WO2025239335A1 - Method for detecting test substance and kit therefor - Google Patents

Abstract

Provided are an immunochromatographic method, and a kit therefor, that enable testing to be carried out with greater sensitivity and/or faster than known methods.　The method for detecting a test substance comprises: simultaneously or sequentially bringing a test substance into contact with a biotin-labeled invisible particle on which there is immobilized a first antibody that undergoes an antigen-antibody reaction with the test substance or an antigen-binding fragment thereof, with a labeled second antibody that undergoes an antigen-antibody reaction with the test substance and can bind by an antigen-antibody reaction to the test substance at the same time as the first antibody or a labeled antigen-binding fragment thereof, and with an avidin-immobilized region where avidin or streptavidin is immobilized; and detecting the label bound to the avidin-immobilized region. In addition, the test kit comprises: a carrier having an avidin-immobilized region where avidin or streptavidin is immobilized; a biotin-labeled invisible particle on which there is immobilized a first antibody that undergoes an antigen-antibody reaction with the test substance, or an antigen-binding fragment thereof; and a labeled second antibody that undergoes an antigen-antibody reaction with the test substance and can bind by an antigen-antibody reaction to the test substance at the same time as the first antibody, or a labeled antigen-binding fragment thereof.

Description

Method for detecting test substance and kit therefor

The present invention relates to a method for detecting a test substance using immunochromatography and a kit therefor.

Conventionally, immunochromatography has been widely used as a test that can be easily performed in medical settings (Point of Care Testing, POCT) to diagnose various infectious diseases.

Among immunochromatographic methods, a method that uses colored particles as labels and visually determines the presence or absence of color is also widely known. Among these, a method that provides an avidin immobilization area on a substrate and uses a biotin-labeled specific antibody to improve the sensitivity and/or speed of the test is known (Patent Document 1).

Japanese Patent Application Publication No. 9-184840

It goes without saying that it would be advantageous to have an immunochromatography method that allows for more sensitive and/or faster testing than known immunochromatography methods.

Therefore, an object of the present invention is to provide an immunochromatography method and a kit therefor that enable testing with higher sensitivity and/or speed than known methods.

As a result of extensive research, the inventors of the present application discovered that by providing an avidin immobilization region on a substrate, immobilizing a first anti-analyte antibody to a biotin-labeled invisible particle, binding the invisible particle to the avidin immobilization region via an avidin-biotin bond, sandwiching the analyte between the first anti-analyte antibody on the invisible particle and a labeled second anti-analyte antibody, and detecting the analyte by detecting the label bound to the avidin immobilization region, it is possible to perform testing with higher sensitivity and/or speed than known methods, and thus completed the present invention.

That is, the present invention provides the following.
(1) A method for detecting a test substance by immunochromatography, comprising:
preparing an immunochromatography strip having a substrate having an avidin immobilization region where avidin or streptavidin is immobilized;
preparing biotin-labeled invisible particles on which a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with the test substance is immobilized;
preparing a second antibody that undergoes an antigen-antibody reaction with the test substance, the second antibody being labeled, or a labeled antigen-binding fragment thereof, which is capable of binding to the test substance through an antigen-antibody reaction simultaneously with the first antibody;
contacting the test substance, the avidin immobilization region, the invisible particles, and the labeled second antibody or labeled antigen-binding fragment thereof simultaneously or sequentially;
A method for detecting a test substance by immunochromatography, comprising the step of detecting the label bound to the avidin immobilization region.

(2) The method according to (1), wherein the label of the labeled second antibody or the labeled antigen-binding fragment thereof is a visible particle.
(3) The method according to (2), wherein the visible particles are colored particles.
(4) The method according to (1), wherein the invisible particles are white particles.
(5) The method according to (1), wherein at least a portion of the first antibody or its antigen-binding fragment immobilized on the invisible particle, or another substance immobilized on the invisible particle, is biotin-labeled.
(6) A method capable of detecting multiple test substances, comprising:
The method according to (1), further comprising the step of preparing a plurality of types of the invisible particles and a plurality of types of the labeled second antibodies or labeled antigen-binding fragments thereof, each corresponding to a respective one of the plurality of test substances, wherein the plurality of types of labeled second antibodies or labeled antigen-binding fragments thereof are distinguishable for each type of test substance, and the type of test substance is identified based on the type of label bound to the avidin immobilization region.
(7) The method according to (6), wherein the label of the labeled second antibody or the labeled antigen-binding fragment thereof is a visible particle.
(8) The method according to (7), wherein the visible particles are colored particles, and the colors are different for each of the plurality of types of test substances.
(9) The method according to any one of (2) to (4), wherein the visible particles and the invisible particles are latex particles.

(10) An immunochromatography kit for carrying out the detection method according to (1), comprising: an immunochromatography strip having a substrate having an avidin-immobilized region on which avidin or streptavidin is immobilized;
a biotin-labeled invisible particle having a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with the test substance immobilized thereon;
An immunochromatography kit comprising a second antibody that undergoes an antigen-antibody reaction with the test substance, and a labeled second antibody or a labeled antigen-binding fragment thereof that is capable of binding to the test substance through an antigen-antibody reaction simultaneously with the first antibody.
(11) The immunochromatography kit according to (10), wherein the label of the labeled second antibody or the labeled antigen-binding fragment thereof is a visible particle.
(12) An immunochromatography kit for carrying out the detection method described in (6), comprising a plurality of types of the invisible particles and a plurality of types of the labeled second antibodies or labeled antigen-binding fragments thereof, each corresponding to a plurality of test substances, and the plurality of types of the labeled second antibodies or labeled antigen-binding fragments thereof are distinguishable by type of test substance. (10)
(13) The immunochromatography kit according to (12), wherein the label of the labeled second antibody or the labeled antigen-binding fragment thereof is a visible particle.
(14) The immunochromatography kit according to (13), wherein the visible particles are colored particles, and the colors are different for each of the plurality of types of test substances.
(15) The immunochromatography kit according to any one of (10) to (14), wherein the visible particles and the invisible particles are latex particles.

[1] A test substance;
a biotin-labeled invisible particle having a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with the test substance immobilized thereon;
a second antibody that undergoes an antigen-antibody reaction with the test substance, the second antibody being labeled and capable of binding to the test substance simultaneously with the first antibody through an antigen-antibody reaction, or a labeled antigen-binding fragment thereof;
an avidin immobilization region in which avidin or streptavidin is immobilized;
simultaneously or sequentially contacting the label with the avidin immobilization region, and detecting the label bound to the avidin immobilization region.
A method for detecting a test substance, comprising:
[2] A plurality of test substances;
a plurality of types of invisible particles, each of which has a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with each of the plurality of test substances immobilized thereon and is biotin-labeled;
a second antibody capable of distinguishing between the types of the plurality of test substances and undergoing an antigen-antibody reaction with each of the plurality of test substances, the second antibody being capable of binding to the test substances simultaneously with the first antibody through an antigen-antibody reaction; and
an avidin immobilization region in which avidin or streptavidin is immobilized;
simultaneously or sequentially contacting the label with the avidin immobilization region, and detecting the label bound to the avidin immobilization region.
A method for detecting a plurality of test substances, comprising:
[3] A carrier having an avidin immobilization region where avidin or streptavidin is immobilized;
a biotin-labeled invisible particle having a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with a test substance immobilized thereon;
A detection kit comprising a second antibody that undergoes an antigen-antibody reaction with the test substance, and a labeled second antibody or a labeled antigen-binding fragment thereof that is capable of binding to the test substance through an antigen-antibody reaction simultaneously with the first antibody.
[4] Invisible particles that are biotin-labeled and have a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with a test substance immobilized thereon.

According to the present invention, a large amount of the first antibody or its antigen-binding fragment can be immobilized on invisible particles, and the invisible particles do not interfere with the visibility of the label, allowing for highly sensitive and/or rapid testing.

FIG. 1 is a schematic diagram for explaining the principle of the present invention. This is a continuation of Figure 1-1.

One embodiment of the present disclosure will be described in detail below, but the scope of the present disclosure is not limited to the embodiment described herein, and various modifications may be made without departing from the spirit of the present disclosure. Each aspect disclosed in this specification can be combined with any other feature disclosed in this specification. When multiple upper and lower limit values are listed for a particular parameter, any of these upper and lower limit values can be combined to form a suitable numerical range. The lower and/or upper limit values of a numerical range described in this disclosure are numerical values within that range and may be replaced with numerical values shown in the examples. The expression "X to Y" indicating a numerical range means "greater than or equal to X and less than or equal to Y." When a specific description given for one embodiment also applies to other embodiments, that description may be omitted in other embodiments.

One embodiment of the present invention relates to invisible biotin-labeled particles onto which a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with a test substance is immobilized. Use of these particles enables highly sensitive and/or rapid testing.

One embodiment of the present invention relates to a method for detecting a test substance, comprising simultaneously or sequentially contacting a test substance with biotin-labeled invisible particles on which a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with the test substance is immobilized, a second antibody that undergoes an antigen-antibody reaction with the test substance, the second antibody or the labeled antigen-binding fragment thereof being capable of binding to the test substance through an antigen-antibody reaction simultaneously with the first antibody, and an avidin immobilization region on which avidin or streptavidin is immobilized, and detecting the label bound to the avidin immobilization region.

One embodiment of the present invention relates to a method for detecting multiple test substances, comprising simultaneously or sequentially contacting multiple test substances with multiple types of invisible particles that are biotin-labeled and have immobilized thereon a first antibody or antigen-binding fragment thereof that undergoes an antigen-antibody reaction with each of the multiple test substances, multiple types of labeled second antibodies or labeled antigen-binding fragments thereof that can be distinguished by type from the multiple test substances and undergo an antigen-antibody reaction with each of the multiple test substances, and that can bind to the test substances via an antigen-antibody reaction simultaneously with the first antibody, with an avidin immobilization region on which avidin or streptavidin is immobilized, and detecting the label bound to the avidin immobilization region.

In the detection method of this embodiment, a channel provided with a carrier having an avidin-immobilized region where avidin or streptavidin is immobilized can be used. Examples of the channel include a membrane and a microchannel.
An example of a device containing a membrane is a strip. Examples of such strips include immunochromatographic strips used in immunochromatography. The basic structure of these strips is the same as that of known immunochromatographic strips, such as those described in Japanese Patent Application Laid-Open No. 2016-217911. That is, the basic structure of a strip is typically a laminate of multiple nitrocellulose membranes or the like, each having a required area, on a backing sheet made of a plastic film. However, the backing sheet is not essential. In this specification and claims, these laminated nitrocellulose membranes or the like are referred to as a "substrate." An immunochromatographic strip includes a determination section on a substrate on which a reagent is immobilized. The immunochromatographic strip typically includes a sample pad, typically made of a nonwoven fabric or the like, onto which a sample is dripped, at the upstream end, and an absorption band, typically made of a nonwoven fabric or the like, that absorbs the sample and developing solution, at the downstream end. In addition, a control section is usually provided to confirm whether the test has been performed normally, and the control section has immobilized thereon a mouse IgG antibody or the like that specifically binds to the constant region of the monoclonal antibody used in the test. A liquid added to a nitrocellulose membrane or the like moves on the immunochromatographic strip by capillary action.

An example of a device that includes a microchannel is a microchannel device. The basic structure of a microchannel device is the same as that of known microchannel devices, such as those described in JP 2017-78664 A. Here, a microchannel refers to a fine channel, specifically a channel with a minimum diameter on the order of micrometers. For example, the depth direction may be on the order of micrometers and the width direction may be on the order of millimeters. A microchannel device refers to a device, such as a microchip or microcapillary, that has a microchannel through which a mixed solution containing a test substance moves, allowing the test substance to be detected. A microchannel device may have a test area, a sample pad, and an absorption band, similar to an immunochromatographic strip.

The principle of the immunochromatography method of the present invention will be explained based on one preferred embodiment with reference to the schematic diagrams shown in Figures 1-1 and 1-2.

The immunochromatographic strip used in the immunochromatographic method of the present invention has an area on a substrate 10 where avidin or streptavidin (hereinafter, in the explanations up to the examples, unless otherwise clear from the context, "avidin" means "avidin or streptavidin") 12 is immobilized (hereinafter, this may be referred to as the "avidin-immobilized area").

Separately, invisible white particles 18 are prepared, onto which a first anti-analyte antibody or its antigen-binding fragment (hereinafter, in the explanations preceding the examples, unless otherwise clear from the context, "antibody" means "antibody or its antigen-binding fragment") 16 is immobilized, which undergoes an antigen-antibody reaction with the analyte 14. Biotin 20 is bound to the first anti-analyte antibody (hereinafter, for convenience, sometimes referred to as the "first antibody"). Note that, although not particularly limited, the particle size of the invisible particles (white particles in this example) is typically approximately 30 to 1,000 nm, preferably approximately 50 to 800 nm, and the amount used is typically approximately 0.08 to 4 μg, preferably approximately 0.4 to 1.5 μg.

Furthermore, colored particles 22 onto which a second anti-analyte antibody (hereinafter, for convenience, sometimes referred to as the "second antibody") 24 is immobilized are prepared. The colored particles 22 are used as labels for the second antibody. The first and second antibodies are selected to be able to simultaneously bind to the analyte through an antigen-antibody reaction with the analyte. Although not particularly limited, the particle size of the colored particles is typically about 30 to 1,000 nm, preferably about 50 to 800 nm, and the amount used is typically about 0.2 to 10 μg, preferably about 2.0 to 5.0 μg.

As shown on the right side of Figure 1-1, the white particles 18, colored particles 22, and analyte 14 are brought into contact with the avidin-immobilized region where avidin 12 is immobilized on the substrate 10. This can be achieved by flowing a developer solution from the upstream side due to capillary action. Figure 1-2 shows the state after further flowing the developer solution to wash away unbound material. As shown in Figure 1-2, the first antibody 16 binds to the avidin 12 via the biotin 20. The first antibody 16 immobilized on the white particles 18 specifically binds to the analyte 14. Furthermore, the first antibody 16 on the white particles 18 specifically binds to the analyte 14. Furthermore, the analyte 14 specifically binds to the second antibody 24 immobilized on the colored particles 22. As a result, the analyte 14 is sandwiched between the first antibody 16 on the white particles 18 and the second antibody 24 on the colored particles 22.

In this way, the colored particles 22 are bound to the avidin immobilization region. Therefore, the analyte 14 can be detected by visually observing the colored particles on the avidin immobilization region. On the other hand, if the sample does not contain the analyte 14, the colored particles 22 will not bind to the avidin immobilization region, and the avidin immobilization region will not be colored. Note that white particles 18 also bind to the avidin immobilization region, but because the white particles are white, they do not interfere with the visibility of the colored particles.

In the above method, the white particles 18 to which the first antibody 16 is immobilized and the colored particles to which the second antibody is immobilized may be mixed with a sample containing the test substance 14 or a diluted version thereof, or regions containing these may be provided upstream of the avidin immobilization region, and during testing, they may be suspended in the sample liquid or developer liquid and moved to the avidin immobilization region by capillary action. In this case, the white particles and colored particles may be placed in the same region or in different regions. Furthermore, if they are placed in different regions, either may be placed upstream.

In the method according to a preferred embodiment of the present invention described above, by using multiple types of white particles onto which multiple types of first antibodies that undergo an antigen-antibody reaction with each of the multiple types of test substances are immobilized, and multiple types of colored particles onto which multiple types of second antibodies that undergo an antigen-antibody reaction with each of the multiple types of test substances are immobilized, it is possible to distinguish and detect multiple types of test substances. In this case, the colored particles onto which the multiple types of second antibodies are immobilized are each made different colors. For example, as described in the examples below, a method can be made that can distinguish and detect influenza A virus and influenza B virus. In this case, white particles onto which an anti-A first antibody that undergoes an antigen-antibody reaction with influenza A virus is immobilized, colored particles onto which an anti-A second antibody that undergoes an antigen-antibody reaction with influenza A virus is immobilized, white particles onto which an anti-B first antibody that undergoes an antigen-antibody reaction with influenza B virus is immobilized, and colored particles onto which an anti-B second antibody that undergoes an antigen-antibody reaction with influenza B virus is immobilized. The colored particles onto which the anti-A second antibody is immobilized are made different colors. For example, the former is red and the latter is blue.

If the sample contains influenza A virus, the avidin immobilization area will turn red, and if the sample contains influenza B virus, the avidin immobilization area will turn blue. In this way, it is possible to distinguish and detect whether the influenza virus contained in the sample is type A or type B. Note that if the sample contains both influenza A virus and influenza B virus (such as in the case of co-infection), the avidin immobilization area will turn purple, a mixture of red and blue.

When detecting multiple types of test substances, all that is required is that multiple types of invisible particles, multiple types of labeled second antibodies, and multiple types of test substances come into contact and react during the reaction. Therefore, for example, multiple types of invisible particles and multiple types of labeled second antibodies may be added to the sample, and any of these may be placed together or separately. In this case, it does not matter which is the upstream side and which is the downstream side.

In the method according to one embodiment of the present invention described above, latex particles such as polystyrene latex can be preferably used as the white particles or colored particles. These are commercially available, so commercially available products can be preferably used.

In addition, in the above-mentioned preferred embodiment of the present invention, white particles are used as the invisible particles, but this is not limited to this. For example, particles of the same color as the substrate or colorless, transparent particles can also be used as the invisible particles.

Furthermore, in the method of the present invention, it is sufficient that the invisible particles are directly or indirectly biotin-labeled. Therefore, in one preferred embodiment of the present invention described above, the first antibody is biotin-labeled, and it is sufficient that at least a portion of the first antibody molecule is biotin-labeled. Furthermore, when an antibody is generally immobilized, a protein such as bovine serum albumin (BSA) or casein is immobilized together with the antibody as a blocking agent to prevent non-specific adsorption, and at least a portion of the protein molecule used as the blocking agent may be biotin-labeled.

In addition, in the above-mentioned preferred embodiment of the present invention, colored particles are used to label the second antibody, but colored metal particles such as gold colloid or platinum colloid, or fluorescent particles can also be preferably used as colored particles. Furthermore, other labels used in conventional immunoassays, such as enzyme labels, fluorescent labels, and chemiluminescent dye labels, can also be used to label the second antibody.

According to the method of the present invention, the substrate and the first antibody are bound by the extremely high-affinity avidin-biotin reaction, and a large number of first antibody molecules can be immobilized on the white particles, thereby improving detection sensitivity and speeding up testing.

One embodiment of the present invention relates to a detection kit comprising: a carrier having an avidin immobilization region where avidin or streptavidin is immobilized; biotin-labeled invisible particles on which a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with the test substance is immobilized; and a second antibody that undergoes an antigen-antibody reaction with the test substance, the second antibody being labeled or the antigen-binding fragment thereof being capable of binding to the test substance through an antigen-antibody reaction simultaneously with the first antibody.

In one embodiment, the detection kit may comprise multiple types of invisible particles and multiple types of labeled second antibodies or labeled antigen-binding fragments thereof, each corresponding to a respective one of multiple test substances.

In one embodiment, the present invention provides an immunochromatography kit for carrying out the above-described method for detecting a test substance by immunochromatography, the kit comprising: an immunochromatography strip having a substrate having an avidin-immobilized region;
a biotin-labeled invisible particle having a first antibody immobilized thereon that undergoes an antigen-antibody reaction with a test substance;
The present invention also provides an immunochromatography kit comprising a second antibody that undergoes an antigen-antibody reaction with the test substance, the second antibody being labeled and capable of binding to the test substance through an antigen-antibody reaction simultaneously with the first antibody.

We also provide an immunochromatography kit for carrying out the above-mentioned method of the present invention for detecting multiple types of test substances, which includes multiple types of invisible particles and multiple types of labeled second antibodies corresponding to each of the multiple test substances, and which can distinguish each type of test substance from the other.

In the method of the present invention, the biological sample to be measured is not particularly limited, but is preferably one that can significantly demonstrate the effect of the present invention, which is to suppress the influence of interfering substances. Preferred examples include body fluids such as serum, plasma, blood, urine, stool, saliva, tissue fluid, cerebrospinal fluid, and swabs, as well as dilutions thereof. Particularly preferred are specimens contaminated with substances derived from the mucous membranes of living organisms, such as sputum, saliva, throat swabs, nasal swabs, nasal aspirates, corneal and conjunctival swabs, and fecal specimens.

In the method using the immunochromatographic test strip of the present invention, the substance to be measured is an antigen or antibody that can be measured by immunoassay, i.e., an assay that utilizes an antigen-antibody reaction. Any antigen can be used as long as it can produce an antibody, including proteins, polysaccharides, lipids, etc. Protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, etc. that contain these substances can also be measured.

The present invention will now be described in detail based on examples and comparative examples. However, the present invention is not limited to the following examples.

Example 1. Test Device of the Present Invention for Detecting Influenza A Virus Antigen (1) Preparation of Avidin-Immobilized Membrane and Avidin-Immobilized Strip A neutral avidin solution (concentration 2 mg/mL) and an anti-mouse IgG antibody solution were applied linearly (application amount 1 μL/cm) to a nitrocellulose membrane backed with a PET film, and then dried to prepare an avidin-immobilized membrane having a test area (avidin-immobilized area) and a control area (anti-mouse IgG antibody-immobilized area). Multiple types of avidin-immobilized membranes were prepared by varying the manufacturer and grade of nitrocellulose membrane. Then, a sample pad was attached to the end of the test area (avidin-immobilized area) of the avidin-immobilized membrane, and an absorption band was attached to the end of the control area (anti-mouse IgG antibody-immobilized area) with a backing sheet to prepare a strip. This is referred to herein as an "avidin-immobilized strip." Multiple types of avidin-immobilized strips were prepared by varying the manufacturer and grade of nitrocellulose membrane.

(2) Preparation of Type A Antibody Immobilized Membrane and Type A Antibody Immobilized Strip An anti-type A influenza virus monoclonal antibody (concentration 2 mg/mL) and an anti-mouse IgG antibody solution were applied linearly (application amount 1 μL/cm) to a nitrocellulose membrane backed with a PET film, and then dried to prepare a Type A antibody immobilized membrane having a determination section (antibody immobilized section) and a control section (anti-mouse IgG antibody immobilized section). A sample pad was then attached to the end of the determination section (antibody immobilized section) of the Type A antibody immobilized membrane, and an absorption band was attached to the end of the control section (anti-mouse IgG antibody immobilized section) with a backing sheet to prepare a strip. This is referred to herein as a "Type A antibody immobilized strip." Several types of Type A antibody immobilized strips were prepared by changing the manufacturer and grade of the nitrocellulose membrane. This immunochromatographic strip is a comparative example because it does not have an avidin immobilization region.

(3) Preparation of biotin-conjugated type A antibody solution The anti-type A influenza virus monoclonal antibody solution used in Example 1 (2) was mixed with the N-hydroxysuccinimide-biotin solution, and then the mixture was reacted overnight at 4° C. The reaction solution was then dialyzed against 50 mM phosphate buffer (pH 7.4), and the resulting solution was used as a biotin-conjugated type A antibody solution.

(4) Preparation of biotin-conjugated type A antibody-immobilized particles The biotin-conjugated type A antibody obtained in (3) was bound to white polystyrene particles, suspended in a buffer solution, and thoroughly dispersed by ultrasonic treatment to obtain biotin-conjugated anti-type A influenza virus monoclonal antibody-immobilized white polystyrene particles. These are referred to herein as "biotin-conjugated type A antibody-immobilized particles." Several types of biotin-conjugated type A antibody-immobilized particles were prepared by varying the particle size of the white polystyrene particles.

(5) Preparation of Type A Antibody-Immobilized Colored Particles An anti-type A influenza virus monoclonal antibody, different from the monoclonal antibody bound to biotin in (3), was bound to colored polystyrene particles, suspended in a buffer solution, and thoroughly dispersed by ultrasonic treatment to obtain anti-type A influenza virus monoclonal antibody-bound colored polystyrene particles. These are referred to herein as "type A antibody-immobilized colored particles." Several types of type A antibody-immobilized colored particles were prepared by varying the particle size of the colored polystyrene particles.

(6) Application and drying of biotin-conjugated antibody A-immobilized particles The biotin-conjugated antibody A-immobilized particles prepared in (4) were applied to a polystyrene nonwoven fabric (application amount 8 μL/cm) and thoroughly dried in a hot air dryer. This is referred to herein as a "biotin-conjugated antibody A-immobilized particle pad." Using multiple types of biotin-conjugated antibody A-immobilized particles with different particle sizes of the white polystyrene particles prepared in (4), multiple types of biotin-conjugated antibody A-immobilized particle pads were prepared in varying amounts.

(7) Application and drying of type A antibody-immobilized colored particles The type A antibody-immobilized colored particles prepared in (5) were applied to a polystyrene nonwoven fabric (application amount 8 μL/cm) and thoroughly dried in a hot air dryer. These are referred to herein as "type A labeled substance pads." Several types of type A labeled substance pads were prepared by varying the particle size of the colored polystyrene particles prepared in (5).

(8) Application and Drying of Biotin-Conjugated Type A Antibody Immobilized Particles and Type A Antibody Immobilized Colored Particles The biotin-conjugated Type A antibody immobilized particles and Type A antibody immobilized colored particles prepared in (4) and (5) were mixed, and the mixture was applied to a polystyrene nonwoven fabric (application amount 8 μL/cm) and thoroughly dried in a hot air dryer. This is referred to as an "A-type mixed labeled pad" in this specification. Several types of A-type mixed labeled pads were prepared by changing the mixing ratio and amount.

(9) Preparation of Type A Test Device The avidin-immobilized membrane prepared in (1) and the A-type mixed label pad prepared in (8) were laminated with other components (backing sheet, absorption band, sample pad) and cut to a width of 5 mm, which was then placed in a plastic molded case to prepare a test device. This device is referred to herein as "Type A test device (i)." The A-type antibody-immobilized membrane prepared in (2) and the A-type label pad prepared in (7) were laminated with other components (backing sheet, absorption band, sample pad) and cut to a width of 5 mm, which was then placed in a plastic molded case to prepare a test device. This device is referred to herein as "Type A test device (ii) (control)." The avidin-immobilized membrane prepared in (1), the biotin-bound A-type antibody-immobilized particle pad prepared in (6), and the A-type label pad prepared in (7) were laminated with other components (backing sheet, absorption band, sample pad) and cut to a width of 5 mm, which was then placed in a plastic molded case to prepare a test device. In this specification, this will be referred to as "Type A test device (iii)." Several types of Type A test devices were produced by changing the bonding order. The members used in Type A test devices (i) to (iii) were the same as those produced in (1) to (8), and several types of Type A test devices were produced for each.

Example 2. Test device of the present invention for detecting influenza B virus antigen (1) Preparation of avidin-immobilized membrane and avidin-immobilized strip Same as Example 1 (1).

(2) Preparation of Type B Antibody Immobilized Membrane and Type B Antibody Immobilized Strip Anti-type B influenza virus monoclonal antibody (concentration 2 mg/mL) and anti-mouse IgG antibody solution were applied linearly to a nitrocellulose membrane backed with a PET film (coating amount 1 μL/cm), and then dried to prepare a Type B antibody immobilized membrane having a determination section (antibody immobilized section) and a control section (anti-mouse IgG antibody immobilized section). A sample pad was then attached to the end of the determination section (antibody immobilized section) of the Type B antibody immobilized membrane, and an absorption band was attached to the end of the control section (anti-mouse IgG antibody immobilized section) with a backing sheet to prepare a strip. This is referred to herein as a "Type B antibody immobilized strip." Several types of Type B antibody immobilized strips were prepared by changing the manufacturer and grade of nitrocellulose membrane.

(3) Preparation of biotin-conjugated type B antibody solution The anti-type B influenza virus monoclonal antibody solution used in Example 2 (2) was mixed with the N-hydroxysuccinimide-biotin solution, and then the mixture was reacted overnight at 4° C. The reaction solution was then dialyzed against 50 mM phosphate buffer (pH 7.4), and the resulting solution was used as a biotin-conjugated type B antibody solution.

(4) Preparation of biotin-conjugated B antibody-immobilized particles The biotin-conjugated B antibody obtained in (3) was bound to white polystyrene particles, suspended in a buffer solution, and thoroughly dispersed by ultrasonic treatment to obtain biotin-conjugated anti-B influenza virus monoclonal antibody-immobilized white polystyrene particles. These are referred to herein as "biotin-conjugated B antibody-immobilized particles." Several types of biotin-conjugated B antibody-immobilized particles were prepared by varying the particle size of the white polystyrene particles.

(5) Preparation of Type B Antibody-Immobilized Colored Particles An anti-type B influenza virus monoclonal antibody, different from the monoclonal antibody bound to biotin in (3), was bound to colored polystyrene particles, suspended in a buffer solution, and thoroughly dispersed by ultrasonic treatment to obtain anti-type B influenza virus monoclonal antibody-bound colored polystyrene particles. These are referred to herein as "type B antibody-immobilized colored particles." Several types of type B antibody-immobilized colored particles were prepared by varying the particle size of the colored polystyrene particles.

(6) Application and drying of biotin-conjugated antibody B immobilized particles The biotin-conjugated antibody B immobilized particles prepared in (4) were applied to a polystyrene nonwoven fabric and thoroughly dried in a hot air dryer. This is referred to herein as a "biotin-conjugated antibody B immobilized particle pad." Using multiple types of biotin-conjugated antibody B immobilized particles with different particle sizes of the white polystyrene particles prepared in (4), and varying the amounts, multiple types of biotin-conjugated antibody B immobilized particle pads were prepared.

(7) Application and drying of type B antibody-immobilized colored particles The type B antibody-immobilized colored particles prepared in (5) were applied to a polystyrene nonwoven fabric (application amount 8 μL/cm) and thoroughly dried in a hot air dryer. These are referred to as "type B labeled substance pads" in this specification. Several types of type B labeled substance pads were prepared by varying the particle size of the colored polystyrene particles prepared in (5).

(8) Application and Drying of Biotin-Conjugated Antibody B-Immobilized Particles and Antibody B-Immobilized Colored Particles The biotin-conjugated antibody B-immobilized particles and antibody B-immobilized colored particles prepared in (4) and (5) were mixed, and the mixture was applied to a polystyrene nonwoven fabric (application amount: 8 μL/cm) and thoroughly dried in a hot air dryer. This is referred to as a "B-mixed labeled pad" in this specification. Several types of B-mixed labeled pads were prepared by varying the mixing ratio and amount.

(9) Preparation of Type B Test Device The avidin-immobilized membrane prepared in (1) and the B-type mixed label pad prepared in (8) were laminated with other components (backing sheet, absorption band, sample pad) and cut to a width of 5 mm, which was then placed in a plastic molded case to prepare a test device. This device is referred to herein as "Type B test device (i)." The B-type antibody-immobilized membrane prepared in (2) and the B-type label pad prepared in (7) were laminated with other components (backing sheet, absorption band, sample pad) and cut to a width of 5 mm, which was then placed in a plastic molded case to prepare a test device. This device is referred to herein as "Type B test device (ii) (control)." The avidin-immobilized membrane prepared in (1), the biotin-bound B-type antibody-immobilized particle pad prepared in (6), and the B-type label pad prepared in (7) were laminated with other components (backing sheet, absorption band, sample pad) and cut to a width of 5 mm, which was then placed in a plastic molded case to prepare a test device. In this specification, this will be referred to as "B-type test device (iii)." Several types of B-type test device (iii) were produced by changing the bonding order. The members used in B-type test devices (i) to (iii) were the same as those produced in (1) to (8), and several types of B-type test devices were produced for each.

Example 3 Test Device of the Present Invention for Detecting Influenza A and B Virus Antigens (1) Preparation of Avidin-Immobilized Membrane and Avidin-Immobilized Strip Same as Example 1 (1).

(2) Preparation of Type A and Type B Antibody Immobilized Membranes and Type A and Type B Antibody Immobilized Strips A linear coating of anti-type A influenza virus monoclonal antibody, anti-type B influenza virus monoclonal antibody, and anti-mouse IgG antibody solution was applied to a nitrocellulose membrane backed with a PET film (coating amount 1 μL/cm), and then dried to prepare Type A and Type B antibody immobilized membranes having a determination section (antibody A immobilized section and type B antibody immobilized section) and a control section (anti-mouse IgG antibody immobilized section). Then, a sample pad was attached to the end of the determination section (antibody A immobilized section and type B antibody immobilized section) of the Type A and Type B antibody immobilized membrane, and an absorption band was attached to the end of the control section (anti-mouse IgG antibody immobilized section) with a backing sheet to prepare a strip. Herein, these are referred to as Type A and Type B antibody immobilized strips. By changing the manufacturer and grade of nitrocellulose membrane, several types of Type A and Type B antibody immobilized strips were prepared.

(3) Application and drying of biotin-conjugated antibody A-immobilized particles and biotin-conjugated antibody B-immobilized particles The biotin-conjugated antibody A-immobilized particles prepared in Example 1 (4) and the biotin-conjugated antibody B-immobilized particles prepared in Example 2 (4) were mixed, and the mixture was applied to a polystyrene nonwoven fabric (application amount 8 μL/cm) and thoroughly dried in a hot air dryer. In this specification, these are referred to as "biotin-conjugated antibody A-immobilized particles" and "biotin-conjugated antibody B-immobilized particle mixed pad."

(4) Application and Drying of Antibody A-Immobilized Colored Particles and Type B Antibody-Immobilized Colored Particles The antibody A-immobilized colored particles prepared in Example 1 (5) and the type B antibody-immobilized colored particles prepared in Example 2 (5) were mixed, and the mixture was applied to a polystyrene nonwoven fabric (application amount 8 μL/cm) and thoroughly dried in a hot air dryer. In this specification, this is referred to as a "type A and type B labeled substance pad."

(5) Application and Drying of Biotin-Conjugated Antibody A Immobilized Particles, Biotin-Conjugated Antibody B Immobilized Particles, Antibody A Immobilized Colored Particles, and Antibody B Immobilized Colored Particles The biotin-conjugated antibody A immobilized particles prepared in Example 1(4) and the biotin-conjugated antibody B immobilized particles prepared in Example 2(4), and the antibody A immobilized colored particles prepared in Example 1(5) and the antibody B immobilized colored particles prepared in Example 2(5) were mixed, and the mixture was applied to a polystyrene nonwoven fabric (application amount 8 μL/cm) and thoroughly dried in a warm air dryer. This is referred to herein as a "A- and B-type mixed labeled body pad."

(6) Preparation of Type A and Type B Simultaneous Detection Test Device The avidin-immobilized membrane prepared in (1) and the Type A and Type B mixed label pad prepared in (5) were bonded to other components (backing sheet, absorption band, sample pad), cut to a width of 5 mm, and placed in a plastic molded case to prepare a test device. This is referred to herein as "Type A and Type B Simultaneous Detection Test Device (i)." The Type A and Type B antibody-immobilized membrane prepared in (2) and the Type A and Type B label pad prepared in (4) were bonded to other components (backing sheet, absorption band, sample pad), cut to a width of 5 mm, and placed in a plastic molded case to prepare a test device. This is referred to herein as "Type A and Type B Test Device (ii) (Control)." The avidin-immobilized membrane prepared in (1), the biotin-bound type A antibody immobilized particle and biotin-bound type B antibody immobilized particle mixed pad prepared in (3), and the type A and type B labeled body pad prepared in (4) were bonded together with other components (backing sheet, absorption band, sample pad), cut to a width of 5 mm, and placed in a plastic molded case to prepare a test device. This is referred to herein as the "type A and type B simultaneous detection test device (iii)." Several types of type A and type B simultaneous detection test device (iii) were prepared by changing the bonding order.

Example 4. Measurement using the test device of the present invention.
(1) Detection of Influenza A Virus Influenza A virus antigen was added to a solution containing a mixture of biotin-conjugated antibody A-immobilized particles (final concentration: 0.00025 w/v%) prepared in Example 1(4) and antibody A-immobilized colored particles (final concentration: 0.002 w/v%) prepared in Example 1(5), and the sample pad side of the avidin-immobilized strip prepared in Example 1(1) was contacted with the solution. On the other hand, as a negative control, the sample pad side of the avidin-immobilized strip was contacted with a mixed solution containing no influenza A virus antigen. After the mixed solution developed to the absorption band of the avidin-immobilized strip by capillary action, the coloring of the test zone (avidin-immobilized zone) was observed. As a result, a clear line of antibody A-immobilized colored particles composed of colored polystyrene particles was observed in the mixed solution containing the influenza A virus antigen, while no line was observed in the test zone (avidin-immobilized zone) in the negative control containing no influenza A virus antigen. These results demonstrate that influenza A virus antigens in a mixed solution can be measured in one form using the immunochromatographic test device of the present invention.

(2) Detection of Influenza B Virus Influenza B virus antigen was added to a solution containing a mixture of biotin-conjugated antibody B immobilized particles (final concentration: 0.00025 w/v%) prepared in Example 2(4) and antibody B immobilized colored particles (final concentration: 0.003 w/v%) prepared in Example 2(5), and the sample pad side of the avidin immobilized strip prepared in Example 2(1) was contacted with the mixture. As a negative control, the sample pad side of the avidin immobilized strip was contacted with a mixed solution containing no influenza B virus antigen. After the mixed solution developed to the absorption band of the avidin immobilized strip by capillary action, the color of the test zone (avidin immobilized zone) was observed. As a result, a clear line of antibody B immobilized colored particles composed of colored polystyrene particles was observed in the mixed solution containing the influenza B virus antigen, whereas no line was observed in the test zone (avidin immobilized zone) in the negative control containing no influenza B virus antigen. These results demonstrate that influenza B virus antigens in a mixed solution can be measured in one form using the immunochromatographic test device of the present invention.

(3) Simultaneous Detection of Influenza A and B Viruses [0043] Influenza A virus antigen or influenza B virus antigen was added to a solution obtained by mixing the biotin-conjugated antibody A-immobilized particles prepared in Example 1(4), the biotin-conjugated antibody B-immobilized particles prepared in Example 2(4) (final concentration: 0.00025 w/v%), the antibody A-immobilized colored particles (red) prepared in Example 1(5) (final concentration: 0.002 w/v%), and the antibody B-immobilized colored particles (blue) prepared in Example 2(5) (final concentration: 0.003 w/v%). The sample pad side of the avidin-immobilized strip prepared in Example 1(1) was then contacted with the mixed solution containing no influenza virus antigen as a negative control. The mixed solution was allowed to develop by capillary action up to the absorption band of the avidin-immobilized strip, and the coloring of the test zone (avidin-immobilized zone) was observed. As a result, a clear line of type A antibody-immobilized colored particles made of red colored polystyrene particles was observed in the mixed solution containing influenza A virus antigen, and a clear line of type B antibody-immobilized colored particles made of blue colored polystyrene particles was observed in the mixed solution containing influenza B virus antigen. On the other hand, no line was observed in the test zone (avidin-immobilized zone) for the negative control containing no influenza virus antigen. These results demonstrate that influenza A virus antigen and influenza B virus antigen in a mixed solution can be simultaneously measured using a single form of the immunochromatographic test device of the present invention.

(4) Comparison with Conventional Methods for Detection of Influenza A Virus Influenza A virus antigens prepared at various dilutions were added to a solution obtained by mixing the biotin-conjugated antibody A-immobilized particles (final concentration: 0.00025 w/v % to 0.00725 w/v %) prepared in Example 1(4) with the antibody A-immobilized colored particles (final concentration: 0.002 w/v %) prepared in Example 1(5), and the sample pad side of the avidin-immobilized strip prepared in Example 1(1) was brought into contact with the solution. After leaving the solution to stand for 5 minutes, the coloring of the test zone (avidin-immobilized zone) was observed, and the presence of color was judged as +, and the absence of color was judged as -. This method is referred to in this section as the "method of the present invention."

Separately, influenza A virus antigen prepared at different dilutions in the same manner as above was added to the solution of type A antibody-immobilized colored particles prepared in Example 1.(5), and the sample pad side of the type A antibody-immobilized strip prepared in Example 1.(2) was brought into contact with the solution. After leaving the solution to stand for 5 minutes, the coloring of the test area (antibody-immobilized area) was observed, and the presence of color was judged as +, and the absence of color was judged as -. This method is referred to in this section as "Conventional Method 1."

Furthermore, influenza A virus antigen prepared at different dilutions in the same manner as above was added to a solution obtained by mixing the biotin-conjugated type A antibody solution prepared in Example 1.(3) with the type A antibody-immobilized colored particles prepared in Example 1.(5), and the sample pad side of the avidin-immobilized strip prepared in Example 1.(1) was brought into contact with the strip. After leaving the strip to stand for 5 minutes, the coloring of the test area (avidin-immobilized area) was observed, and the presence of color was judged as +, and the absence of color was judged as -. This method is referred to in this section as "Conventional Method 2."

Using the method of the present invention, conventional method 1, and conventional method 2, the minimum detectable dilution ratio for influenza A virus antigen was compared by changing the membrane type and white latex particle size. The results are summarized in the table below.

For both membrane types, the method of the present invention resulted in a higher minimum detectable dilution factor for influenza A virus antigen compared to conventional methods 1 and 2. It was also confirmed that the minimum detectable dilution factor increased in proportion to the particle size of the white polystyrene particles used.

Furthermore, in general immunochromatography, the detection sensitivity of the substance being measured tends to decrease as the membrane flow rate increases, i.e., as the membrane pore size increases; however, compared to conventional method 1, the method of the present invention showed almost no decrease in sensitivity even with a membrane with a fast flow rate.

These results demonstrate that in one embodiment using the immunochromatographic test device of the present invention, the method of the present invention has higher sensitivity than conventional methods, and that this sensitivity can be maintained even when using a membrane with a high flow rate in POCT products that require results to be determined in a shorter time.

(5) Comparison with Conventional Methods for Detection of Influenza B Virus Influenza B virus antigens prepared at various dilutions were added to a solution obtained by mixing the biotin-conjugated antibody B immobilized particles (final concentration 0.00025 to 0.00725 w/v%) prepared in Example 2(4) and the antibody B immobilized colored particles (final concentration 0.003 w/v%) prepared in Example 2(5), and the sample pad side of the avidin-immobilized strip prepared in Example 2(1) was brought into contact with the solution. After leaving the solution to stand for 5 minutes, the coloring of the test area (avidin-immobilized area) was observed, and the presence of color was judged as +, and the absence of color was judged as -. This method is referred to in this section as the "method of the present invention."

Separately, influenza B virus antigen prepared at different dilutions in the same manner as above was added to a solution of type B antibody-immobilized colored particles (final concentration 0.003 w/v%) prepared in Example 2(5), and the sample pad side of the type B antibody-immobilized strip prepared in Example 2(2) was brought into contact with the solution. After leaving the solution to stand for 5 minutes, the coloring of the test area (antibody-immobilized area) was observed, and the presence of color was judged as +, and the absence of color was judged as -. This method is referred to in this section as "Conventional Method 1."

Furthermore, influenza B virus antigen prepared at different dilutions in the same manner as above was added to a solution (final concentration 0.003 w/v%) made by mixing the biotin-conjugated type B antibody solution prepared in Example 2(3) with the type B antibody-immobilized colored particles prepared in Example 2(5), and the sample pad side of the avidin-immobilized strip prepared in Example 2(1) was brought into contact with the solution. After leaving the solution to stand for 5 minutes, the coloring of the test area (avidin-immobilized area) was observed, and the presence of color was judged as +, and the absence of color was judged as -. This method is referred to in this section as "Conventional Method 2."

Using the method of the present invention, conventional method 1, and conventional method 2, the minimum detectable dilution ratio for influenza B virus antigen was compared by changing the membrane type and white latex particle size. The results are summarized in the table below.

For both membrane types, the method of the present invention resulted in a higher minimum detectable dilution factor for influenza B virus antigens than conventional methods 1 and 2. It was also confirmed that the minimum detectable dilution factor increased in proportion to the particle size of the white polystyrene particles used.

Furthermore, in general immunochromatography, the detection sensitivity of the substance being measured tends to decrease as the membrane flow rate increases, i.e., as the membrane pore size increases; however, compared to conventional method 1, the method of the present invention showed almost no decrease in sensitivity even with a membrane with a fast flow rate.

These results demonstrate that in one embodiment using the immunochromatographic test device of the present invention, the method of the present invention has higher sensitivity than conventional methods, and that this sensitivity can be maintained even when using a membrane with a high flow rate in POCT products that require results to be determined in a shorter time.

(6) Comparison with Conventional Methods in Simultaneous Detection of Influenza A and B Viruses To a solution obtained by mixing the biotin-conjugated antibody A-immobilized particles (final concentration 0.00025 to 0.00725 w/v%) prepared in Example 1(4), the antibody A-immobilized colored particles (red) (final concentration 0.002 w/v%) prepared in Example 1(5), the biotin-conjugated antibody B-immobilized particles (final concentration 0.00025 to 0.00725 w/v%) prepared in Example 2(4), and the antibody B-immobilized colored particles (blue) (final concentration 0.003 w/v%) prepared in Example 2(5), influenza A virus antigen or influenza B virus antigen prepared at various dilutions was added, and the sample pad side of the avidin-immobilized strip prepared in Example 1(1) was brought into contact with the solution. After leaving the mixture to stand for 5 minutes, the color of the test area (avidin-immobilized area) was observed, and the presence of red coloring was rated as A+, the presence of blue coloring was rated as B+, and the absence of blue coloring was rated as -. This method is referred to as the "method of the present invention" in this section.

In addition, influenza A virus antigen or influenza B virus antigen prepared at different dilutions in the same manner as above was added to a mixed solution of type A antibody-immobilized colored particles (red) (final concentration 0.002 w/v%) prepared in Example 1(5) and type B antibody-immobilized colored particles (blue) (final concentration 0.003 w/v%) prepared in Example 2(5), and the sample pad side of the type A and type B antibody-immobilized strips prepared in Example 3(2) was brought into contact with the mixture. After leaving the mixture to stand for 5 minutes, the coloring of the test zones (antibody A immobilized zone and type B antibody immobilized zone) was observed. If the type A antibody immobilized zone was colored, it was judged as A+; if the type A antibody immobilized zone was colored, it was judged as B+; if there was no coloring, it was judged as -. This method is referred to in this section as "conventional method 1."

Using the method of the present invention, conventional method 1, and conventional method 2, the minimum detectable dilution ratios for influenza A virus antigen and influenza B virus antigen were compared by changing the membrane type and white latex particle size. The results are summarized in the table below.

For both membrane types, the method of the present invention resulted in higher minimum detectable dilution factors for influenza A virus antigen and influenza B virus antigen compared to conventional method 1. It was also confirmed that the minimum detectable dilution factor increased in proportion to the particle size of the white polystyrene particles used.

Furthermore, in general immunochromatography, the detection sensitivity of the substance being measured tends to decrease as the membrane flow rate increases, i.e., as the membrane pore size increases; however, compared to conventional method 1, the method of the present invention showed almost no decrease in sensitivity even with a membrane with a fast flow rate.

These results demonstrate that, in one embodiment using the immunochromatographic test device of the present invention, the method of the present invention has higher sensitivity than conventional methods, and that this sensitivity can be maintained even when using a membrane with a high flow rate in POCT products that require shorter determination times. Furthermore, by changing the color of the colored polystyrene particles used, it is possible to distinguish and measure two types of antigens using just one determination area (avidin immobilization area).

Example 4. Measurement using the test device of the present invention.
(1) Detection of Influenza A Virus Example 1. A test solution prepared by adding influenza A virus antigen to an antigen dilution solution containing a buffer, surfactant, etc. was added dropwise to the various types of type A test devices prepared in (9). As a negative control, an antigen dilution solution without influenza A virus antigen was added dropwise to the type A test device. After the solution developed by capillary action to the absorption band of the avidin-immobilized strip, the color of the test zone (avidin-immobilized zone) was observed. The results are summarized in the table below.

In all of the groups containing influenza A virus antigen, coloring was observed in the test area (avidin-immobilized area), while in the non-additive group containing influenza A virus antigen, no coloring was observed in the test area (avidin-immobilized area). Furthermore, even when the biotin-bound antibody A-immobilized particles and the antibody A-immobilized colored particles were placed on separate pads and their order was reversed, no difference was observed compared to the mixed type A test device (i). These results demonstrate that influenza A virus antigen in a test liquid can be measured in one form of the immunochromatographic test device of the present invention, which is equipped with pads on which biotin-bound antibody A-immobilized particles and antibody A-immobilized colored particles have been dried.

(2) Detection of Influenza B Virus Example 2. A test solution prepared by adding influenza B virus antigen to an antigen dilution solution containing a buffer, surfactant, etc. was added dropwise to the various types of type B test devices prepared in (9). As a negative control, an antigen dilution solution without influenza B virus antigen was added dropwise to the type B test device. After the solution developed by capillary action to the absorption band of the avidin-immobilized strip, the color of the test area (avidin-immobilized area) was observed. The results are summarized in the table below.

In all of the groups containing influenza B virus antigen, coloring was observed in the test area (avidin immobilized area), while in the non-addition group containing influenza B virus antigen, no coloring was observed in the test area (avidin immobilized area). Furthermore, even when the biotin-bound type A antibody immobilized particles and type A antibody immobilized colored particles were placed on separate pads and their order was reversed, no difference was observed compared to the mixed type B test device (i). These results demonstrate that influenza B virus antigen in a test solution can be measured in one form of the immunochromatographic test device of the present invention, in which biotin-bound type B antibody immobilized particles and type B antibody immobilized colored particles are mixed and dried.

(3) Simultaneous Detection of Influenza A and B Viruses Example 3. A test solution prepared by adding influenza A virus antigen or influenza B virus antigen to an antigen dilution solution containing a buffer, surfactant, etc., was added dropwise to the multiple types of simultaneous A and B detection test devices prepared in (6). Separately, as a negative control, an antigen dilution solution without influenza virus antigen was added dropwise to the simultaneous A and B detection test device. After the solution developed by capillary action to the absorption band of the avidin-immobilized strip, the color of the test zone (avidin-immobilized zone) was observed. A+ was evaluated when a red line originating from the A-antibody-immobilized colored particles made of red polystyrene particles was observed; B+ was evaluated when a blue line originating from the B-antibody-immobilized colored particles made of blue polystyrene particles was observed; and - was evaluated when no coloration was observed. The results are summarized in the table below.

In all of the groups containing influenza A virus antigen, a clear red line was observed in the test area (avidin immobilized area), while in all of the groups containing influenza B virus antigen, a clear blue line was observed in the test area (avidin immobilized area). No coloring was observed in the test area (avidin immobilized area) in the non-additive group containing influenza virus antigen. Furthermore, when biotin-conjugated type A antibody immobilized particles, biotin-conjugated type B antibody immobilized particles, and type A antibody immobilized colored particles and type B antibody immobilized colored particles were placed on separate pads, no difference was observed when the order was reversed compared to the simultaneous type A and type B detection test device (i) in which all were mixed. These results demonstrate that one form of the immunochromatographic test device of the present invention, equipped with pads on which biotin-conjugated type A antibody immobilized particles, biotin-conjugated type B antibody immobilized particles, type A antibody immobilized colored particles, and type B antibody immobilized colored particles were dried, can simultaneously measure influenza A virus antigen and influenza B virus antigen in a test solution.

(4) Comparison with Conventional Methods for Simultaneous Detection of Influenza A and B Viruses Example 3. Test solutions prepared by adding influenza A virus antigens or influenza B virus antigens prepared at various dilutions to an antigen dilution solution consisting of a buffer, surfactant, etc., and mixing them were added dropwise to the multiple types of test devices (i) and (ii) (control) for simultaneous detection of influenza A and B viruses prepared in (6). After leaving the devices to stand for 5 minutes, the coloring of the test zones (avidin immobilized zones or antibody A immobilized zones and antibody B immobilized zones) was observed. A red line resulting from the red colored particles immobilized with the antibody A was judged as A+; a blue line resulting from the blue colored particles immobilized with the antibody B was judged as B+; and no coloring was judged as -. The results are summarized in the table below.

For both membrane types, the method of the present invention resulted in higher minimum detectable dilution factors for influenza A virus antigen and influenza B virus antigen compared to conventional method 1. It was also confirmed that the minimum detectable dilution factor increased as the particle size of the white polystyrene particles used increased.

Furthermore, in general immunochromatography, the detection sensitivity of the substance being measured tends to decrease as the membrane flow rate increases, i.e., as the membrane pore size increases; however, compared to conventional method 1, the method of the present invention showed almost no decrease in sensitivity even with a membrane with a fast flow rate.

These results demonstrate that even in one form of the immunochromatographic test device of the present invention, which is equipped with pads on which biotin-conjugated antibody type A immobilized particles, biotin-conjugated antibody type B immobilized particles, and antibody type A immobilized colored particles and antibody type B immobilized colored particles have been dried, the method of the present invention has higher sensitivity than conventional methods, and that this sensitivity can be maintained even when using a membrane with a high flow rate in POCT products that require shorter determination times.Furthermore, by changing the color of the colored polystyrene particles used, it is possible to distinguish and measure two types of antigens using just one determination area (avidin immobilized area).

Example 5 Test Device for Detecting Influenza A Virus Antigen (1) Preparation of Avidin-Immobilized Microfluidic Device A neutral avidin solution was applied to an imprinted sheet, which was then dried to prepare an avidin-immobilized imprinted sheet having a test area (avidin-immobilized area).
Then, a microchannel sheet including a drop section was attached so that the channel overlapped the avidin immobilized section of the avidin-immobilized imprinted sheet, and a sample pad was attached to the drop section with adhesive tape, and an absorption band was attached to the opposite side of the drop section, thereby producing an avidin-immobilized microchannel device.

(2) Preparation of Type A Antibody-Immobilized Imprinted Sheet An anti-type A influenza virus monoclonal antibody was applied to an imprinted sheet, which was then dried to prepare a Type A antibody-immobilized imprinted sheet having a detection section (antibody-immobilized section). A microchannel sheet including a drop section was then attached to the Type A antibody-immobilized imprinted sheet so that the channel overlapped with the Type A antibody-immobilized section, and an absorption band was attached with adhesive tape to the side opposite the drop section to prepare a Type A antibody-immobilized microchannel device.

(3) Preparation of biotin-conjugated type A antibody solution The anti-type A influenza virus monoclonal antibody solution used in (2) was mixed with the N-hydroxysuccinimide-biotin solution, and then reacted overnight at 4° C. The reaction solution was then dialyzed against 50 mM phosphate buffer (pH 7.4), and the resulting solution was used as a biotin-conjugated type A antibody solution.

(4) Preparation of biotin-conjugated type A antibody-immobilized particles The biotin-conjugated type A antibody obtained in (3) was bound to white polystyrene particles, which were then suspended in a buffer solution and thoroughly dispersed by ultrasonic treatment to obtain biotin-conjugated anti-type A influenza virus monoclonal antibody-immobilized white polystyrene particles. These are referred to herein as biotin-conjugated type A antibody-immobilized particles.

(5) Preparation of Type A Antibody-Immobilized Colored Particles An anti-type A influenza virus monoclonal antibody, different from the monoclonal antibody bound to biotin in (3), was bound to colored polystyrene particles, which were then suspended in a buffer solution and thoroughly dispersed by ultrasonic treatment to obtain anti-type A influenza virus monoclonal antibody-bound colored polystyrene particles. These are referred to herein as Type A antibody-immobilized colored particles.

Example 6 Test Device for Detecting Influenza B Virus Antigen (1) Preparation of an Avidin-Immobilized Microfluidic Device The same as in Example 5 (1) was used.

(2) Preparation of Type B Antibody-Immobilized Imprinted SheetAn anti-type B influenza virus monoclonal antibody was applied to the imprinted sheet, which was then dried to prepare a Type B antibody-immobilized imprinted sheet having a determination area (antibody immobilized area).A microchannel sheet including a dropping section was then attached to the Type B antibody-immobilized imprinted sheet so that the flow channel overlapped the Type B antibody-immobilized area.A sample pad was attached to the dropping section and an absorption band was attached to the opposite side of the dropping section with adhesive tape, thereby preparing a Type B antibody-immobilized microchannel device.

(3) Preparation of biotin-conjugated type B antibody solution The anti-influenza type B virus monoclonal antibody solution used in (2) was mixed with the N-hydroxysuccinimide-biotin solution, and then reacted overnight at 4° C. The reaction solution was then dialyzed against 50 mM phosphate buffer (pH 7.4), and the resulting solution was used as a biotin-conjugated type B antibody solution.

(4) Preparation of biotin-conjugated B antibody-immobilized particles The biotin-conjugated B antibody obtained in (3) was bound to white polystyrene particles, which were then suspended in a buffer solution and thoroughly dispersed by ultrasonic treatment to obtain biotin-conjugated anti-B influenza virus monoclonal antibody-immobilized white polystyrene particles. These are referred to herein as biotin-conjugated B antibody-immobilized particles.

(5) Preparation of Type B Antibody-Immobilized Colored Particles An anti-type B influenza virus monoclonal antibody, different from the monoclonal antibody bound to biotin in (3), was bound to colored polystyrene particles, suspended in a buffer solution, and thoroughly dispersed by ultrasonic treatment to obtain anti-type B influenza virus monoclonal antibody-bound colored polystyrene particles. These are referred to herein as Type B antibody-immobilized colored particles.

Example 7. Measurement Using the Test Device of the Present Invention (1) Detection of Influenza A Virus The biotin-conjugated antibody A-immobilized particles prepared in Example 5(4) and the antibody A-immobilized colored particles prepared in Example 5(5) were mixed with a solution containing a buffer and a surfactant, and an influenza A virus antigen was added thereto. After standing for 10 minutes, the mixture was dropped onto the sample pad of the avidin-immobilized microfluidic device prepared in Example 5(1). As a negative control, a mixed solution without the influenza A virus antigen was similarly dropped onto the sample pad of the avidin-immobilized microfluidic device. After the mixed solution was absorbed by capillary action up to the absorption band of the avidin-immobilized microfluidic device, the flow channel was washed with a cleaning solution, and the color of the test zone (avidin-immobilized zone) was observed. As a result, a clear signal of the antibody A-immobilized colored particles made of colored polystyrene particles was observed for the mixed solution containing the influenza A virus antigen, while no signal was observed on the test zone (avidin-immobilized zone) for the negative control without the influenza A virus antigen. These results demonstrate that influenza A virus antigens in a mixed solution can be measured in one form using the immunochromatographic test device of the present disclosure.

(2) Detection of Influenza B Virus The biotin-conjugated antibody B immobilized particles prepared in Example 6(4) and the antibody B immobilized colored particles prepared in Example 6(5) were mixed with a solution containing a buffer and a surfactant, and the mixture was added with an influenza B virus antigen. After allowing to stand for 10 minutes, the mixture was dropped onto the sample pad of the avidin-immobilized microfluidic device prepared in Example 6(1). As a negative control, a mixed solution without the influenza B virus antigen was similarly dropped onto the sample pad of the avidin-immobilized microfluidic device. After the mixed solution was absorbed by capillary action up to the absorption band of the avidin-immobilized microfluidic device, the flow channel was washed with a cleaning solution, and the color of the test zone (avidin-immobilized zone) was observed. As a result, a clear signal of the antibody B immobilized colored particles composed of colored polystyrene particles was observed for the mixed solution containing the influenza B virus antigen, whereas no signal was observed on the test zone (avidin-immobilized zone) for the negative control without the influenza B virus antigen. These results demonstrate that influenza B virus antigens in a mixed solution can be measured in one form using the immunochromatographic test device of the present invention.

(3) Comparison with conventional methods for detecting influenza A virus The biotin-conjugated antibody A immobilized particles prepared in Example 5(4) and the antibody A immobilized colored particles prepared in Example 5(5) were mixed with a solution consisting of a buffer and a surfactant, and influenza A virus antigens prepared at various dilutions were added. After leaving the mixture to stand for 10 minutes, the mixture was dropped onto the sample pad of the avidin-immobilized microfluidic device prepared in Example 5(1). After the mixed solution was absorbed into the absorption band, the flow path was washed with a washing solution, and the coloring of the determination section (avidin immobilized section) was observed. The presence of coloring was judged as +, and the absence of coloring was judged as -.

Separately, influenza A virus antigen prepared at different dilutions in the same manner as above was added to the solution of antibody A-immobilized colored particles prepared in Example 5.(5). After leaving it to stand for 10 minutes, the mixture was dropped onto the sample pad of the antibody A-immobilized microfluidic device prepared in Example 5.(2). After the mixed solution was absorbed into the absorption band, the flow path was washed with a cleaning solution, and the coloring of the test area (avidin-immobilized area) was observed. The presence of coloring was judged as +, and the absence of coloring was judged as -. This method is referred to in this section as the conventional method.

The method of the present disclosure and the conventional method were used to compare the minimum detectable dilution ratio of influenza A virus antigen.
The results are summarized in the table below.

Compared to conventional methods, the method disclosed herein resulted in a higher minimum detectable dilution factor for influenza A virus antigens.

(4) Comparison with Conventional Methods for Detection of Influenza B Virus The biotin-conjugated type B antibody-immobilized particles prepared in Example 6 (4) and the type B antibody-immobilized colored particles prepared in Example 6 (5) were mixed with a solution consisting of a buffer and a surfactant, and type B virus antigen prepared at various dilutions was added. After leaving the mixture to stand for 10 minutes, the mixture was dropped onto the sample pad of the avidin-immobilized microfluidic device prepared in Example 6 (1). After the mixed solution was absorbed into the absorption band, the flow path was washed with a washing solution, and the color of the determination section (avidin-immobilized section) was observed. The presence of color was judged as +, and the absence of color was judged as -.

Separately, influenza B virus antigen prepared at different dilutions in the same manner as above was added to the solution of type B antibody-immobilized colored particles prepared in Example 6 (5), and after leaving to stand for 10 minutes, the mixture was dropped onto the sample pad of the type B antibody-immobilized microfluidic device prepared in Example 6 (2). After the mixed solution was absorbed into the absorption band, the flow path was washed with a cleaning solution, and the coloring of the test area (avidin-immobilized area) was observed. The presence of color was judged as +, and the absence of color was judged as -. This method is referred to in this section as the conventional method.

The method of the present disclosure and the conventional method were used to compare the minimum detectable dilution ratio of influenza B virus antigen.
The results are summarized in the table below.
The method of the present disclosure resulted in a higher minimum detectable dilution factor for influenza B virus antigens than the conventional method.

Claims (17)

A test substance;
a biotin-labeled invisible particle having a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with the test substance immobilized thereon;
a second antibody that undergoes an antigen-antibody reaction with the test substance, the second antibody being labeled and capable of binding to the test substance simultaneously with the first antibody through an antigen-antibody reaction, or a labeled antigen-binding fragment thereof;
an avidin immobilization region in which avidin or streptavidin is immobilized;
simultaneously or sequentially contacting the label with the avidin immobilization region, and detecting the label bound to the avidin immobilization region.
A method for detecting a test substance, comprising: The method of claim 1, wherein the label of the labeled second antibody or labeled antigen-binding fragment thereof is a visible particle. The method of claim 2, wherein the visible particles are colored particles. The method of claim 1, wherein the invisible particles are white particles. The method of claim 1, wherein at least a portion of the first antibody or antigen-binding fragment thereof immobilized on the invisible particle, or another substance immobilized on the invisible particle, is biotin-labeled. A plurality of test substances;
a plurality of types of invisible particles, each of which has a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with each of the plurality of test substances immobilized thereon and is biotin-labeled;
a second antibody capable of distinguishing between the types of the plurality of test substances and undergoing an antigen-antibody reaction with each of the plurality of test substances, the second antibody being capable of binding to the test substances simultaneously with the first antibody through an antigen-antibody reaction; and
an avidin immobilization region in which avidin or streptavidin is immobilized;
simultaneously or sequentially contacting the label with the avidin immobilization region, and detecting the label bound to the avidin immobilization region.
A method for detecting a plurality of test substances, comprising: The method of claim 6, wherein the label of the labeled second antibody or labeled antigen-binding fragment thereof is a visible particle. The method of claim 7, wherein the visible particles are colored particles, each having a different color for each of the multiple types of test substances. The method of claim 2 or 7, wherein the visible particles and the invisible particles are latex particles. a carrier having an avidin immobilization region where avidin or streptavidin is immobilized;
a biotin-labeled invisible particle having a first antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with a test substance immobilized thereon;
A detection kit comprising a second antibody that undergoes an antigen-antibody reaction with the test substance, and a labeled second antibody or a labeled antigen-binding fragment thereof that is capable of binding to the test substance through an antigen-antibody reaction simultaneously with the first antibody. The detection kit of claim 10, wherein the label of the labeled second antibody or labeled antigen-binding fragment thereof is a visible particle. The detection kit of claim 10, comprising multiple types of invisible particles and multiple types of labeled second antibodies or labeled antigen-binding fragments thereof, each corresponding to a respective one of multiple test substances. The detection kit of claim 12, wherein the label of the labeled second antibody or labeled antigen-binding fragment thereof is a visible particle. The detection kit of claim 13, wherein the visible particles are colored particles, each having a different color for each of the plurality of test substances. The detection kit described in any one of claims 11, 13, and 14, wherein the visible particles and the invisible particles are latex particles. Invisible particles that are biotin-labeled and have a first antibody or its antigen-binding fragment immobilized thereon, which undergoes an antigen-antibody reaction with the test substance. The particles described in claim 16, wherein the invisible particles include latex particles.