Method for improving visibility of test line in immunochromatography

Abstract

To provide a method for improving the visibility of a test line in immunochromatography.SOLUTION: Forming a complex of a detection target substance capturing substance, a detection target substance, and a substance that specifically binds to the labeled detection target substance on the test line on the instrument on which the detection target substance capturing substance is solid-phased, using a substance that specifically binds to a detection target substance labeled with a labeling substance that is a colored particle; An immunochromatographic measurement instrument for detecting a substance to be detected by coloring a test line with a labeling substance, wherein the test line contains at least one compound selected from the group consisting of sodium cholate, CHAPS, digitonin, non-surface-active sulfobetaine (NDSB), guanidine thiocyanate, a copolymer containing a 2-methacryloyloxyethylphosphocholine polymer as a monomer unit, and ethanol.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for preventing uneven color development or blurring of a test line for observing color development in immunochromatography, and for improving the visibility of the coloration of the test line. [Background technology]

[0002] In immunochromatography, a capture reagent for the analyte, such as an antibody, capable of capturing the analyte to be measured is immobilized on a test line on an immunochromatography measuring device, and the analyte is captured by the capture reagent for the analyte. Furthermore, a substance that binds to the analyte, such as an antibody, labeled with colored particles, such as gold colloids, is bound to the analyte, and the colored particles are accumulated on the test line. The color of the colored particles is visually recognized, and the analyte is detected (Patent Document 1).

[0003] In this case, the test line may become unevenly colored or blurred, which may impair its visibility. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2019-012912 Summary of the Invention [Problem to be solved by the invention]

[0005] In immunochromatography, there was a problem of uneven color development and blurring of the test line, which impaired the visibility of the test line.

[0006] An object of the present invention is to provide a method for improving the visibility of a test line in immunochromatography. [Means for solving the problem]

[0007] The present inventors have discovered that by adding any of the following to the immunochromatographic test line: sodium cholate, CHAPS, digitonin, non-surfactant sulfobetaine (NDSB), guanidine thiocyanate, copolymers containing 2-methacryloyloxyethylphosphocholine polymer as a monomer unit (Lipidure BL-402, Lipidure BL-405), or ethanol, the occurrence of uneven color development and blurring of the test line can be suppressed and the visibility of the test line can be improved, leading to the completion of the present invention.

[0008] That is, the present invention is as follows. [1] An immunochromatographic measuring instrument that includes a membrane having a test line on which a detectable substance capture substance capable of capturing the detectable substance is immobilized, and that uses a substance that specifically binds to the detectable substance labeled with a labeling substance that is a colored particle to form a complex of the detectable substance capture substance-detectable substance-substance that specifically binds to the labeled detectable substance on the test line on the instrument on which the detectable substance capture substance is immobilized, thereby detecting the detectable substance by coloring the test line with the labeling substance, wherein the test line contains at least one compound selected from the group consisting of sodium cholate, CHAPS, digitonin, non-detergent sulfobetaine (NDSB), guanidine thiocyanate, a copolymer containing 2-methacryloyloxyethylphosphocholine polymer as a monomer unit, and ethanol. [2] The immunochromatography measuring device according to [1], wherein the substance that captures the analyte and the substance that specifically binds to the analyte are antibodies. [3] The immunochromatography measurement device according to [1], wherein the copolymer containing 2-methacryloyloxyethylphosphocholine polymer as a monomer unit is Lipidure (registered trademark) BL-402 or Lipidure (registered trademark) BL-405. [4] An immunochromatographic measurement method that includes a membrane having a test line on which a detectable substance capture substance capable of capturing the detectable substance is immobilized, and that uses a substance that specifically binds to the detectable substance labeled with a labeling substance that is a colored particle to form a complex of the detectable substance capture substance-detectable substance-substance that specifically binds to the labeled detectable substance on the test line on the instrument on which the detectable substance capture substance is immobilized, and detects the detectable substance by coloring the test line with the labeling substance, characterized in that the visibility of the coloring on the test line is improved by including in the test line at least one compound selected from the group consisting of sodium cholate, CHAPS, digitonin, non-detergent sulfobetaine (NDSB), guanidine thiocyanate, a copolymer containing 2-methacryloyloxyethylphosphocholine polymer as a monomer unit, and ethanol. [5] The immunochromatographic measurement method according to [4], wherein the substance that captures the analyte and the substance that specifically binds to the analyte are antibodies. [6] The immunochromatographic measurement method according to [4], wherein the copolymer containing 2-methacryloyloxyethylphosphocholine polymer as a monomer unit is Lipidure (registered trademark) BL-402 or Lipidure (registered trademark) BL-405. [Effects of the Invention]

[0009] The method of the present invention can prevent uneven color development or blurring of the test line used to observe color development in immunochromatography, thereby improving the visibility of the coloration on the test line. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described in detail below. The present invention is a method for immunochromatography (also called immunochromatography method) for detecting a substance to be detected, in which a specific compound is contained in the test line, which is the detection area on a membrane, which is a membrane carrier, thereby preventing uneven color development and blurring in the test line and improving the visibility of the coloration in the test line, thereby increasing the detection sensitivity.

[0011] Immunochromatography can be performed using an immunochromatography measuring instrument that includes a support having a test line (detection region) on which an analyte capture substance such as an antibody that captures an analyte such as an antigen (called antibody 1 in the case of an antibody), a label region containing a substance that specifically binds to the analyte such as a mobile antibody labeled with an appropriate labeling substance such as colored polystyrene particles or gold colloid (called antibody 2 in the case of an antibody), a sample pad onto which a sample liquid that may contain the analyte is added dropwise, and an absorption band that absorbs the sample liquid developed on the support. In this method, a liquid containing the prepared analyte is added dropwise to the sample pad, and capillary action is used to develop and migrate a complex of the analyte and a substance that specifically binds to the analyte (labeled reagent) labeled with an appropriate labeling substance such as colored polystyrene particles or gold colloid onto the support on which the analyte capture substance that captures the analyte is immobilized. As a result, a complex consisting of the immobilized analyte capture substance, the analyte, and a substance that specifically binds to the labeled analyte is formed on the test line on the support. The analyte can be detected by detecting the signal of the labeling substance emitted from the complex due to coloring with the labeling reagent (in the case of gold colloid, the test line on the support where the substance capable of binding to the analyte antigen is immobilized turns red). The analyte is an antigen or an antibody. When the analyte is an antigen, the analyte capture substance that captures the analyte is an antibody (antibody 1), and the substance that binds to the analyte and specifically binds to the analyte is a mobile substance labeled with an appropriate labeling substance, such as colored polystyrene particles or gold colloid, is also an antibody (antibody 2). When the analyte is an antibody, the analyte capture substance that captures the antibody analyte and the substance that specifically binds to the analyte can be an antibody against the antibody analyte, or an antigen that binds to the antibody analyte.

[0012] Immunochromatography measurement devices are described in more detail below. The following description focuses on immunochromatography measurement devices that detect antigens using antibodies (substances that capture analytes) immobilized on a support and labeled antibodies (substances that specifically bind to analytes). Note that immunochromatography measurement devices are also sometimes called immunochromatographic test pieces or immunochromatographic measurement strips.

[0013] The support is a base material capable of immobilizing antibodies that bind to and capture the target substance (antigen), and does not impede the horizontal flow of liquid. It is preferably a porous thin film with capillary action, capable of transporting liquid and its dispersed components by absorption. The material forming the support is not particularly limited, and examples include cellulose, nitrocellulose, cellulose acetate, polyvinylidene difluoride (PVDF), glass fiber, nylon, and polyketone. Of these, a thin film made of nitrocellulose is more preferred. A membrane with an immobilized antibody is called an antibody solid-phase membrane or antibody-immobilized membrane. The support is also called a carrier.

[0014] The label region includes a label that binds to the analyte. Here, the label refers to a second analyte capture substance, such as a mobile antibody labeled with a label. The label is preferably a labeled antibody. The label used to label the analyte can be selected from the group consisting of gold colloid, platinum colloid, colored polystyrene particles (also called colored latex particles), fluorescent polystyrene particles, colored magnetic particles, colored silica particles, fluorescent silica particles, colored cellulose particles, fluorescent cellulose particles, fluorescent dye labels, and enzyme labels, or a combination of two or more of these. Labeling can be performed by known methods.

[0015] The test line (detection area) refers to a region on a support where an antibody that binds to and captures the substance to be detected (antigen) is immobilized. The detection area is a region where an antibody for capturing the antigen is immobilized, and at least one detection area must be provided on the support. The test line may be included on the support, and the antibody may be immobilized on the support.

[0016] The number of test lines and the type of labeled antibody contained in the labeled region are not limited to one; by using antibodies corresponding to multiple objects to be measured, two or more antigens can be detected using the same immunochromatography measurement device.

[0017] The sample pad is a porous material and is used to supply a specimen sample that may contain a substance to be detected. Commonly used materials such as filter paper, glass fiber, and nonwoven fabric can be used for this purpose. In order to use a large amount of specimen sample in an immunoassay, a pad with a thickness of approximately 0.2 mm to 5 mm is preferable. The specimen may also include a sample prepared using the specimen, such as a sample obtained by suspending the specimen in another solution. The specimen sample may be supplied by, for example, dropwise addition. The sample pad is also called the specimen supplying portion.

[0018] The absorption band is a component for absorbing the sample developed on the support and the labeled substance that did not participate in the reaction. The material can be a filter paper or sponge made of a general natural or synthetic polymer compound with high water retention, but a highly water-absorbent material is preferred to promote the development of the sample. The absorption band is also called a sample absorption pad.

[0019] The backing sheet is a component for attaching and fixing all of the aforementioned materials, i.e., the support, sample pad, label region, absorption band, etc., with partial overlap. The backing sheet is not necessarily required as long as these materials are arranged and fixed at optimal intervals, but it is generally preferable to use one for convenience in manufacturing and use. The backing sheet is preferably made of plastic.

[0020] The immunochromatography measuring device of the present invention may further include a control indicator. The control indicator is a portion that indicates that the test has been performed correctly. For example, the control indicator is located downstream of the test line and emits a signal by coloring or the like when the sample passes through the test line and reaches the control indicator. The control indicator may have immobilized thereon a substance that binds to the label, or a reagent such as a pH indicator that changes color when the sample reaches the control indicator. When the antibody to which the label is bound is a mouse monoclonal antibody, an anti-mouse IgG antibody may be used.

[0021] In the immunochromatography measurement device of the above type, the sample passes through a porous flow path formed by a series of connections of the sample pad, label region, support, test line, absorption band, etc. Therefore, in this type, all of these constitute the sample migration region.

[0022] The size of the immunochromatography measuring instrument is not limited, but is, for example, about several centimeters to a dozen centimeters in length and several millimeters to several centimeters in width.

[0023] The entire immunochromatographic measurement device may be covered with a clear plastic laminate.

[0024] Immunochromatographic measurement devices are also called immunochromatographic test pieces or immunochromatographic test strips. The immunochromatographic measurement device may be placed in a container, which is called a test device case.

[0025] Measurement of an analyte using an immunochromatography measurement device can be carried out at 5 to 35°C, preferably at room temperature, and pretreatment with a sample treatment solution may also be carried out within this temperature range.

[0026] When manufacturing the immunochromatography measurement device, a target substance capture substance, such as an antibody for capturing the target substance, is immobilized on a test line on a support. Methods for immobilizing a target substance capture substance on a support include known methods such as physical adsorption or chemical bonding. Specifically, a solution containing the capture substance is applied to the support and dried to immobilize the capture substance and form a test line. The capture substance is immobilized on the support in a linear shape to form the test line, but the shape of the test line is not limited to a linear shape and can be either circular or rectangular. In the present invention, the site on the support where the capture substance is immobilized is referred to as the test line, regardless of its shape.

[0027] In the present invention, the test line on the support is formed by the addition of sodium cholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate), digitonin (C 56 H 92 O 29The test line contains at least one compound selected from the group consisting of ethanol, non-surfactant sulfobetaine (NDSB), guanidine thiocyanate, copolymers containing 2-methacryloyloxyethylphosphocholine (MPC) polymer as a monomer unit (Lipidure® BL-402, Lipidure® BL-405), and ethanol. The test line may contain multiple types of these compounds, for example, two, three, four, five, six, or seven types. These compounds are called solid-phase addition reagents. The amount of the above compound per immunochromatography measurement device is, for example, 0.0275 to 5.5 (nl), preferably 0.0275 to 2.75 (nl), in the case of a copolymer containing 2-methacryloyloxyethylphosphocholine (MPC) polymer as a monomer unit (Lipidure (registered trademark) BL-402, Lipidure (registered trademark) BL-405), for example, 0.055 to 5.5 (nl), preferably 0.275 to 1.1 (nl), in the case of ethanol, and is, for example, 0.055 to 5.5 (nl), preferably 0.275 to 1.1 (nl), in the case of sodium cholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate), digitonin (C 56 H 92 O 29 In the case of surfactants such as guanidine thiocyanate (NDSB), non-surfactant sulfobetaine (NDSB), and guanidine thiocyanate, the amount is, for example, 0.055 to 5.5 μg, and preferably 0.165 to 2.75 μg. If the amount exceeds the upper limit, the adsorption of the antibody to the membrane may decrease, while if the amount is below the lower limit, the color development improving effect of the additive may decrease.

[0028] In the present invention, when a substance for capturing an analyte such as an antibody is immobilized on a test line on a support, a solution containing the substance for capturing an analyte is added with sodium cholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate), digitonin (C 56 H 92 O 29The composition contains at least one compound selected from the group consisting of methylcellulose, non-surfactant sulfobetaine (NDSB), guanidine thiocyanate, copolymers containing 2-methacryloyloxyethylphosphocholine (MPC) polymer as a monomer unit (Lipidure® BL-402, Lipidure® BL-405), and ethanol. The composition may contain multiple types of these compounds, for example, two, three, four, five, six, or seven types.

[0029] The concentration of the compound added to the solution containing the analyte capture substance is, for example, 0.005 to 1.0 (v / v)%, preferably 0.005 to 0.5 (v / v)% in the case of a copolymer containing 2-methacryloyloxyethylphosphocholine (MPC) polymer as a monomer unit (Lipidure (registered trademark) BL-402, Lipidure (registered trademark) BL-405), and is, for example, 0.01 to 1.0 (v / v)%, preferably 0.05 to 0.2 (v / v)% in the case of ethanol, and is, for example, 0.01 to 1.0 (v / v)%, preferably 0.05 to 0.2 (v / v)% in the case of sodium cholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate), digitonin (C 56 H 92 O 29 In the case of non-surfactant sulfobetaine (NDSB) and guanidine thiocyanate, the concentration is, for example, 0.01 to 1.0 (w / v)%, preferably 0.03 to 0.5 (w / v)%.

[0030] The substance to be detected using the immunochromatography measurement device of the present invention is not limited, and can be any substance detected by an antigen-antibody reaction and contained in a sample such as a biological sample. For example, proteins, sugars, other compounds, or substances of biological origin can be used. The substance to be detected can also be an antibody. It can also be a complex of these, such as a pathogenic microorganism such as a bacterium or virus, or a biological substance in a living organism or a substance in the environment. Specific examples include viral antigens such as influenza virus antigen, adenovirus antigen, respiratory syncytial virus antigen, HA antigen, HBc antigen, HCV antigen, HIV antigen, EBV antigen, and NLV antigen; bacterial antigens such as Chlamydia trachomatis antigen, streptococcus antigen, Bordetella pertussis antigen, Helicobacter pylori antigen, Leptospira antigen, Treponema pallidum antigen, Toxoplasma gondii antigen, Borrelia antigen, Bacillus anthracis antigen, and MRSA antigen; mycoplasma antigens such as Mycoplasma pneumoniae; peptide hormones such as human chorionic gonadotropin; steroids such as steroid hormones; bioactive amines such as epinephrine and morphine; vitamins such as vitamin B; prostaglandins; antibiotics such as tetracycline; toxins produced by bacteria, and various tumor markers. Preferably, the method of the present invention is directed to viral antigens such as influenza virus antigen, adenovirus antigen, respiratory syncytial virus antigen, HA antigen, HBc antigen, HCV antigen, HIV antigen, EBV antigen, and NLV antigen; and bacterial antigens such as Chlamydia trachomatis antigen, Streptococcus antigen, Bordetella pertussis antigen, Helicobacter pylori antigen, Leptospira antigen, Treponema pallidum antigen, Toxoplasma gondii antigen, Borrelia antigen, Bacillus anthracis antigen, and MRSA antigen.

[0031] The test sample may be a body fluid such as blood, serum, plasma, urine, stool, saliva, tissue fluid, cerebrospinal fluid, swab, or a dilution thereof, and is preferably blood, serum, plasma, urine, stool, cerebrospinal fluid, or a dilution thereof.

[0032] The immunochromatography measurement device used in the present invention may also be a device for measuring glycan antigens in a sample. To measure glycan antigens in a sample, nitrite is mixed with acid to generate nitrous acid, the glycan antigens in the sample are extracted with the nitrous acid, the acid is neutralized, and the glycan antigens are bound to antibodies immobilized on the immunochromatography measurement device to measure the glycan antigens. In this method, the following methods (A) and (B) can be used to extract and measure glycan antigens.

[0033] (A) A sample is mixed with an acidic solution in advance and added to a sample pad of an immunochromatography measurement device that has been impregnated with nitrite and a neutralizing reagent. When the mixture reaches the area impregnated with nitrite, the nitrite reacts with the acid to generate nitrous acid, which extracts the carbohydrate antigens in the sample. The carbohydrate antigen extract is neutralized in the area on the immunochromatography measurement device that has been impregnated with a neutralizing reagent, and the carbohydrate antigens bind to the antibodies immobilized on the immunochromatography measurement device and can be detected. Examples of acidic solutions used in this method include acetic acid, hydrochloric acid, malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

[0034] (B) The sample and nitrite solution are mixed in advance and added to the sample pad of the immunochromatography measurement device, which is impregnated with an acidic reagent and a neutralizing reagent. When the mixture reaches the area impregnated with the acidic reagent, the nitrite reacts with the acid, generating nitrous acid, which extracts the carbohydrate antigens in the sample. The carbohydrate antigen extract is neutralized in the area on the immunochromatography measurement device impregnated with the neutralizing reagent, and the carbohydrate antigens bind to the antibodies immobilized on the immunochromatography measurement device, allowing them to be detected.

[0035] In either method, extraction and neutralization of carbohydrate antigens using nitrous acid are performed on the immunochromatography measurement device. To perform extraction of carbohydrate antigens using nitrous acid on the immunochromatography measurement device, an acidic reagent or nitrite may be impregnated on the immunochromatography measurement device. To perform neutralization on the immunochromatography measurement device, a neutralizing reagent may be impregnated on the immunochromatography measurement device.

[0036] In the immunochromatography measurement device of the present invention for carrying out the above-mentioned method (A) or (B), an immunochromatography measurement device in which nitrite is impregnated upstream of the label region (upstream of the sample flow, on the side where the sample pad is located), i.e., in the sample pad or between the sample pad and the label region, can be used for the above-mentioned method (A). Furthermore, an immunochromatography measurement device in which an acidic reagent is impregnated in the sample pad or between the sample pad and the label region can be used for the above-mentioned method (B). Note that a solid acidic reagent is used as the acidic reagent.

[0037] The solid acidic reagent or nitrite may be impregnated into a sample pad, or into a pad made of a porous material such as a nonwoven fabric, separate from the sample pad, and the resulting solid acidic reagent-impregnated porous material or nitrite-impregnated porous material may be placed between the sample pad and the label region, i.e., upstream of the label region. Here, the region impregnated with the solid acidic reagent or nitrite may or may not be in contact with the sample pad or the label region.

[0038] The neutralizing reagent is disposed downstream of the region impregnated with the solid acidic reagent or nitrite. The neutralizing reagent may be impregnated into a support, or a pad made of a porous material such as a nonwoven fabric other than the support, and the resulting neutralizing reagent-impregnated porous material may be disposed between the region impregnated with the solid acidic reagent or nitrite and the labeled substance region. That is, a region impregnated with the neutralizing reagent is disposed upstream of the labeled substance region, and a region impregnated with the solid acidic reagent or nitrite is disposed upstream of the region impregnated with the neutralizing reagent.

[0039] The solid acidic reagent is solid at room temperature and does not volatilize at high temperatures. Preferred solid acidic reagents include malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

[0040] Nitrite salts used in the present invention include sodium nitrite and potassium nitrite. The amount of solid acidic reagent or nitrite used, i.e., the amount impregnated into the immunochromatographic measurement device, is not particularly limited, but is typically about 0.01 μg to 1 mg, and preferably about 0.1 μg to 0.1 mg, per test piece of the immunochromatographic measurement device. However, it is preferable to select an optimal amount that will produce the desired effect depending on the type of solid acidic reagent or nitrite salt used, the composition of the sample suspension, and the amount added.

[0041] To impregnate a sample pad or porous material with a solid acidic reagent or nitrite, the solid acidic reagent or nitrite is first dissolved, applied, and then dried. For example, a 0.1 to several M solution of the solid acidic reagent or nitrite may be applied and then dried.

[0042] Neutralizing reagents are solid at room temperature and do not volatilize at high temperatures. Neutralizing reagents are also called basic reagents. Examples of preferred neutralizing reagents include Tris base (trishydroxymethylaminomethane), sodium hydroxide, dipotassium hydrogen phosphate, trisodium citrate, and Good's buffer, which has buffering capacity in the alkaline range. The amount of neutralizing reagent used, i.e., the amount impregnated into the immunochromatographic measurement device, is not particularly limited, but is typically about 0.01 μg to 1 mg, and preferably about 0.1 μg to 0.1 mg, per test piece of the immunochromatographic measurement device. However, it is preferable to select an optimal amount that will produce the desired effect depending on the type of neutralizing reagent used, the composition of the sample suspension, and the amount added.

[0043] The structure of an instrument for measuring glycan antigens in a sample and a method for measuring glycan antigens using the instrument are described, for example, in JP 2019-012912 A.

[0044] In measuring carbohydrate antigens in a sample using an instrument for measuring carbohydrate antigens, a solution containing the substance to be detected is mixed with a sodium nitrite solution, etc., but the solution containing the capture substance is added with sodium cholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate), digitonin (C 56 H 92 O 29 The effect of improving the visibility of the test line by a compound selected from the group consisting of methylcellulose, non-surfactant sulfobetaine (NDSB), guanidine thiocyanate, copolymers containing 2-methacryloyloxyethylphosphocholine (MPC) polymer as a monomer unit (Lipidure (registered trademark) BL-402, Lipidure (registered trademark) BL-405), and ethanol is equivalent to that of an immunochromatographic measurement method in which a solution containing the substance to be detected is not mixed with a sodium nitrite solution or the like. [Example]

[0045] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0046] In the following examples, % indicates w / v % unless otherwise specified.

[0047] [Example 1] Example of immunochromatographic test strip for measuring group A streptococci The following nitrites, solid acidic reagents, and neutralizing reagents were used. Nitrite (liquid reagent) 3.0M sodium nitrite Solid acidic reagent (impregnated into immunochromatographic test strips) 1.0M citric acid Neutralizing reagent (impregnated into immunochromatographic test strips) 3.0M Tris base (Trizma BASE)

[0048] 1. Immobilization of anti-Streptococcus pyogenes (group A beta-hemolytic streptococcus) antibodies onto nitrocellulose membrane (support) A diluted solution of anti-Streptococcus pyogenes antibody and anti-rabbit IgG antibody were prepared, and 0.55 μL of the anti-Streptococcus pyogenes antibody solution was applied linearly to a site near the sample pad side of a PET film-backed nitrocellulose membrane, and 0.55 μL of the anti-rabbit IgG antibody solution was applied linearly to the absorption band side to form a test line. The nitrocellulose membrane was then dried to obtain an anti-Streptococcus pyogenes antibody-immobilized membrane. This membrane is referred to as the "antibody-immobilized membrane" in this example. The immobilized anti-Streptococcus pyogenes antibody corresponds to the "analyte capture substance" of this invention.

[0049] In this case, the following compositions were added to a diluted solution of anti-Streptococcus pyogenes antibody at the concentrations shown in Examples 2 to 6, and the anti-Streptococcus pyogenes antibody solution was applied to a nitrocellulose membrane and then dried to form a test line, and the visibility tests of Examples 2 and onward were performed.

[0050] Sodium deoxycholate (DOC), sodium taurodeoxycholate (TDOC), sodium cholate, CHAPS, digitonin, non-detergent sulfobetaine (NDSB), guanidine thiocyanate, copolymers containing 2-methacryloyloxyethylphosphocholine polymer as monomer units (Lipidure BL-402, Lipidure BL-405), ethanol, and PEG (polyethylene glycol) 600.

[0051] 2. Immobilization of anti-Streptococcus pyogenes antibodies on colored polystyrene particles (production of a substance that specifically binds to a target substance labeled with a labeling substance that is a colored particle) Colored polystyrene particles were added to a diluted solution of anti-Streptococcus pyogenes antibody, and after stirring, carbodiimide was added and further stirred. The supernatant was removed by centrifugation, and the particles were resuspended in a buffer solution to obtain a suspension of anti-Streptococcus pyogenes antibody-bound colored polystyrene particles. In this example, these particles are referred to as "antibody-immobilized particles (colored polystyrene)." In the present invention, the anti-Streptococcus pyogenes antibody-bound colored polystyrene particles correspond to "a substance that specifically binds to a detection target substance labeled with a labeling substance that is a colored particle."

[0052] 3. Coating and drying of anti-Streptococcus pyogenes antibody-immobilized colored polystyrene particles A predetermined amount of the antibody-immobilized colored polystyrene particle suspension prepared in 2 was applied to a nonwoven fabric (glass fiber) and allowed to dry. The resulting nonwoven fabric is referred to as "C. Pad (Conjugate Pad)" in this example.

[0053] 4. Application of neutralizing reagent (basic reagent) The above neutralizing reagent (basic reagent) was applied to a filter paper.

[0054] 5. Preparation of Solid Acidic Reagent-impregnated Nonwoven Fabric The solid acidic reagent was applied to the nonwoven fabric, which was then immediately dried to obtain a solid acidic reagent-impregnated nonwoven fabric as a solid acidic reagent-impregnated region.

[0055] 6. Preparation of immunochromatographic test strips for detecting Streptococcus pyogenes First, an antibody-immobilized membrane (support) was attached to the backing sheet of the strip at a position 20 mm downstream. An absorption band was attached downstream of the membrane to absorb the liquid. A 10 mm wide glass fiber (C.Pad) coated with antibody-bound colored polystyrene particles was attached upstream of the membrane, with a 2 mm overlap between the membrane and the fiber. The neutralizing reagent-impregnated pad (neutralizing reagent-impregnated area) was attached so that the overlap between the C.Pad and the pad impregnated with the neutralizing reagent was 4 mm. A top laminate sheet (length 60 mm) made of PET was attached to the top of the immunochromatographic test piece. Finally, the pad impregnated with the solid acidic reagent (solid acidic reagent impregnated area) was attached to the upstream (lower end) of the immunochromatographic test strip on top of the pad impregnated with the neutralizing reagent, with the top laminate sheet interposed between them, and so that the pad impregnated with the solid acidic reagent and the pad impregnated with the neutralizing reagent were in contact with each other.

[0056] 7. Fabrication of Immunochromatographic Test Device The immunochromatographic test strip for detecting Streptococcus pyogenes prepared in 6 was placed in a test device case (a resin case for storing immunochromatographic test strips).

[0057] [Example 2] The immunochromatographic test device prepared in Example 1 was inoculated with 1.1 x 10 Streptococcus pyogenes in physiological saline. 7 A solution suspended in cfc / mL was diluted 16-fold with 2M sodium nitrite solution, and 80 to 90 μL of the sample was dropped onto the sample. After 5 minutes, the visibility of the coloration on the test line was compared.

[0058] The results are shown in Table 1. The result was evaluated as "good" when the colored line spread in the upstream and downstream directions, and the blurring at both ends of the line was improved.

[0059] [Table 1]

[0060] As shown in Table 1, improved visibility was observed when sodium cholate and guanidine thiocyanate were used, but no improvement in visibility was observed when DOC or TDOC was used.

[0061] [Example 3] The immunochromatographic test device prepared in Example 1 was inoculated with 1.1 x 10 Streptococcus pyogenes in physiological saline. 7 A solution suspended in cfc / mL was diluted 4 times with 2M sodium nitrite solution, and 80 to 90 μL of the sample was dropped onto the sample. After 5 minutes, the visibility of the coloration on the test line was compared.

[0062] The results are shown in Table 2.

[0063] [Table 2]

[0064] As shown in Table 2, improved visibility was observed when CHAPS, NDSB, and guanidine thiocyanate were used, but no improvement in visibility was observed when DOC and TDOC were used.

[0065] [Example 4] The immunochromatographic test device prepared in Example 1 was inoculated with 1.1 x 10 Streptococcus pyogenes in physiological saline. 7 A solution suspended in cfc / mL was diluted 16-fold with 2M sodium nitrite solution, and 80 to 90 μL of the sample was dropped onto the sample. After 5 minutes, the visibility of the coloration on the test line was compared.

[0066] The results are shown in Table 3.

[0067] [Table 3]

[0068] As shown in Table 3, improved visibility was observed when NDSB and guanidine thiocyanate were used, but no improvement in visibility was observed when DOC and PEG600 were used.

[0069] [Example 5] The immunochromatographic test device prepared in Example 1 was inoculated with 1.1 x 10 Streptococcus pyogenes in physiological saline. 7 A solution suspended in cfc / mL was diluted 4 times with 2M sodium nitrite solution, and 80 to 90 μL of the sample was dropped onto the sample, and the visibility of the coloration on the test line was compared after 5 minutes.

[0070] The results are shown in Table 4.

[0071] [Table 4]

[0072] As shown in Table 4, improved visibility was observed when guanidine thiocyanate and Lipidure BL-402 were used, but no improvement in visibility was observed when DOC or PEG600 was used.

[0073] [Example 6] The immunochromatographic test device prepared in Example 1 was inoculated with 1.1 x 10 Streptococcus pyogenes in physiological saline. 7 A solution suspended in cfc / mL was diluted 16-fold with 2M sodium nitrite solution, and 80 to 90 μL of the sample was dropped onto the sample, and the visibility of the coloration on the test line was compared after 5 minutes.

[0074] The results are shown in Table 5.

[0075] [Table 5]

[0076] As shown in Table 5, improved visibility was observed when guanidine thiocyanate, Lipidure BL-405, and ethanol were used, but no improvement in visibility was observed when DOC was used. [Industrial Applicability]

[0077] The method of the present invention can be used to measure target substances such as pathogens and disease markers by immunochromatography.

Claims

1. An immunochromatographic measuring device includes a membrane having a test line on which a detectable substance capture substance capable of capturing the detectable substance is immobilized, and a substance that specifically binds to the detectable substance labeled with a labeling substance that is a colored particle is used to form a complex of the detectable substance capture substance-detectable substance-substance that specifically binds to the labeled detectable substance on the test line on the device where the detectable substance capture substance is immobilized, thereby detecting the detectable substance by coloring the test line with the labeling substance. The immunochromatographic measuring device is characterized in that the test line contains at least one compound selected from the group consisting of sodium cholate, CHAPS, digitonin, non-detergent sulfobetaine (NDSB), guanidine thiocyanate, a copolymer containing 2-methacryloyloxyethylphosphocholine polymer as a monomer unit, and ethanol.

2. 2. The immunochromatographic measuring device according to claim 1, wherein the analyte capture substance and the substance that specifically binds to the analyte are antibodies.

3. 2. The immunochromatographic measurement device according to claim 1, wherein the copolymer containing 2-methacryloyloxyethylphosphocholine polymer as a monomer unit is Lipidure (registered trademark) BL-402 or Lipidure (registered trademark) BL-405.

4. An immunochromatographic measurement method comprising: a membrane having a test line on which a detectable substance capture substance capable of capturing the detectable substance is immobilized; and a substance that specifically binds to the detectable substance labeled with a labeling substance that is a colored particle is used to form a complex of the detectable substance capture substance-detectable substance-substance that specifically binds to the labeled detectable substance on the test line on the instrument on which the detectable substance capture substance is immobilized; and the detectable substance is detected by coloring the test line with the labeling substance. The immunochromatographic measurement method is characterized in that the visibility of the coloring on the test line is improved by including in the test line at least one compound selected from the group consisting of sodium cholate, CHAPS, digitonin, non-detergent sulfobetaine (NDSB), guanidine thiocyanate, a copolymer containing 2-methacryloyloxyethylphosphocholine polymer as a monomer unit, and ethanol.

5. 5. The immunochromatographic assay method according to claim 4, wherein the analyte capture substance and the substance that specifically binds to the analyte are antibodies.

6. The immunochromatographic measurement method according to claim 4, wherein the copolymer containing 2-methacryloyloxyethylphosphocholine polymer as a monomer unit is Lipidure (registered trademark) BL-402 or Lipidure (registered trademark) BL-405.

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