JPS63253255A - Allergen inspecting means and allergen inspecting method - Google Patents

Abstract

PURPOSE:To facilitate production and to permit inspection of plural allergens with one application of a liquid to be inspected by fixing plural kinds of the allergens by covalent bonding to each of the plural blocks on a water insoluble film. CONSTITUTION:A nylon film having a carboxyl group on the surface is activated by trichloro-s-triazone and is cut to a disk shape of about 3cm diameter. The disk is equally divided to 6 parts in the circumferential direction. Solns. contg. 1): mite extract, 2): household dust extract, 3): cat dandruff extract, 4): extract or cedar pollen, and 5): anti-IgE human antibody are coated to the approximate central parts of the respective blocks and are dried. A peroxidase labeled antihuman IgE antibody is coated as a contrast on the 6th block. This film is then immersed in an aq. goat milk seine soln. to block the film surface..

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an allergen testing tool and an allergen testing method using the testing tool.

[Background of the invention] As a method for testing allergy-causing substances (allergens),
Conventionally, methods have been used in which allergens are brought into contact with the skin through injections, cuts, etc., and allergic reactions are observed. However, this method has the problem of causing pain to the subject (patient) and, in some cases, leaving aftereffects.

Therefore, recently, a method of confirming the presence of immunoglobulin E (IgE) in the blood, which is produced by the immune system in response to allergens that are antigens, has been commonly used to diagnose allergies, particularly type I allergies. . And I
To confirm the presence of gE, for example, an anti-human IgE antibody that specifically binds to IgE is used to bind IgE, and the bound IgE is treated with 1. Enzyme immunoassay (enzyme immunoassay: E I A
) is used. In addition, to the above bound IgE,
A method of further binding an anti-human IgE antibody with a radioactive label (radioimmunoassay: RIA) is also used.

When carrying out the method described in , for example, an allergen measuring device in which an allergen is immobilized on the surface of a cellulose sheet by a reaction using CNBr is prepared, and a body fluid in which the presence of IgE is predicted is attached to the device to detect the allergen and IgE. IgE is immobilized on the allergen by a binding reaction with
Fixing the gE antibody and contacting it with a coloring agent solution,
An operation for detecting the presence or absence of IgE bound to an allergen through color development is known.

However, the above method has a problem in that in order to monitor allergic reactions to multiple allergens, measuring instruments corresponding to the number of allergens are required. In other words, in order to see allergic reactions in subjects, it is necessary to observe reactions to many types of allergens. become. Also.

In this case, there is a problem that the amount of body fluid required is considerable. Also, depending on the type of allergen, CNBr
There are some allergens that are not dissolved in the carbonate aqueous solution used in the reaction for immobilization using this method, and there is a problem that such allergens cannot be immobilized on the carrier by this immobilization method. Furthermore, when preparing this allergen measuring device,
As mentioned above, it is necessary to use highly toxic CNBr,
When industrially manufacturing a large quantity of allergen measuring devices, there is a problem in productivity.

On the other hand, an allergen test tool in which the cellulose sheet as a carrier is replaced with a nitrocellulose sheet and multiple allergens are adsorbed and fixed on the surface of the nitrocellulose sheet is published by J. Immunol, Methods.

66.99 (1984). This allergen measuring device measures multiple allergens on one carrier sheet.
Although it has the advantage of being able to immobilize allergens, it has the drawback of requiring a long period of time to immobilize the allergen.

Furthermore, since physical adsorption is used to immobilize allergens, the applied allergens tend to diffuse, making it difficult to immobilize allergens at high concentrations at predetermined locations on the carrier surface. In addition, cleaning of sheets during allergen detection operations and P of various liquids applied to the surface
Allergens that have been adsorbed and fixed by H and the like tend to move or desorb from predetermined positions, and therefore the test results cannot be said to be fully satisfactory in terms of quantitativeness, reliability, reproducibility, etc.

Also, in Japanese Patent Publication No. 58-501637.

As a test method for allergic reactions with multiple allergens, different allergens are immobilized on each thread such as cotton thread using cyanogen halide such as CNBr, and these are bundled to perform tests for multiple allergens at once. An operational method is disclosed. However, even with this method, there are problems with the above-mentioned allergen fixation operation using CNBr, and it has been reported that it is difficult to detect the color development between the allergen and IgE, especially in rapid quantitative testing.

[Structure of the Invention] The main object of the present invention is to provide an allergen test tool that is easy to manufacture and that allows highly accurate allergen tests for multiple allergens to be performed in a single test liquid application operation. It is about providing.

The present invention provides an allergen test device characterized in that two or more different allergens are immobilized on each of two or more partitioned areas on the surface of a water-insoluble film via covalent bonds. It is in.

In the allergen test device of the present invention, nine body fluids in which the presence of IgE is predicted are attached to the surface on which the allergen is immobilized, and then an enzyme-labeled anti-IgE antibody solution is brought into contact with the surface. It can be used for highly accurate allergen testing by exposing it to a color former solution after washing and detecting the presence or absence of IgE bound to the allergen.

The carrier for allergen immobilization used in the manufacture of the 13 unreleased allergen testing tools is a film that is substantially insoluble in water and contains functional groups (such as carboxyl It is a film that has a group (group) on its surface. Such films are known as carriers for immobilizing enzymes to be subjected to enzymatic reactions (for example, Methods of Ee
zymology, 44° 114 (1976)) and commercially available polyamide films for enzyme immobilization, polyfluorocarbon films for enzyme immobilization, etc., can be used.

For example, these have a functional group such as a carboxyl group or ti on the surface of a polyamide film, and an active compound such as dicyclohexylcarbodiimide or trichloro-3-)riazine is bonded to the carboxyl group to form an active group. By forming it, it becomes easier to bind to proteins. By attaching an aqueous solution or aqueous dispersion of an allergen (most allergens have protein as their main component) to the back side of a film whose surface has been activated in this way, the allergen can be easily immobilized by covalent bonds. do.

In the allergen test device of the present invention, the allergen is immobilized on each of two or more partitioned areas on the surface of the film carrier. That is, when testing multiple allergens, each allergen needs to be fixed in a pre-designated area, and each allergen needs to be fixed in separate areas from each other. For this purpose, for example, a disc-shaped carrier film can be divided into multiple sections in the circumferential direction (e.g., 4 sections, 5 sections, 6 sections, 1 section, 12 sections).
Methods such as dividing the area into sections and fixing the allergen in each section are used. The allergen is usually fixed in the shape of a circle with a diameter of 1 to 5 mm in the center of each compartment at a high concentration without diffusing much, but there are no particular restrictions on its position, shape, or size. , the relationship with the allergen fixed thereon may be marked by printing letters, symbols, etc. on the film carrier itself, or
One body may be housed in a fixed positional relationship in a mount that indicates the relationship between each compartment of the carrier and the allergen fixed thereon.

The film carrier is usually disc-shaped or rectangular (including square), but its shape is not particularly limited, and the area for fixing the allergen can be determined by any method. It can be done by

The allergen test tool of the present invention can immobilize most of the hitherto known allergens whose main components are proteins. Examples of such allergens include plant pollen, fungi, animal dirt, milk, pickles or hair, insect toxins, dust, plant ingredients, certain drugs, and the like.

In addition, in the allergen test tool of the present invention, anti-IgE is applied to the area on the film surface where the allergen is immobilized and another area.
The antibody and/or labeled IgE antibody may be immobilized via covalent bonds, thus, on the surface of the test device,
When the anti-IgE antibody is immobilized via a covalent bond, the presence of IgE in the sample solution can be confirmed, the presence or absence of allergic disease can be investigated, and the labeled IgE antibody When immobilized via covalent bonds, these antibodies can be used as markers to confirm the location of each allergen on the film (which is usually colorless and its location cannot be detected visually) or as a colored marker. It can be used as a positive control.

The allergen test tool of the present invention may be used as is, or may be housed in a mount made of plastic material or the like.

The allergen test tool of the present invention can be used for allergen testing, for example, based on the following operations.

First, on the surface of the film where the allergen is fixed,
A body fluid in which IgE is expected to be present is attached. Examples of body fluids used include blood, serum, plasma, nasal mucus,
Body fluids such as bone marrow fluid and saliva are usually collected using a micropipette in an amount of 10 to 5007ti.
The six body fluids that are dropped or spotted on the film surface may be applied to each allergen fixation position, or they may be applied near the center of the film and diffused around the fixed allergen. A method of contacting the entire surface of the film, a method of applying body fluid to the entire surface of the film, etc. may be used.

By the operation described in L, IgE in the body fluid that reacts with the immobilized allergen binds to the allergen and is immobilized at that position.

Next, if desired, the film surface is washed using a buffer such as a phosphate buffer. This washing operation removes body fluids. No particular change occurs in allergens to which no corresponding IgE is attached in this operation.On the other hand, since the allergen itself is fixed to the film surface by covalent bonds, the IgE bound to the allergen
is fixed on the film surface and does not move or be removed from the film even when washed.

Next, an enzyme-labeled anti-IgE antibody solution is brought into contact with the surface of the film (the surface to which body fluid has been applied). The anti-IgE antibodies provided to the film surface by this operation are
Combine with E.

Next, the film surface is washed using a buffer such as a phosphate buffer. by this cleaning operation.

Body fluids (if the washing operation was not performed before the contacting operation with the enzyme-labeled anti-IgE antibody solution) and the enzyme-labeled anti-IgE antibody solution are removed. The corresponding IgE
No particular change occurs in allergens that are not attached to the allergen. On the other hand, since the allergen itself is covalently fixed to the film surface, the IgE bound to the allergen and the enzyme-labeled anti-Ig bound to the IgE
The E antibody is also immobilized on the film surface and does not move or be removed from the film even by washing. In other words, the enzyme-labeled anti-IgE antibody is immobilized only at the position of the allergen to which IgE is bound. It will remain.

Examples of enzymes used in the enzyme-labeled anti-IgE antibody described in 1. include peroxidase, lactate dehydrogenase, β-galactosidase, and alkaline phosphatase. However, it is also possible to use s-substances used in known enzyme-labeled anti-IgE antibodies.

The position of the enzyme-labeled anti-IgE antibody is detected by contacting the surface of the film treated as described in L with a solution containing a coloring agent that develops color by reaction with an enzyme, and at the same time,
Allergens bound to IgE can be detected.

There are various known coloring agents that develop color upon contact with enzymes, and any of these coloring agents can be used. Examples of such color formers are described below.

Examples of coloring agents that develop color upon contact with peroxidase include 2.2'-azinobis(3-ethylbenzthiazolinesulfonic acid), tetramethylbenzidine, diaminobenzidine, 4-chloro-1-naphthol, 2. Mention may be made of 7-diamifluorene, O-phenylenediamine, aminoantipyrine, and luminol.

An example of a coloring agent that develops color upon contact with lactate dehydrogenase is 3-(4°,5°-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium.
Bromide [MTT], 2-(p-iodophenyl)-
3-(p-nitrophenyl)-5-phenyltetrazolium chloride [INT], and 2.2°-di(p-
Nitrophenyl)-5,5'-diphenyl-3,3'-
(3,3'-dimethoxy4,4'-diphenylene)ditetrazolium chloride [NET] can be mentioned.

An example of a coloring agent that develops color upon contact with alkaline phosphotase is p-nitrophenyl phosphate.

Examples of coloring agents that develop color upon contact with galactosidase include p-nitrophenyl-β-D-galactopyranoside and 5-bromo-4-chloro-3-indolyl-
Mention may be made of β-D-galactopyranosite.

Through a series of simple operations as described above, the presence or absence of IgE in body fluids sensitized by multiple allergens immobilized on the film of the allergen testing device of the present invention can be easily and semi-quantitatively determined. be able to. In addition, by performing colorimetric measurements, even more quantitative tests can be performed.

Next, examples of the present invention will be given.

[Example 1] Preparation of allergen-immobilized film A nylon membrane having carboxyl groups on its surface (trade name Dicarboxidyne, manufactured by Ball Co., Ltd., USA) was coated with trichloro-3-
The triazine-activated membrane is cut out into a disk shape with a diameter of 3 cm, and the disk is divided into six equal sections (angle of 60 degrees) in the circumferential direction, and approximately in the center of each section is placed 1: mite extract, 2: Household dust extract, 3: Cat dander extract, 4:
A solution (10% by weight solution) containing cedar pollen extract and 5: anti-IgE human antibody was added using a microsyringe.
1LJ1 was applied and dried at room temperature. In the sixth section,
As a control (positive control), a peroxidase-labeled anti-human IgE antibody was applied to the membrane.Next, this membrane was immersed in a 2% goat milk casein aqueous solution to block the membrane surface.

The allergen-fixed film prepared as described above was stored in a heparin-containing phosphate buffer (pH 7, 2, 50 M).

Allergen detection procedure: Remove the allergen-fixed film mentioned above from the buffer solution.
Without drying the film surface, 100 p of blood collected from a patient (added with an anticoagulant) was applied uniformly onto the surface, and then left at room temperature for 3 hours.

Next, the film was soaked in phosphate buffer (pH 7, 2, 50mM).
, containing 0.05% nonionic surfactant) three times, and then peroxidase-labeled anti-H) I on the film surface.
Label a gE antibody solution (pH 7, 2, 10 mM phosphate buffer with anti-human Ig prepared from antiserum obtained by sensitizing a goat to human IgE by binding peroxidase to the goat antibody using a known method). The amount of protein is 1
100 pl (containing 0 pg/mJ1) was applied uniformly over the entire surface and left at room temperature for 10 minutes.

After washing the film described in I-1 with phosphate buffer one to three times in the same manner as above, the film surface was coated with a coloring solution (0.02% solution of tetramethylbenzidine, acetate buffer pH 4 containing 1mM hydrogen peroxide). , 4) and leave it for 2 minutes,
When washed with water, clear color development was seen at the allergen location. By comparing this with a colorimetric standard color, the presence of IgE could be determined semi-quantitatively.

Claims (1)

[Claims] 1. Two or more different allergens are fixed to each of two or more partitioned regions on the surface of a water-insoluble film via covalent bonds. Allergen testing tool. 2. Claim 1, characterized in that the covalent bond between the film surface and the allergen is formed by a reaction between the allergen and an active group bonded to a functional group exposed on the film surface. Allergen test tool listed. 3. Claim 1, characterized in that the covalent bond between the film surface and the allergen is formed by a reaction between the allergen and an active group bonded to a carboxyl group exposed on the film surface. Allergen test tool listed. 4. The allergen test device according to claim 1, wherein the film is a film mainly composed of polyamide. 5. Claim 1, characterized in that the film is a film whose main component is polyfluorocarbon.
Allergen test tool listed in section. 6. An anti-IgE antibody and/or a labeled IgE antibody is immobilized via a covalent bond in an area other than the area where the allergen is immobilized on the film surface, according to claim 1. Allergen test tool. 7. In a demarcated area on the surface of the water-insoluble film,
Ig is applied to the surface of an allergen test tool to which two or more different allergens are each immobilized via covalent bonds.
After attaching a body fluid in which the presence of E. coli is predicted to be present, the surface is brought into contact with an enzyme-labeled anti-IgE antibody solution, and after the surface is washed, a coloring agent solution is brought into contact with the surface to remove IgE bound to the allergen. An allergen testing method that consists of detecting the presence or absence. 8. The allergen testing method according to claim 7, wherein the enzyme of the enzyme-labeled anti-IgE antibody is peroxidase, lactate dehydrogenase, β-galactosidase, or alkaline phosphatase.