plurality of detection bands is saturated, so as to capture the antigen by the downstream detection band. The present invention relates to the above-mentioned immunoassay. More specifically, the present invention provides a method wherein each detection zone has an amount of color generated by capturing a labeled antigen to which a labeled antibody and an antigen are bound, such that the concentration of coloration no longer reflects the amount of the captured labeled antigen. The above-mentioned immunological test body, wherein the antibody is fixed in an amount that saturates with the amount of antigen.

Further, the present invention provides a method wherein the solution in which an antigen is dissolved is subjected to chromatographic development using the above-mentioned immunoassay, and the position or the number of detection bands colored by binding to a labeled antigen is
The present invention relates to an allergen test method for measuring the amount of antigen dissolved in the liquid. Furthermore, the present invention relates to an allergen test kit comprising a sample collection section, an antigen solution storage section, and the above-mentioned immunoassay. In addition, the present invention relates to the above-mentioned allergen sample kit, which is detachable from the sample collection unit and can be attached to a predetermined portion of the kit and measure the sample after the sample is collected by the sample collection unit.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The immunoassay according to the present invention is characterized in that the detection section is formed of a plurality of detection zones separated along the back of the chromatograph. Thereby, after the antigen-capturing ability of the immobilized antibody forming the upstream detection band among the plurality of detection bands is saturated by capturing the antigen, the antigen is captured by the downstream detection band. There is. Using the immunoassay of the present invention, the solution in which the antigen was dissolved was chromatographed, and the position of the detection band colored by binding to the labeled antigen was observed to determine the amount of the antigen dissolved in the solution. Can be measured.

In the present invention, the expression that the detection portion is formed by a plurality of detection bands separated along the back of the chromatograph means that a detection complex is bound to a labeled antigen bound to a labeled antibody to form a sandwich complex. This means that the detection section on which the antibody capable of immobilization is formed is formed not as a single zone but as a plurality of zones, and the respective detection bands are arranged side by side in the direction of chromatographic development.

The present invention will be described below with reference to the drawings. FIG.
Indicates an immunological test article of the present invention. In FIG. 2, reference numeral 3 denotes an antigen detection unit composed of a plurality of detection bands 4 (4-1, 4-2, 4-3,..., 4-n). An immersion section 6, a labeled antibody holding section 2, a detection section 3, a test end confirmation section 5, and a liquid absorption section 7 are provided on a base material 8 of the immunological test body 1 in order from the upstream. When the immersion part 6 of the present immunological test specimen is immersed in a solution in which the antigen has been dissolved,
The dissolving solution is sucked up by the base material 8 and chromatograph development is started. When the lysate comes to position 2 where the labeled antibody is retained, the antigen binds to the labeled antigen and becomes a labeled antigen,
Move further downstream. When the labeled antigen reaches the position where the immobilized antibody is immobilized, that is, reaches the detection unit 3, it binds to the immobilized antibody, is captured there, and colors the detection unit. When the lysate (generally, this medium also serves as a developing solution) reaches the labeled antibody, a part of the labeled antibody is directly carried downstream by the lysate without binding to the antigen. Since the labeled antibody itself cannot bind to the immobilized antibody, it passes through the detection unit and reaches the test completion confirmation unit 5 provided further downstream. Since the test completion confirmation section is formed by fixing an antibody capable of binding to the labeled antibody, the transported labeled antibody is captured here and the test completion confirmation section is colored. This confirms that the test has been performed normally and has been completed.

In the test body based on the conventional immunochromatography method shown in FIG. 1, the antigen detecting section 3 has a single part. The detection unit is colored by capturing the labeled antigen, and the concentration of the color depends on the amount of the captured labeled antigen.
Although the amount of the captured antigen can be visually determined to some extent, the coloring state changes depending on the flow of the antigen solution, making it difficult to use a color sample and making it impossible to clearly indicate the amount of the antigen.

In the present invention, as shown in FIG. 2, the antigen detecting section 3 is made up of a plurality of detection zones 4 and a first detection zone 4-1.
In the case where there is an amount of antigen such that the color change can no longer be discriminated, the first detection zone is designed to saturate the ability to capture the labeled antigen. Therefore, the labeled antigen sent beyond the point in time at which a sufficient color change corresponding to the amount of the labeled antigen captured in the first detection zone cannot be obtained passes through the first detection zone and the second detection zone. It will be captured by obi 4-2. That is, the second detection band becomes colored. The limit capture amount is set in the same manner in the second and subsequent detection bands. Therefore, as the amount of the present antigen increases, the number of the detection bands colored in order from the upstream increases, whereby the range of the abundance of the antigen can be grasped.

The size of each detection band may be different or the same, but may be the same unless it is necessary to make a difference. Also, the arrangement intervals of the respective detection bands may be different or the same, but may be the same unless there is a particular need. This interval is preferably provided as close as possible within a range in which it can be easily identified as another detection band so as not to make the size of the detection object excessive. Further, the shape of each detection band may be a line as shown in FIG. 2, or may be a circle, a rectangle, or another shape. It is preferable that the difference be as small as possible as long as the difference in color can be easily identified.

A cover 9 having an open display window may be applied to the detecting section and the test end confirming section, which can make the appearance beautiful, but is not always necessary.

In such an immunochromatography method, if the amount of the labeled antibody is not sufficiently large with respect to the amount of the existing antigen, the amount of the antigen that reaches the immobilized antibody without being bound to the labeled antibody and is captured by the immobilized antibody is present. I will be. In this case, since the immobilized antibody is consumed without coloring, the evaluation result is smaller in the amount of antigen than in the case where it is not. This applies not only to the conventional method but also to the present invention. Therefore, the amount of the labeled antibody retained on the chromatographic substrate must be sufficiently larger than the amount of the antigen. Alternatively, when using the immunological test article of the present invention, it is preferable to reduce the amount of the antigen in the sample and perform the measurement in consideration of the amount of the labeled antibody retained on the substrate.

In the detection section, if the rate at which the labeled antigen reacts with the immobilized antibody is low, some may pass through the detection band and reach the downstream detection band before the upstream detection band is saturated. This results in a color change, which may result in erroneous measurement results. In order to avoid this, it is necessary to appropriately set the size of the label portion, the density of the antibody immobilized on the detection portion, the reaction constant of the antibody-antigen reaction, the developing speed of the antigen solution, and the like. This can be properly established by experiment.

In the present invention, the antibody constituting the detection section and the antibody forming the labeled antibody are antibodies capable of binding to different epitopes of the antigen to be evaluated,
For example, if the antigen is an antigen of a tick allergen,
For example, the detection section antibody uses an anti-mite allergen antibody, and the labeled antibody uses an anti-mite allergen antibody different from the detection section antibody. In addition, as the antibody immobilized on the test completion confirmation section, for example, an anti-IgG antibody can be used.

The substrate is selected from those suitable for chromatographic development. For example, glass filters, polyethylene-based nonwoven fabrics, polypropylene-based nonwoven fabrics and the like are preferable. Glass filters are generally preferred because they have negligible affinity for organic substances. When a glass filter is used, its density and fineness are appropriately selected depending on the required chromatographic development speed. For the immersion part and the liquid absorption part, a cotton-like substance or a porous material having open cells can be used, and preferably a filter paper, a membrane filter, or a glass filter is used.

[0021]

The present invention will be specifically described below with reference to examples. Example 1 (1) Preparation of immunoassay 1) Preparation of labeled antibody Antibody (anti-de) was added to colored polystyrene latex (manufactured by Bangs Laboratories).
rp antibody) was mixed and incubated to immobilize the antibody on the polymer particles.

2) Preparation of immunological test specimen A glass filter (manufactured by Whatman) having a size of 1 cm × 15 cm (0.7 mm in thickness) was prepared as a chromatographic substrate.
1 μl of anti-mite allergen antibody solution (anti-mite allergen antibody dissolved in physiological saline) serving as a fixed antibody for antigen detection is applied to a portion corresponding to the downstream portion of the chromatographic development.
Three lines having a thickness of 3 mm were dropped with a pipetteman at 0 mm intervals, and then 1 μl of an anti-IgG antibody solution that reacted with a labeled antibody and served as a confirmation of the end of the test was dropped further down the pipetman. Put this glass filter at 50 ℃
For 1 minute to fix two types of antibodies on a glass filter. Next, 15 μl of the labeled antibody prepared above was dropped on the upstream portion of the glass filter. The glass filter to which the labeled antibody had been dropped was dried at 50 ° C. for 5 minutes to hold the labeled antibody on the glass filter. In the above,
First of labeled antibody holding part and anti-Der p1 antibody fixing part
Are 3 cm and 4 cm from the end of the glass filter on the sample supply side, respectively. The second and third detection bands are provided at 1 cm and 2 cm from the first detection band. An anti-IgG antibody-immobilized portion was formed at a position 2 cm further downstream from the detection band.

[0023] (2) waste that was collected over a period of 1 minute vacuum cleaner to the futon of use example 1m ² of immunoassay body 2
The mixture was mixed with 0 ml of the antigen solution and filtered to remove solids, thereby preparing a sample solution. The immersion part, which is the uppermost stream of the immunological test specimen prepared above, was immersed in this sample liquid, and left standing for 5 seconds with the immunological test specimen held vertically. After 5 seconds, the first and second detection bands of the detection unit and the test completion confirmation unit were colored deep red based on the labeled fine particles, and the third detection band was slightly colored red. No discoloration was observed in the fourth and subsequent detection bands.

[0024]

The immunoassay of the present invention is an improvement of the quantitative property, which was the limit of the conventional immunoassay for detecting a subject by an antigen-antibody reaction, and the range of the amount of the existing analyte is limited. Can be detected. Therefore, by measuring allergens such as mites using this immune test specimen, the amount of allergens present can be ascertained, and more appropriate measures can be selected.

[Brief description of the drawings]

FIG. 1 is a schematic view showing a configuration of a conventional immunological test body, (a) a side view, and (b) a plan view.

FIG. 2 is a schematic view showing the configuration of the immunological test body of the present invention, (a) a side view, and (b) a plan view.

[Explanation of symbols]

1: immunological specimen, 2: labeled antibody holding part,
3: detection unit, 3 ': detection display window,
4: Detection band 4 ': Detection band display window
4-1: first detection band, 4-2: second detection band, 4-n: nth detection band, 5: test end confirmation section, 5 ': test end confirmation section display window, 6: Immersion part, 7: liquid absorption part 8: base material, 9: cover.

Claims (7)

[Claims] 1. One of them comprises two types of antibody pairs which are fixed to a chromatographically expandable base material and the other are bound to colored fine particles and held on the base material in a form which can be dissolved before chromatographic development. Wherein the detection section is formed by a plurality of detection bands separated along the back of the chromatograph. 2. The method according to claim 1, wherein after the antigen-capturing ability of the immobilized antibody forming the upstream detection band among the plurality of detection bands is saturated by capturing the antigen, the antigen is captured by the downstream detection band. Item 7. The immunological test according to Item 1. 3. The amount of antigen in each detection zone is smaller than that in which the concentration of color produced by capturing the labeled antigen in which the labeled antibody and the antigen are bound no longer reflects the amount of the captured labeled antigen. The immunoassay according to claim 2, wherein an amount of the antibody that saturates with is fixed. 4. A solution in which an antigen is dissolved is subjected to chromatographic development using the immunoassay according to claim 2 or 3, and the solution is determined from the position or the number of detection bands colored by binding to a labeled antigen. A method for testing allergens that measures the amount of antigens present in them. 5. An allergen test kit comprising a sample collection section, an antigen solution storage section, and the immunological test body according to claim 1, 2, or 3. 6. The allergen sample kit according to claim 5, wherein after the sample is collected by the sample collection unit, the sample collection unit can be attached to and detached from a predetermined portion of the kit to measure the sample. 7. The allergen sample kit according to claim 5, wherein at least the sample collection section can be used by being attached to a vacuum cleaner, an air purifier, an air conditioner, or a futon dryer.