JP2002286716A - Immunochromatograph ic method capable of simultaneous analysis of a plurality of items and strip for immunochromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunochromatographic method and a strip for immunochromatography capable of making quantitative analysis quickly and simply. SOLUTION: The immunochromatographic method is to analyze each of the signals of labels originating from immuno-complex bodies each containing (1) a specific immuno-reacting substance capable of being coupled peculiarly with only one of the substances to be analyzed and fixed on a support individually, (2) the applicable substance to be analyzed, and (3) a common immuno- reacting substance capable of being coupled with all substances to be analyzed and labeled with a labeling substance, and the labeled common immuno-reacting substance to be used becomes substantial from individual labeled common immuno-reacting substance containing only one unit of common immuno-reacting substance. A strip according to the invention includes a specimen-added region, a region holding the labeled common immuno-reacting substance, and a region where the specific immuno-reacting substance is fixed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunochromatographic method capable of simultaneously analyzing a plurality of items and a strip for immunochromatography.

[0002]

2. Description of the Related Art In a clinical test using an immunoadsorption reaction, when analyzing a plurality of types of components contained in the same test sample, it is common to divide the test sample and analyze each item individually. It is a target. However, when the number of test samples is small or when an urgent test is required, a simple method is to simultaneously measure a plurality of types of components simultaneously without dividing or subdividing the same test sample. Means are eagerly sought from medical practice.

[0003] In recent years, means for simultaneously measuring a plurality of items has been developed using immunochromatography, which has become popular as a simple measuring method. For example, a plurality of antigens such as viruses in diarrhea in infants are simultaneously measured. A method has been disclosed (JP-A-2000-292427).
In this method, a plurality of analytes (eg, rotavirus antigen, calcivirus antigen, coronavirus antigen,
Adenovirus antigen, enterovirus antigen, etc.)
, A plurality of types of labeled immunoreactive substances that individually bind (eg, labeled anti-rotavirus antigen antibodies, calcivirus antigen antibodies, coronavirus antigen antibodies, adenovirus antigen antibodies, and enterovirus antigen antibodies)
Are simultaneously used in the same immunochromatographic reagent to enable simultaneous quantification of a plurality of components by immunochromatography. However, in this immunochromatography method, a plurality of types of analytes (for example, a plurality of types of allergen-specific Ig such as mite, cedar, mold, or egg white) are used.
When a common labeled immunoreactive substance (for example, a labeled anti-IgE antibody) was used for the E antibody), analysis could not be performed with high quantitativeness.

[0004] Hereinafter, the analysis will be described in detail using an example of analysis of an allergen-specific IgE antibody. In recent years, allergic diseases represented by atopic dermatitis or hay fever have been increasing mainly in developed countries, and it is said that the proportion of people who have any allergic predisposition exceeds 30% of Japanese people. . Type I allergy, a common allergic reaction,
From severe, occasional deaths such as anaphylactic shock and bronchitis asthma, to chronic diseases such as atopic dermatitis, allergic rhinitis, or hay fever, to those that threaten daily life, and transient urticaria And eczema, mild rhinitis, etc. I represented by these diseases
For the development of type allergy, a protein component collectively called an allergen is indispensable.

[0005] Allergens are known to be present in a large amount in general living spaces. The main ones are mites such as Dermatophagoides pteronyssinus and Dermatophagoides farinae, Candida, Alternaria, Aspergillus and Cladosporium. Mold fungi, cedar, cypress, birch,
Examples thereof include pollen of plants such as duckweed, harugaya, ragweed or mugwort, epithelium (dandruff) of animals such as dogs, cats and hamsters, and foods such as eggs, milk, soybean, wheat, rice and the like. Foods such as bee venom and peanuts or buckwheat are also known as dangerous allergens that cause anaphylactic shock and often lead to fatal accidents.

[0006] As described above, allergens are present everywhere in daily life, but pathogenic viruses and bacteria,
Alternatively, unlike highly toxic chemicals, they are not dangerous to everyone, and it is practically difficult to eliminate the allergen from daily living space as a whole society. Therefore, it is a basic and fundamental treatment for allergic diseases that the allergic disease patient knows what the allergen is as a substance causing allergy to the person and avoids the allergen as much as possible in daily life.

[0007] As described above, it is very important to examine allergen substances that are a cause of allergic diseases. As an allergen search method, an allergen load test called a skin test has been performed for a long time.
Scratch the skin, apply several types of allergen aqueous solution,
If the skin becomes red and swollen, it is a method to determine that the skin is allergic to the allergen. However, this method has a heavy patient burden, and the test itself may cause severe allergic symptoms.

Therefore, recently, measurement of allergen-specific IgE antibodies in serum has become widespread as an alternative test. An allergen-specific IgE antibody is an in vivo component essential for binding to the allergen and causing the type I allergic reaction. Therefore, by measuring the abundance of IgE antibodies in the blood specific to individual allergens, it is possible to predict how much allergen a patient will exhibit with allergens, as in the skin test. At present, several companies in Japan alone have allergen-specific IgE antibody measurement reagents (for example, Pharmacia CA
P, Yatron Alastat, Hitachi Chemical MAST, Shionogi Lumiward, etc.) are commercially available. In general, when measuring a plurality of allergen-specific IgE antibodies,
The test sample must be divided for each allergen item, but Hitachi Chemical MAST is a unique product that can simultaneously measure multiple allergen items without dividing the test sample. Hitachi Chemical MAST does not use immunochromatography but is a modification of ELISA (enzyme-linked immunosorbent assay). More specifically, a plurality of yarns (each having different allergens immobilized on each yarn) ing)
After reacting a test sample containing an allergen-specific IgE antibody in a reaction vessel carrying the same in the same vessel (primary reaction), washing is performed, and then a labeled antibody specifically reacting with IgE is removed. After the reaction (secondary reaction), finally, the signal derived from the label is analyzed. As described above, the Hitachi Chemical MAST is completely different in principle from the immunochromatography method, and has a drawback that it requires a measurement day and night.

[0009]

Accordingly, an object of the present invention is to provide a labeled immunoreaction in which a plurality of analytes (for example, a plurality of allergen-specific IgEs) react in common with all the analytes. Substances (eg, labeled anti-Ig
An object of the present invention is to provide an immunochromatographic method for analyzing using an E antibody), which is quantitative and can be performed simply and in a short time without dividing a test sample.

[0010]

According to the present invention, there is provided a test sample which may contain a plurality of analytes, and (1) only one of the above-mentioned respective analytes, Each specific immunoreactive substance capable of specifically binding and separately immobilized on a support; (2) each of the analytes; and (3) the plurality of analytes. By analyzing a signal of a label derived from each immune complex including a common immunoreactive substance labeled with a labeling substance and being able to bind to all of the substances, the In the immunochromatography method for analyzing each analyte, the labeled common immunoreactive substance used is substantially different from each labeled common immunoreactive substance containing only one unit of the common immunoreactive substance. Characterized in that, it can be solved by immunochromatography.

[0011] The present invention also provides a method in which: (1) a sample-added region; (2) all of a plurality of analytes can be bound on a support on which a liquid can be spread; A labeled common immunoreactive substance holding region that holds a labeled common immunoreactive substance;
And (3) immobilization of specific immunoreactive substances in which specific immunoreactive substances capable of specifically binding only to any one of the above-described analyte substances are immobilized in a state of being isolated from each other. Labeling regions are arranged in this order with the test sample addition region being upstream, and the labeled common immunoreactive substances to be used are individually labeled common immunoreactive substances each containing only one unit of the common immunoreactive substance. The present invention relates to an immunochromatographic strip, which is substantially composed of an immunoreactive substance.

In the present specification, the term “immunochromatography” is not particularly limited.
The method includes a step of using a chromatographic method (particularly, a thin layer chromatographic method or a paper chromatographic method) during the step, and at least a part of one or a plurality of antigen-antibody reactions is performed on a thin film support for the chromatograph. An immunological analysis performed while developing may be mentioned. “Analysis” in this specification includes both “measurement” for quantitatively or semi-quantitatively determining the amount of an analyte and “detection” for determining the presence or absence of an analyte.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present invention, a common immunoreactive substance, that is, an immunoreactive substance capable of immunologically binding to all of a plurality of analyte substances, is an individual immunoreactive substance containing only one unit of the common immunoreactive substance. It consists essentially of a labeled common immunoreactive substance.

The substance to be analyzed which can be analyzed by applying the method of the present invention is not particularly limited as long as it is a substance in which a common immunoreactive substance is present. Can be mentioned. Examples of the antibody include IgE and autoantibodies. Examples of the antigen include glycoproteins and
Alternatively, a protein composed of subunits can be used.

When the substance to be analyzed is an antibody, for example, when the substance to be analyzed is a plurality of allergen-specific IgE, an anti-IgE antibody can be used as the common immunoreactive substance. The analyte is a plurality of autoantibodies (provided that the γ globulin class is IgG), for example, anti-DNA antibody, anti-ENA (extractable).
When the antibody is a nuclear antigen (soluble nuclear antigen) antibody, an anti-cardiolipin antibody, an anti-mitochondrial antibody, or an anti-smooth muscle antibody, an anti-IgG antibody can be used as a common immunoreactive substance. Furthermore, when the analyte is a plurality of anti-DNA antibodies (eg, IgG, IgM, IgE, Ig
D or IgA), a specific DNA commonly recognized by each of the above-mentioned anti-DNA antibodies can be used as the common immunoreactive substance.

On the other hand, if the analyte is an antigen, for example,
Elephant substance contains different sugar chains and common protein
If there are multiple glycoproteins, common immunoreactivity
As a substance, an antibody recognizing the common protein portion
Can be used. Also, the analytes are different
Multiple including subunit part and common subunit part
Is a common immunoreactive substance.
Using an antibody that recognizes the common subunit portion
Can be In the present invention, a common immune response
The antibody itself can be used as the responsive substance
Or a fragment thereof, for example, F
(Ab ') _Two, Fab, Fab 'or Fv
Can also be.

Each of the labeled common immunoreactive substances included in the group of labeled common immunoreactive substances used in the present invention contains only one unit of the common immunoreactive substance,
Contains one or more units of a labeling substance. The group of labeled common immunoreactive substances used in the present invention is substantially composed of labeled common immunoreactive substances containing only one unit of such a common immunoreactive substance. That is, the labeled common immunoreactive substance containing two or more units of the common immunoreactive substance is not included in an amount that affects the quantitative property of the method of the present invention.

Here, one unit of the common immunoreactive substance means a unit of an antigen or an antibody which performs one antigen-antibody immunoreaction. For example, an antibody means a molecule having substantially only one antigen binding site, and an antigen means a molecule having substantially only one epitope. Specifically, in Fab, Fab ', or Fv, one molecule of each antibody fragment corresponds to one unit of the common immunoreactive substance. In addition, an antibody itself (for example, an entire IgG molecule) or F (ab ′) ₂ has two antigen-binding sites in one molecule, but if the molecular weight of the substance to be analyzed is sufficiently large, the three-dimensional Due to structural obstacles, it is considered that the number of antigen binding sites is substantially one.
Molecule or F (ab ') ₂ molecule is a common immunoreactive substance 1
Equivalent to unit. In addition, one unit of the labeling substance is a minimum unit that exhibits a function as the labeling substance.

The labeling substance used in the present invention is designed such that each labeled common immunoreactive substance contains only one unit of the common immunoreactive substance and one or more units of the labeling substance bound thereto. A labeling substance capable of labeling the common immunoreactive substance with the labeling substance,
Although not particularly limited, examples thereof include a radioisotope, a fluorescent substance, or an enzyme, and a plastid having a diameter of 5 nm or less (preferably 3 nm or less).

As the radioisotope, for example, ¹²⁵
I. In a known labeling method using a radioisotope, a common immunoreactive substance is not usually polymerized. Therefore, according to the known labeling method, only one unit of the common immunoreactive substance and one radioisotope are used. A labeled common immunoreactive substance containing more than one unit bound can be prepared.

Examples of the fluorescent substance include europium, fluorescein isocyanate, dichlorotriazinyl fluorescein, and tetramethylrhodamine isocyanate. In a known labeling method using a fluorescent substance, the common immunoreactive substance is not usually polymerized. Therefore, according to the known labeling method, only one unit of the common immunoreactive substance and one fluorescent substance are used.
A labeled common immunoreactive substance containing more than one unit bound can be prepared.

Examples of the enzyme include alkaline phosphatase, peroxidase, β-galactosidase, and glucose-6-phosphate dehydrogenase. When alkaline phosphatase is used as an enzyme, 5-bromo-4-chloro-3-indolic acid (BCIP) can be used as a chromogenic substrate, and when peroxidase is used as an enzyme,
Tetramethylbenzidine (TMB) can be used as a chromogenic substrate.

By using these enzymes, each labeled common immunoreactive substance can contain only one unit of the common immunoreactive substance and can contain one or more units of the labeling substance in combination. A method for labeling a common immunoreactive substance is as follows:
Although not particularly limited, for example, a maleimide method utilizing the reactivity of a maleimide group with a thiol group or an amino group can be used. When the antibody is labeled with an enzyme, a method of binding the enzyme to the thiol group of Fab ′ obtained by pepsin treatment can also be used. In the maleimide method, a maleimide group is introduced into a thiol group or an amino group of an enzyme, and then reacted with a thiol group of an antibody or an antigen, whereby one molecule of the enzyme and one molecule of a common immunoreactive substance (for example, an antibody) are combined. To give a labeled common immunoreactive substance.

In general, a cross-linking reagent is used to label an antibody or an antigen with an enzyme. The main ones are carbodiimide, isocyanate, diazo compound, benzoquinone, glutaraldehyde, periodic acid,
Maleimide compounds and pyridyl disulfide compounds (enzyme immunoassay, 3rd edition Eiji Ishikawa). For example, the enzyme labeling method using one-stage glutaraldehyde reported in 1969 is a widely used method, but this method has many nonspecific chemical reactions, and antibodies or antigens or enzymes are polymerized. Resulting in. A so-called homopolymerization phenomenon is observed, and in recent years, it is rarely used as an enzyme labeling method. In the present invention, such an enzyme labeling technique is not suitable. Also, a two-stage glutaraldehyde method is known as another enzyme labeling method. In the two-stage glutaraldehyde method, the enzyme is first treated with excess glutaraldehyde, and then glutaraldehyde is removed.
React with antibody or antigen. However, even in the case of an enzyme such as peroxidase and alkaline phosphatase, which is relatively less polymerized by glutaraldehyde treatment, a plurality of antibodies or antigens often bind via the enzyme,
In the present invention, the two-stage glutaraldehyde method is also not suitable.

Examples of the chromophore in the chromophore having a diameter of 5 nm or less that can be used as a labeling substance include, for example, latex, gold, silver, iron, copper, selenium, sulfur, tellurium, and carbon colloid. Can be.
These plastids can generally be linked to a common immunoreactive substance by a physical bond, such as an electrostatic bond, a hydrophobic bond, or a coordination bond.

The molecular weight of a typical mouse monoclonal IgG Fab fragment as a common immunoreactive substance is about 75,000, and its maximum size is about 8 nm. When a plastid having a diameter exceeding 5 nm is used, it is considered that a plurality of IgG Fab fragments bind to one plastid molecule. When an IgG Fab fragment is used as the common immunoreactive substance in the present invention, at most one IgG Fab fragment is bound to one plastid molecule by using a plastid having a diameter of 5 nm or less. Can be done.
When the plastid is extremely small (for example, 1 nm or less in diameter), a plurality of plastids bind to one Fab fragment molecule of IgG. However, since only one unit of the common immunoreactive substance is contained, the quantitative property is maintained. There is no problem. In addition, as a common immunoreactive substance, a substance having a significantly smaller molecular weight than the IgG Fab fragment [for example, Fa
When a b ′ fragment, a virus antigen protein, or an allergen protein (molecular weight = tens of thousands to thousands)] is used, it is necessary to use a plastid having a smaller diameter.
The diameter of the plastid can be appropriately determined according to the molecular weight and / or the maximum length of the common immunoreactive substance to be used.

The immunochromatography method of the present invention is characterized in that the labeled immunoreactive substance is substantially composed of individual labeled common immunoreactive substances each containing only one unit of the common immunoreactive substance. Except for using a reactive substance, the reaction can be carried out in the same manner as in a normal immunochromatography method.

In general, a test sample containing a substance to be analyzed is
When a part of a support (for example, a thin-film support) is brought into contact with the support by a method such as dropping, it is absorbed by the support and diffused by capillary action. At this time, the diffusion direction is arbitrary in the horizontal direction of the thin film, but usually, the test sample is brought into contact with the vicinity of the cut side of the support, and the cellulose that absorbs a large amount of liquid near the cut side on the opposite side. Absorbent carriers such as are adhered to each other to absorb the test sample solution after the analyte has been immobilized and removed on the support by an immunological reaction. This naturally limits the direction of diffusion in the support to one direction. In addition, among various immunochromatography methods, there is a method (JP-A-11-69996) in which the test sample is brought into contact with the support not at the vicinity of the cut side of the support but at the center thereof. The substrate liquid is also contact-absorbed from the vicinity of the cut side of the support, and the absorbing carrier is arranged on the opposite side, so that the diffusion direction converges toward the absorbing carrier. Hereinafter, for convenience, a side closer to the place where the test sample that may contain the analyte may be contacted and absorbed will be referred to as a support upstream, and a side closer to the absorption carrier will be referred to as a downstream.

The immunochromatography method of the present invention is not particularly limited, but can be carried out, for example, using the immunochromatographic strip of the present invention. Hereinafter, the structure of the immunochromatographic strip of the present invention will be described mainly with reference to FIG. The immunochromatographic strip 10 shown in FIG. 1 includes a sample addition pad 1 functioning as a sample addition area, a labeled immunoreactive substance, and a sample addition pad, which extends from upstream to downstream in the developing direction (the direction indicated by arrow A in FIG. 1). A labeled common immunoreactive substance holding pad 2 functioning as a holding area, an immobilized membrane 3 functioning as an immunoreactive substance immobilizing area, and an absorption pad 4 functioning as a development promoting area are arranged in this order.
On the pressure-sensitive adhesive sheet 5 as a base material layer, for example, it is stuck and arranged with an adhesive. The immunochromatographic strip of the present invention is not particularly limited as long as it includes at least a test sample addition region, a labeled immunoreactive substance holding region, and an immunoreactive substance immobilized region.
It is preferable to further include a deployment promoting region.

The immobilized membrane 3 can specifically bind only to any one of a plurality of analytes (for example, analyte A, analyte B, and analyte C). Each immunoreactive substance (hereinafter, referred to as
(Referred to as specific immunoreactive substances) are separately immobilized, which are detection zones 3a, 3b, and 3c. For example, a specific immunoreactive substance A that binds to the analyte A but does not bind to either the analyte B or the analyte C is immobilized in the detection zone 3a. Has immobilized thereon a specific immunoreactive substance B which binds to the analyte B but does not bind to either the analyte C or the analyte A, and the detection zone 3c contains The specific immunoreactive substance C that binds but does not bind to either the analyte A or the analyte B is immobilized.

Each specific immunoreactive substance used in the present invention comprises:
It can be appropriately determined according to the substance to be analyzed. For example, when the substance to be analyzed is a plurality of allergen-specific IgE, each allergen can be used as each specific immunoreactive substance. When the substance to be analyzed is a plurality of autoantibodies, for example, an anti-DNA antibody, an anti-ENA antibody, an anti-cardiolipin antibody, an anti-mitochondrial antibody, or an anti-smooth muscle antibody, the autoantibody is used as each specific immunoreactive substance. Can be used. When the substance to be analyzed is a plurality of anti-DNA antibodies composed of different gamma globulin classes, each specific immunoreactive substance is an antibody capable of recognizing each gamma globulin class (for example, an anti-IgG antibody, an anti-IgG antibody). IgM antibody, anti-IgE
Antibodies, anti-IgD antibodies, or anti-IgA antibodies) can be used. When the analyte is a plurality of glycoproteins containing different sugar chain portions and a common protein portion, each antibody that recognizes each of the above sugar chain portions may be used as each specific immunoreactive substance. Can be. When the substance to be analyzed is a plurality of proteins including different subunit portions (hereinafter, referred to as specific subunit portions) and a common subunit portion, each of the specific subunits is used as a specific immunoreactive substance. Each antibody that recognizes a portion can be used. In the present invention,
As the specific immunoreactive substance, the antibody itself can be used, or a fragment thereof, for example, F (a
b ′) ₂ , Fab, Fab ′, or Fv can also be used.

In the immunochromatographic strip of the present invention, the arrangement pattern of each specific immunoreactive substance in the immunoreactive substance-immobilized region is determined by each immunocomplex formed on each detection zone in the analysis step described later. When analyzing the signal of the label derived from, there is no particular limitation as long as each signal is isolated so as to be distinguishable. For example, as shown in FIG. 1, they are sequentially arranged in series in the deployment direction from upstream to downstream (one lane
(Multi-row type), or they can be arranged side by side in the developing direction (multi-lane, single-row type), or they can be combined and arranged in a grid pattern [that is, a grid pattern]. Arranged on each intersection (multi-lane / multi-row type)]
However, in order to make the analytical components in the test sample immunoreactive with the immunoreactive substance on the immobilized region on the membrane as efficiently as possible, they can be sequentially connected in series with the developing direction. It is preferable to arrange them in Also, the shape of each detection zone is not particularly limited, for example,
It can be dot-shaped or band-shaped.

The support in the immunochromatographic strip of the present invention is not particularly limited as long as the support is capable of developing a liquid. Examples thereof include polyamide fiber, glass fiber, cellulose, nitrocellulose, and the like. A porous body (eg, filter paper or porous polymer) made of cellulose acetate, vinylidene fluoride resin, polytetrafluoroethylene, or the like can be given. In the immunochromatographic strip of the present invention, a substrate layer, for example, a glass plate, an aluminum plate, or a plastic plate may be provided on the back surface of the support.

The immunochromatography method of the present invention is not particularly limited. For example, (1) a test sample containing a plurality of analytes and a labeled common immunoreactive substance may be used. Developing on a support and contacting with each immobilized specific immunoreactive substance immobilized on the support (hereinafter referred to as a contacting step); and (2) each of the immobilized specific immunoreactions Analyzing a signal of a label derived from each immune complex consisting of an active substance, an analyte bound thereto, and a labeled common immunoreactive substance bound thereto (hereinafter referred to as an analysis step). Is called)
Can be included.

Hereinafter, the case where the immunochromatographic strip 10 shown in FIG. 1 is used (that is, the case where the analytes are three types of the analyte A, the analyte B, and the analyte C) will be described. First, the contacting step and the analyzing step will be described. When a test sample containing the analyte A, the analyte B, and the analyte C is dropped on the sample addition pad 1 of the immunochromatographic strip 10 shown in FIG. The development of the test sample is started. When each of the three kinds of analytes contained in the test sample arrives at the holding pad 2 holding the labeled common immunoreactive substance, a 1: 1 immune complex with the labeled common immunoreactive substance is obtained. To form That is, each of the analyte A, the analyte B, and the analyte C immunologically binds to the labeled common immunoreactive substance to form three types of immune complexes. Hereinafter, a 1: 1 immunocomplex of the analyte A and the labeled common immunoreactive substance is referred to as a two-component immunocomplex A. The 1: 1 immune complex is called a two-component immune complex B, and the 1: 1 immune complex of the analyte C and the labeled common immunoreactive substance is called a two-component immune complex C.

As the development proceeds further, each of the two-component immune complexes (ie, two-component immune complex A, two-component immune complex B, and two-component immune complex C) is immobilized on the immobilized membrane 3.
Reaches the detection zone 3a. In the detection zone 3a, the specific immunoreactive substance A that binds to the analyte A but does not bind to either the analyte B or the analyte C is immobilized. Of the complexes, only the two-component immune complex A binds to the immobilized specific immunoreactive substance A and forms a three-component immune complex (hereinafter, referred to as a three-component immune complex A) on the detection zone 3a. I do.
On the other hand, the two-component immune complex B and the two-component immune complex C
It passes through the detection zone 3a and reaches the next detection zone 3b.

In the detection zone 3b, a specific immunoreactive substance B which binds to the analyte B but does not bind to either the analyte C or the analyte A is immobilized. Of the two-component immune complex, only the two-component immune complex B binds to the immobilized specific immunoreactive substance B, and the three-component immune complex (hereinafter, referred to as the three-component immune complex B) is detected in the detection zone 3b. Formed above. The remaining two-component immune complex C passes through the detection zone 3b and reaches the next detection zone 3c. In the detection zone 3c, the analyte C
Since the specific immunoreactive substance C that binds to but does not bind to either the analyte A or the analyte B is immobilized, the last remaining two-component immune complex C is also immobilized. The three-component immune complex (hereinafter, referred to as three-component immune complex C) binds to the reactive substance C and is detected in the detection zone 3
formed on c. As described above, in the contact step in the immunochromatography method of the present invention, even in a single common lane (developing zone), even if a test sample in which a plurality of analytes are mixed is provided, each sample provided for each analyte is provided. On the detection zone, a three-component immune complex can be formed independently of each other without impairing the quantitative property.

Although the contact step has been described above for the case where there are three kinds of the analytes, the same number of the same number of the analytes can be similarly applied when the number of the analytes is two or four or more. By providing a detection zone of
On each of these detection zones, a three-component immune complex can be formed independently without impairing the quantification.

In the immunochromatography method of the present invention, a labeled common immunoreactive substance holding pad 2 can be arranged downstream of a sample addition pad 1 like an immunochromatography strip 10 shown in FIG. ,
A labeled common immunoreactive substance addition pad can be arranged upstream of the sample addition pad, or a test sample can be prepared in advance without separately providing the sample addition pad and the labeled common immunoreactive substance holding pad. The development can also be started by dropping in a mixed liquid state in which the mixture is mixed with a labeled common immunoreactive substance.

After the completion of the contacting step, in the analysis step, a signal derived from the label in each three-component immune complex is analyzed for each detection zone. In the analysis step, a signal derived from the label can be analyzed by an analysis means known per se in accordance with the type of the labeling substance to be used. For example, when a plastid is used as a labeling substance, it can be analyzed visually or by a color measuring instrument, and when a radioisotope is used as a labeling substance, it can be analyzed by a radiometric instrument, and as a labeling substance, When a fluorescent substance is used, it can be analyzed visually or by a fluorescence analyzer. When an enzyme is used as a labeling substance, after the completion of the contacting step, the chromogenic substrate is developed again from the upstream, whereby the chromogenic substrate can react with the enzyme in each immune complex to develop color. In this case, the color development can be analyzed visually or by a color measuring device.

[0041]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 << Alkaline phosphatase labeled anti-IgE
Preparation of Immunochromatographic Strip Using Antibody Fab '>> (1) Preparation of Allergen-Immobilized Membrane A nitrocellulose membrane (HF180; manufactured by Millipore) was used
It was cut into a size of mm × 25 mm. At a position of 15 mm and 20 mm from one end (upstream direction) of the membrane, a mite allergen-extracted protein aqueous solution (Dermatophagoides pteronyssinus; Glia) [concentration 1 mg / mL; 5 mmol
/ L borate buffer (pH 8.5), dialyzed aqueous solution] and egg white allergen extracted protein aqueous solution [concentration 1 mg / mL; 5 mmol / L borate buffer (pH 8.5.
Aqueous solution dialyzed after dilution in 5)] was applied in a linear shape having a width of 1 mm. The mixture was allowed to stand at room temperature for 1 hour, and then allowed to stand at 37 ° C. for 30 minutes to immobilize allergen-extracted proteins. By storing in a silica gel desiccator and drying overnight at room temperature, an allergen-immobilized membrane A (arranged in order of mite allergen and egg white allergen from upstream to downstream) was produced.

On the other hand, 15 mm and 20 mm from one end of the film
, Respectively, except that the egg white allergen-extracted protein aqueous solution and the mite allergen-extracted protein aqueous solution are applied in a linear shape having a width of 1 mm, by repeating the above-described operation for producing an allergen-immobilized membrane A. And an allergen-immobilized membrane B (arranged in the order of egg white allergen and mite allergen from upstream to downstream). In addition, except that the mite allergen-extracted protein aqueous solution was applied in a linear shape having a width of 1 mm at a position 15 mm from one end of the membrane, the above-described operation for producing the allergen-immobilized membrane A was repeated, whereby allergen immobilization was performed. A film C (only mite allergen was arranged) was prepared. Further, except that the egg white allergen-extracted protein aqueous solution was applied in a linear form having a width of 1 mm at a position 15 mm from one end of the membrane, the above-described operation for producing the allergen-immobilized membrane A was repeated, whereby allergen immobilization was performed. Membrane D
(Only the egg white allergen was arranged).

(2) Alkaline phosphatase labeled anti-I
Preparation of gE Antibody Fab ′ An anti-human IgE mouse monoclonal antibody was treated with pepsin, then reduced with 2-mercaptoethylamine, and 1 mg of purified Fab ′ fragment was prepared by gel filtration. On the other hand, separately from this, 2 mg of bovine intestinal alkaline phosphatase was diluted with a phosphate buffer to a concentration of 2 mg / mL, and dialyzed to give N-succinimidyl 3- (2-pyridylthiol) adjusted to 7.5 mmol / L. ) 100 μL of propionate (SPDP) was added. After reaction at 4 ° C. for 5 hours, the mixture was dialyzed with a phosphate buffer to prepare pyridyldithiopropionate (PDP) -introduced alkaline phosphatase. The anti-IgE antibody Fab 'and the P
After mixing equal amounts of DP-introduced alkaline phosphatase in 1 mL portions, 1 mol / L hydroxylamine 40 μL
Was added. After reacting at 4 ° C. for 3 days, the mixture was subjected to gel filtration using a gel filtration column (TSKgel G3000SW; manufactured by Tosoh Corporation), and unreacted anti-IgE antibody Fab ′ and P
The DP-introduced alkaline phosphatase was removed to prepare an alkaline phosphatase (AP) -labeled anti-IgE antibody Fab ′ aqueous solution.

(3) Preparation of AP-Labeled Antibody Pad The aqueous solution of the AP-labeled anti-IgE antibody Fab ′ prepared in Example 1 (2) was adjusted to 5.0 μg so that the amount of antibody would be 1 μg.
% Sucrose in 5 mmol / L phosphate buffer (pH
After dilution in 7.2), 10 μL of the diluted solution was added to 5 mm ×
It was applied to an absorption pad (PREM1420; manufactured by Pall) cut into 5 mm, and dried in a silica gel desiccator at room temperature overnight under reduced pressure (≦ 100 mmHg) to prepare an AP-labeled antibody pad.

(4) Preparation of Sample Addition Pad The glass fiber pad was cut into 5 mm × 18 mm,
0.5% sucrose, 0.2% Tween 20, and 0.1
% Polyvinyl alcohol (degree of polymerization = approximately 500), and then immersed in an aqueous solution.
A sample addition pad was prepared.

(5) Preparation of Absorbent Pad A cellulose pad (AP25; manufactured by Millipore) was cut into a size of 5 mm × 20 mm to obtain an absorbent pad.

(6) Preparation of strip for immunochromatography A plastic adhesive sheet (manufactured by BioDot) cut into a size of 5 mm × 60 mm was prepared in Example 1 (4) from the upstream side to the downstream side in the developing direction. Sample addition pad, AP prepared in Example 1 (3) above
The labeled antibody pad, the allergen-immobilized membrane A prepared in Example 1 (1), and the absorbent pad prepared in Example 1 (5) were arranged in this order by 1 m with a member adjacent to each end.
By laminating and adhering about m, an immunochromatographic strip (1) was produced. The above operation is repeated except that the allergen-immobilized membrane A is replaced with the allergen-immobilized membrane B, allergen-immobilized membrane C, or allergen-immobilized membrane D prepared in Example 1 (1). , Immunochromatographic strip (2), immunochromatographic strip (3),
And a strip for immunochromatography (4).

[0048]

Comparative Example 1 << Preparation of Immunochromatographic Strip Using Colloidal Gold (Diameter = 20 nm) Labeled Anti-IgE Antibody >> (1) Preparation of Colloidal Gold (Diameter = 20 nm) Labeled Anti-IgE Antibody Anti-Human IgE Mouse 1 mg of the monoclonal antibody was added to 0.
The phosphate buffer solution (2 mmol / mL) was adjusted to 1 mg / mL.
L, pH 7.0) and dialyzed. Subsequently, a gold colloid suspension (GOLD COLLOID 20; B)
ritish BioCell International
nal) (100 mL) while stirring the anti-human Ig.
10 mL of an E mouse monoclonal antibody aqueous solution was slowly added dropwise, followed by stirring at room temperature for 10 minutes. Further, 10% bovine serum albumin (A-7888; Sigma
a); hereinafter, referred to as BSA) 10 mL of an aqueous solution was slowly dropped, and the mixture was further stirred at room temperature for 10 minutes. To this aqueous solution, 15 mL of an aqueous solution containing 5% sucrose and 0.2% Tween 20 was added, and mixed.
After centrifugation at 00 × g for 1 hour, the supernatant was removed.

The precipitated colloidal gold-labeled antibody was added to 0.5
% Sucrose and 0.02% Tween 20 in 100 mL of Borax buffer (pH 8.0), 10 ° C.
For 1 hour at 16,000 × g, and the supernatant was removed. Further, the precipitated gold colloid-labeled antibody was
% Sucrose and 0.02% Tween 20 in 100 mL of Borax buffer (pH 8.0), 10 ° C.
For 1 hour at 16,000 × g, and the supernatant was removed. Finally, the precipitated colloidal gold-labeled antibody was added to 0.1 mL.
After resuspension in Borax buffer (pH 8.0) containing 5% sucrose and 0.02% Tween 20, the particle density was about 10 ¹³ / mL (absorbance at 520 nm wavelength = about 1).
4) Adjust the concentration so that it becomes
It was E antibody suspension.

(2) Preparation of Colloidal Gold-Labeled Antibody Pad An AP-labeled anti-IgE antibody aqueous solution prepared in Comparative Example 1 (1) was used instead of the AP-labeled anti-IgE antibody aqueous solution. By repeating the procedure of Example 1 (3), a gold colloid-labeled antibody pad was prepared.

(3) Preparation of strip for immunochromatography Instead of the AP-labeled antibody pad, the comparative example 1 (2) was used.
The procedure of Example 1 (5) was repeated, except that the gold colloid-labeled antibody pad prepared in Step 4 was used, so that four types of immunochromatographic strips (5) were used.
To (8). The immunochromatographic strip having the allergen-immobilized membrane A is referred to as an immunochromatographic strip (5).
The respective immunochromatographic strips having the allergen-immobilized membranes B, C, or D are referred to as immunochromatographic strips (6), (7), and (8), respectively.

[0052]

[Evaluation] Four types of immunochromatographic strips (1) to (4) prepared in Example 1 and four types of immunochromatographic strips (5) to (8) prepared in Comparative Example 1 )
Each with the sample addition pad down,
L was inserted into a microplate well which had been dispensed in advance and leaned, and the sample was developed by chromatography by capillary action. Immunochromatographic strip (1) ~
About (4), 10 minutes after the start of the chromatographic development, 200 μL of 5-bromo-4-chloro-3-indolic acid (BCIP; manufactured by Boehringer Mannheim)
Was further developed by chromatography.

As the sample, sample 1 (mite-specific IgE = 0 IU / mL, egg white-specific IgE = 0 IU)
/ ML), Sample 2 (mite-specific IgE = 3 IU / mL,
Egg white specific IgE = 3 IU / mL, sample 3 (mite specific IgE = 10 IU / mL, egg white specific IgE = 10 I)
U / mL) and Sample 4 (mite-specific IgE = 30 IU)
/ ML, egg white specific IgE = 30 IU / mL). Amount of mite-specific IgE and egg white-specific Ig in each sample
The amount of E was determined by a conventional quantitative measurement method (Alastat specific IgE).
Antibody measurement kit; sold by Yatron Co., Ltd., manufactured by DPC in the United States) and expressed in IU / mL, which is an international standard unit.

After completion of the chromatographic development, the degree of coloring in the allergen-immobilized region (1 mm wide band) was visually determined. Each immunochromatographic strip (1) ~
The configuration of (8) is shown in FIG. 1, and the determination result is shown in FIG. A
Regarding the immunochromatographic strips (1) to (4) using the P-labeled anti-IgE antibody Fab ′, the degree of blue coloration when the specimen 3 was measured using the immunochromatographic strip (3) was defined as “+”, "++" indicates that the degree of coloring is stronger than that, and "±" indicates that it is weaker.
Indicated by The case where no coloring is confirmed is indicated by “−”. On the other hand, with respect to the immunochromatographic strips (5) to (8) using the gold colloid-labeled anti-IgE antibody, the degree of red coloring when the specimen 3 was measured using the immunochromatographic strip (7) was “+”. The case where the degree of coloring is stronger is indicated by “++”, and the case where the degree of coloring is weak is indicated by “±”. The case where no coloring is confirmed is indicated by “−”.

<< Table 1 >> Immunochromatography 1 2 3 4 5 6 7 8 7 8 F Strip Allergen Immobilization Membrane ABCD ABCD Upstream Mite Egg White Mite Egg White Mite Egg White Mite Egg White Lower Egg White Mite--Egg White Mite - - labeled antibody pad AP-labeled antibody colloidal gold-labeled antibody

<< Table 2 >> Immunochromatography 1 2 3 4 5 6 7 8 8 F Strip Allergen Amount (IU / mL) Sample Mite 0 ------1 Egg White 0------ Sample Dust 3 ± ± ± ± ± ± 2 egg white 3 ± ± ±-± ± sample mite 10 + + + + ± + 3 egg white 10 + + + ± + + sample mite 30 + + + + + + + + + + + + 4 egg white 30 + + + + + + + + + + + +

Gold colloid (particle size = 20 nm) labeled anti-Ig
When the E antibody was used, the degree of coloring of the allergen band immobilized at the downstream position of the immunochromatographic membrane was lower than that of the upstream allergen or single allergen solid phase. on the other hand,
When the AP-labeled anti-IgE antibody Fab ′ was used,
The upstream and downstream of the membrane were distinguished and the allergen solid phase was equally colored, singly or in combination. Therefore,
In order to simultaneously measure a plurality of items of allergen-specific IgE by immunochromatography, in the case of this example, it was found that alkaline phosphatase labeling suitable for single labeling was suitable.

[0058]

According to the immunochromatography method of the present invention, quantitatively and without dividing a test sample,
It is possible to analyze a plurality of substances to be analyzed simply and in a short time. For example, in the hitherto known Hitachi Chemical MAST, in order to carry out an antigen-antibody reaction in a reaction vessel holding a plurality of threads on which an allergen is immobilized, even if the reaction vessel is shaken, the allergen carried on the threads And the chance of contact with the allergen-specific IgE antibody to be analyzed in the test sample is low, requiring a long reaction time (for example, all day long). Further, the dilution of the test sample and the addition of the test sample into the reaction vessel also caused the contact opportunity to be further reduced. In contrast, in the immunochromatography method of the present invention, for example, by providing an absorption pad,
The test sample can be forcibly moved toward the absorption pad in a short period of time, so that most of the analyte (eg, allergen-specific IgE) in the test sample is immobilized on the immobilized specific immunity. Reactive substances (eg allergens)
Can be contacted for a short time and forcefully,
The antigen-antibody reaction can be completed in a short time together with the improvement in sensitivity.

According to the immunochromatography method of the present invention, it is possible to collect a sample such as a patient's blood at the time of a doctor's consultation, and to identify the allergic disease-causing substance on the spot. To reduce the burden on patients who had to go to the hospital again at a later date in order to obtain allergen-specific IgE antibody test results, and to immediately provide allergen removal treatment and guidance by doctors based on allergen-specific IgE antibody test results Since it can be performed, it leads to improvement of medical efficiency. This responds to the recent increase in the number of patients with allergic diseases, and contributes to curbing medical costs.

[Brief description of the drawings]

FIG. 1 is a plan view schematically showing one embodiment of an immunochromatographic strip that can be used in the immunochromatographic method of the present invention.

[Explanation of symbols]

1 ... sample addition pad; 2 ... labeled common immunoreactive substance holding pad; 3 ... immobilized membrane; 4 ... absorption pad;
..Adhesive sheet; 10: Strip for immunochromatography.

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Shiro Matsuura 1-11-4 Higashikanda, Chiyoda-ku, Tokyo Inside Yatron Co., Ltd.

Claims (4)

[Claims] 1. A test sample which may contain a plurality of substances to be analyzed: (1) Only one of the above-mentioned respective substances to be analyzed can be specifically bound, and Each of the specific immunoreactive substances separately immobilized on the body; (2) each of the analytes; (3) all of the plurality of analytes can be bound; By analyzing a signal of a label derived from each immune complex including a common immunoreactive substance labeled with a labeling substance, an immunochromatographic method for analyzing each analyte in the test sample, An immunochromatographic method, characterized in that the labeled common immunoreactive substances used each consist essentially of individual labeled common immunoreactive substances containing only one unit of the common immunoreactive substance. . 2. The method according to claim 1, wherein the labeling substance is a radioisotope, a fluorescent substance, an enzyme, or a plastid having a diameter of 5 nm or less. 3. The method according to claim 1, wherein the plurality of analytes are a plurality of allergen-specific IgE antibodies. 4. A support on which a liquid can be developed, (1) an area to which a test sample is added; (2) a substance which can bind to all of a plurality of analytes and is labeled with a labeling substance. A labeled common immunoreactive substance holding region holding a common immunoreactive substance;
And (3) immobilization of specific immunoreactive substances in which specific immunoreactive substances capable of specifically binding only to any one of the above-described analyte substances are immobilized in a state of being isolated from each other. Labeling regions are arranged in this order with the test sample addition region being upstream, and the labeled common immunoreactive substances to be used are individually labeled common immunoreactive substances each containing only one unit of the common immunoreactive substance. An immunochromatographic strip, consisting essentially of an immunoreactive substance.