IMMUNOCHROMATOGRAPHIC ASSAY METHOD, AND TEST STRIP USED IN SAID IMMUNOCHROMATOGRAPHIC ASSAY METHOD
20220187290 · 2022-06-16
Assignee
Inventors
Cpc classification
G01N33/54393
PHYSICS
G01N33/5306
PHYSICS
International classification
Abstract
The invention aims to suppress non-specific reaction effectively in an immunochromatographic assay method for an analyte. The object can be achieved by an immunochromatographic assay method for an analyte, the method including using a test strip including the following (1) to (3): (1) a sample pad containing a sample application region for applying a biological sample that may contain the analyte; (2) an insoluble membrane containing at least one detection region to which a binding partner immunologically reactive with the analyte is immobilized, wherein the binding partner is immobilized to a label; and (3) a conjugate containing a binding partner immunologically reactive with the analyte, wherein the binding partner is immobilized to a label.
Claims
1. An immunochromatographic assay method for an analyte, the method comprising using a test strip comprising following (1) to (3): (1) a sample pad containing a sample application region for applying a biological sample that may contain the analyte; (2) an insoluble membrane containing at least one detection region to which a binding partner immunologically reactive with the analyte is immobilized, wherein the insoluble membrane contains a cationic substance; and (3) a conjugate containing a binding partner immunologically reactive with the analyte, wherein the binding partner is immobilized to a label.
2. The immunochromatographic assay method according to claim 1, wherein the analyte is a respiratory tract virus.
3. The immunochromatographic assay method according to claim 1, wherein the cationic substance is a cationic peptide or a cationic amino acid.
4. The immunochromatographic assay method according to claim 1, characterized in that the conjugate is contained in a conjugate region between the sample application region and the detection region in the (1) or (2).
5. The immunochromatographic assay method according to claim 1, wherein the cationic substance is contained at a concentration of 0.05 to 10% by mass in a solid-phase antibody application solution to be applied to the detection region in preparation of the test strip.
6. The immunochromatographic assay method according to claim 1, wherein the biological sample is respiratory secretions.
7. The immunochromatographic assay method according to claim 1, wherein the analyte is influenza B virus.
8. An immunochromatographic test strip comprising following (1) to (3): (1) a sample pad containing a sample application region for applying a biological sample that may contain an analyte; (2) an insoluble membrane containing at least one detection region to which a binding partner immunologically reactive with the analyte is immobilized, wherein the insoluble membrane contains a cationic substance; and (3) a conjugate containing a binding partner immunologically reactive with the analyte, wherein the binding partner is immobilized to a label.
9. The immunochromatographic test strip according to claim 8, wherein the analyte is a respiratory tract virus.
10. The immunochromatographic test strip according to claim 8, wherein the cationic substance is a cationic peptide or a cationic amino acid.
11. The immunochromatographic test strip according to claim 8, characterized in that the conjugate is contained in a conjugate region between the sample application region and the detection region in the (1) or (2).
12. The immunochromatographic test strip according to claim 8, wherein the cationic substance is contained at a concentration of 0.05 to 10% by mass in a solid-phase antibody application solution to be applied to the detection region in preparation of the test strip.
13. The immunochromatographic test strip according to claim 8, wherein the biological sample is respiratory secretions.
14. The immunochromatographic test strip according to claim 8, wherein the analyte is influenza B virus.
15. A method of suppressing non-specific reaction in an immunochromatographic assay method for an analyte, the method comprising using a test strip comprising following (1) to (3): (1) a sample pad containing a sample application region for applying a biological sample that may contain the analyte; (2) an insoluble membrane containing at least one detection region to which a binding partner immunologically reactive with the analyte is immobilized, wherein the insoluble membrane contains a cationic substance; and (3) a conjugate containing a binding partner immunologically reactive with the analyte, wherein the binding partner is immobilized to a label.
16. The method of suppressing non-specific reaction according to claim 15, wherein the cationic substance is a cationic peptide or a cationic amino acid.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0018]
[0019]
[0020]
DESCRIPTION OF EMBODIMENTS
(Analyte)
[0021] In the invention, examples of the analyte include viruses, and physiologically active substances such as proteins that can be generally measured using antigen-antibody reaction. Examples of the viruses include influenza viruses such as influenza A virus and influenza B virus, hepatitis B virus, hepatitis C virus, and human immunodeficiency virus. Examples of the proteins include human hemoglobin, hepatitis B virus antibodies, hepatitis C virus antibodies, and human immunodeficiency virus antibodies.
[0022] The analyte in the invention is preferably a respiratory tract virus. The respiratory tract virus in the present description is a virus that causes respiratory disease. The respiratory tract virus in the invention is not limited as long as it is a virus that can be detected using antigen-antibody reaction, and examples of the respiratory tract virus include coronaviruses such as coronavirus 229E, coronavirus OC43, and coronavirus NL63; influenza viruses such as influenza A virus, influenza B virus, influenza C virus, parainfluenza virus 1, parainfluenza virus 2, and parainfluenza virus 3; RS viruses such as RS virus A and RS virus B; adenovirus; rhinovirus; metapneumovirus; cytomegalovirus; and bocavirus. In particular, the analyte is preferably an influenza virus, more preferably influenza B virus. Most preferably, the later-described detection region is formed at a plurality of positions (for example, two positions) to detect influenza A virus and influenza B virus.
[0023] In the description, the “respiratory tract” is a general term for the organs involved in the respiration, and means the organs positioned from the nasal vestibule to the alveoli (lung), including the nasal cavity, pharynx, larynx, trachea, bronchi, and bronchioli.
(Biological Sample)
[0024] Examples of the biological sample that may contain an analyte, used in the invention include substances mainly derived from the living body, such as body fluids; and extracts prepared by extraction of the analyte from these substances. Specific examples of the substances derived from the living body include blood, urine, stool, and respiratory secretions. In cases where the analyte is a respiratory tract virus, the biological sample is preferably respiratory secretions, such as nasal discharge derived from the nares, nasal cavity, pharynx, nasopharynx, or the like; sputum; or saliva. The living body-derived substances and the extracts thereof may be used as they are as samples, or may be appropriately diluted with a sample diluent to provide samples. They may also be appropriately filtered to provide samples.
(Cationic Substance)
[0025] In the description, the cationic substance means a substance that exhibits cationic properties when the substance is dissolved or dispersed in water. A cationic surfactant, a cationic lipid, a cationic peptide, a cationic amino acid, or the like may be used as the cationic substance. A cationic peptide or a cationic amino acid is preferably used.
[0026] The cationic peptide means a peptide positively charged as a whole molecule under neutral conditions, for example, under conditions where the peptide is dissolved or dispersed in water. The cationic peptide may contain a large number of basic amino acid residues (arginine (Arg), lysine (Lys), and/or histidine (His)) as amino acid residues constituting the peptide. More specifically, the content of basic amino acid residues in the total amino acid residues constituting the cationic peptide is 40% or more, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more. Most preferably, the content of basic amino acid residues is 100%. In the cationic peptide, the content of acidic amino acid residues (aspartic acid (Asp), glutamic acid (Glu), cysteine (Cys), and/or tyrosine (Tyr)) is preferably at a certain value or less in the total amino acid residues constituting the peptide. More specifically, the content of acidic amino acid residues in the total amino acid residues is 20% or less, preferably 15% or less, more preferably 12% or less, still more preferably 10% or less, still more preferably 8% or less, still more preferably 6% or less, still more preferably 4% or less, still more preferably 2% or less, still more preferably 1% or less. Most preferably, the content of acidic amino acid residues is 0%.
[0027] The cationic peptide is not limited as long as it is a peptide positively charged as a whole peptide under neutral conditions. Examples of the cationic peptide that may be used include cationized derivatives of protein degradation products derived from the living body; and polyarginine, polylysine, and polyhistidine, which are polymers of the basic amino acid residues described above. Among the cationized derivatives of protein degradation products derived from the living body, derivatives containing a quaternary ammonium group bound to the N-terminus of a peptide of a protein degradation product derived from the living body are especially preferred. Hydroxypropyltrimonium hydrolyzed soy protein (trade name, Promois WS-HQ; manufactured by Seiwa Kasei Co, Ltd.) or cocodimonium hydroxypropyl hydrolyzed keratin (trade name, Promois WK-HCAQ; manufactured by Seiwa Kasei Co, Ltd.) is still more preferably used. As long as the effect of the invention can be obtained, a salt of a cationic peptide may be used. Promois is a registered trademark.
[0028] The cationic amino acid means arginine (Arg), lysine (Lys), or histidine (His). As the cationic amino acid, an L-isomer is preferably used. As long as the effect of the invention can be obtained, a salt of a cationic amino acid, such as a hydrochloride may be used. L-Lysine hydrochloride is especially preferably used.
[0029] These cationic substances may be used individually, or two or more thereof may be used in combination. As the cationic substance, a cationic substance selected from the group consisting of L-Lysine hydrochloride, hydroxypropyltrimonium hydrolyzed soy protein (trade name, Promois WS-HQ; manufactured by Seiwa Kasei Co, Ltd.), cocodimonium hydroxypropyl hydrolyzed keratin (trade name, Promois WK-HCAQ; manufactured by Seiwa Kasei Co, Ltd.), and combinations thereof is most preferably used.
[0030] The concentration of the cationic substance may be appropriately adjusted depending on the type of the cationic substance, and the analyte. For example, from the viewpoint of the effect for suppressing the non-specific reaction, the concentration of the cationic substance in terms of the concentration in the solid-phase antibody application solution to be applied to the detection region in the preparation of the test strip is 0.001 to 10% by mass, preferably 0.01 to 10% by mass, more preferably 0.02 to 10% by mass, still more preferably 0.05 to 10% by mass, most preferably 0.1 to 10% by mass. The upper limit of the concentration may be 8% by mass, 5% by mass, 3% by mass, or 2% by mass.
(Conjugate)
[0031] The conjugate contains a binding partner immunologically reactive with the analyte. The binding partner is immobilized to a label. Regarding the mode of presence of the conjugate, the conjugate may be present as a conjugate pad in the state impregnated in a pad, provided separately from a sample pad, a third pad, and an insoluble membrane, is impregnated with the conjugate (type A), may be present as a conjugate region in a part of a sample pad or a part of an insoluble membrane (type B), or may be present alone as a conjugate reagent which is to be mixed with the biological sample (type C).
[0032] As the label used in the invention, a known label may be used. For example, the label is preferably colloidal metal particles such as colloidal gold particles or colloidal platinum particles; colored latex particles; magnetic particles; or the like. The label is especially preferably colloidal gold particles.
[0033] In the case of detecting influenza virus, the conjugate is preferably a conjugate in which an anti-influenza virus monoclonal antibody is immobilized on colloidal gold particles.
[0034] The following describes a test strip in which the mode of presence of the conjugate is type A.
[0035] From upstream to downstream of the direction of the sample flow, a sample pad, a conjugate pad, a third pad, and an insoluble membrane are placed in this order. Each layer is placed such that at least part of the layer overlaps with the upstream/downstream layer(s).
[0036] When a sample containing an analyte is applied to the sample pad of the test strip, the analyte flows through the sample pad to the conjugate pad located downstream. In the conjugate pad, the analyte contacts the conjugate, and passes through the pad while forming a first complex (aggregate). Thereafter, the first complex passes through a porous third pad placed in contact with the lower side of the conjugate pad, followed by development into the insoluble membrane.
[0037] On the part of the insoluble membrane, a binding partner immunologically reactive with the analyte is immobilized. Here, immune reaction occurs to cause the first complex to bind to the binding partner immunologically reactive with the analyte, resulting in the formation of an immobilized second complex. The immobilized second complex is then detected by means that detects a signal such as the absorbance, reflected light, fluorescence, or magnetism derived from the conjugate.
[0038] The following describes a test strip in which the mode of presence of the conjugate is type B.
[0039] The difference from the test strip of type A is that the sample pad or the insoluble membrane is integrated with the conjugate pad.
[0040] The sample application region is a region where the sample containing the analyte is applied, and the conjugate region is a region containing the conjugate. The sample application region is located upstream of the conjugate region.
[0041] The conjugate region is formed on the sample pad or an insoluble membrane such that the conjugate region is located downstream of the sample application region. The conjugate region is preferably placed to forma line shape in the direction perpendicular to the direction of sample development, that is, perpendicular to the line connecting the center of the sample application region in the sample pad and the center of the downstream end of the later-mentioned insoluble membrane. In other words, the line-shaped conjugate region is preferably placed to form a line shape in the direction perpendicular to the longitudinal direction of the sample pad and the insoluble membrane. Although the conjugate region may be present in the insoluble membrane such that the conjugate region is located upstream of the detection region, it is preferably formed in the sample pad. In this case, the downstream side of the conjugate region in the sample pad does not necessarily need to be entirely occupied by the conjugate region, and the downstream side of the conjugate region may have a porous material portion which does not contain the conjugate.
[0042] The position of the conjugate region can be appropriately adjusted to an appropriate length by those skilled in the art. The centerline of the line of the line-shaped conjugate region is preferably positioned at least 3 mm upstream from the downstream end of the sample pad. In the description, the “centerline” of the conjugate region or the detection region means the centerline drawn in the direction perpendicular to the longitudinal direction of the sample pad, and does not mean the centerline drawn in the direction parallel to the longitudinal direction of the sample pad.
[0043] The sample containing the analyte applied to the sample application region migrates from upstream to downstream. In the conjugate region, the analyte and the conjugate forma first complex. The first complex migrates further downstream. The first complex then forms a second complex with a binding partner immobilized to the detection region, in which binding partner is immunologically reactive with the analyte. The second complex immobilized to the detection region is then detected by means that detects a signal such as the absorbance, reflected light, fluorescence, or magnetism derived from the conjugate.
[0044] The following describes a test strip in which the mode of presence of the conjugate is type C.
[0045] The difference from the test strip of type A is that there is no conjugate pad, and that the conjugate is present as a separate conjugate reagent. For example, a filter tip having a filter containing the conjugate therein is employed. An analyte is passed through the filter tip to bind the conjugate to the analyte, thereby forming a first complex (aggregate). By applying the resulting first complex to a test strip which is the same as type A except for the absence of the conjugate pad, a second complex can be formed in the detection region to which a binding partner immunologically reactive with the analyte is immobilized. The immobilized second complex is then detected by means that detects a signal such as the absorbance, reflected light, fluorescence, or magnetism derived from the conjugate.
(Binding Partners Immunologically Reactive with Analyte)
[0046] The binding partners immunologically reactive with the analyte, used in the invention are preferably antibodies capable of binding to the analyte. The binding partners immunologically reactive with the analyte are immobilized to the label and the detection region.
[0047] In cases where the binding partners are antibodies, the antibodies immobilized to the label and the detection region may be the same, but different antibodies are preferably used for the label and the detection region. By using different antibodies as the antibody immobilized to the label and the antibody immobilized to the detection region, the competition between
[0048] the reaction between the analyte bound to the conjugate and the antibody in the detection region, and
[0049] the reaction between the analyte bound to the conjugate and the unreacted conjugate
can be suppressed. Further, the reactivity between the analyte bound to the conjugate and the antibody in the detection region can be increased. As a result, the immunochromatographic test strip can have a good sensitivity. The different antibodies means that the types of the antibodies are different, that is, the antibodies recognize different epitopes. The antibodies immobilized to the label and the detection region are preferably monoclonal antibodies. By the use of monoclonal antibodies, the specificity of the reaction can be increased.
[0050] Other than the whole molecules of these antibodies, functional fragments of the antibodies having antigen-antibody reaction activity are similarly regarded as the antibodies in the invention. Examples of the functional fragments of the antibodies include those obtained through the process of immunization of an animal, those obtained using genetic recombination techniques, and chimeric antibodies. Examples of the functional fragments of the antibodies include F(ab′).sub.2 and Fab′. These functional fragments can be produced by treating the antibodies with a protease (such as pepsin or papain).
(Sample Pad)
[0051] The sample pad is layered on the conjugate pad or the later-mentioned insoluble membrane such that the lower side of the downstream end of the sample pad is in contact with the upper side of the conjugate pad or the insoluble membrane. Examples of the porous material constituting the sample pad include pads made of non-woven fibers such as paper, cellulose mixture, nitrocellulose, polyester, acrylonitrile copolymer, glass, and rayon. The pad is especially preferably made of glass fibers (a glass fiber pad).
(Third Pad)
[0052] In a test strip of type A, for the purpose of removing non-specific reacting substances in the biological sample, the third pad is placed between the conjugate pad and the insoluble membrane. The third pad is preferably placed when necessary depending on the properties of the sample and/or the like. The third pad is not limited as long as the complex of the analyte in the sample and the conjugate can pass through the third pad.
(Insoluble Membrane)
[0053] The insoluble membrane that may be used in the invention contains at least one detection region to which a binding partner immunologically reactive with the analyte is immobilized. The binding partner immunologically reactive with the analyte may be immobilized to the insoluble membrane by a known method. For example, a solution containing the binding partner at a predetermined concentration is prepared. Thereafter, the solution is applied to the insoluble membrane to form a line shape using, for example, an apparatus having a mechanism capable of traveling in the horizontal direction while discharging the solution from a nozzle at a constant rate. By drying the applied solution, the antibody can be immobilized to the insoluble membrane.
[0054] The solution containing the binding partner at a predetermined concentration can be prepared by adding the binding partner to a buffer. Examples of the type of the buffer include conventionally used buffers such as phosphate buffer, tris buffer, and Good's buffer. The buffer has a pH within the range of preferably 6.0 to 9.5, more preferably 6.5 to 8.5, still more preferably 7.0 to 8.0. The buffer may also contain: salts such as sodium chloride; stabilizers such as sugars; preservatives; and antiseptics such as ProClin. Examples of the salts include not only those added for adjusting the ionic strength, such as sodium chloride, but also those added for the purpose of adjusting the pH of the buffer, such as sodium hydroxide.
[0055] After the immobilization of the binding partner to the insoluble membrane, the portion other than the site of antibody immobilization may be blocked by coating with a solution or vapor of a conventionally used blocking agent.
[0056] The detection region provided on the insoluble membrane contains a cationic substance. Regarding the means of including the cationic substance in the detection region, it is preferred to prepare an application solution containing the cationic substance, and containing the binding partner at a predetermined concentration, and then to apply the application solution to the insoluble membrane, followed by drying and immobilization thereon.
[0057] Examples of the membrane constituting the insoluble membrane that may be used in the invention include known membranes that have been conventionally used as insoluble membranes for immunochromatographic test strips. Examples of the membrane include membranes constituted by fibers composed of polyethylene; polyethylene terephthalate; nylon; glass; a polysaccharide such as cellulose or cellulose derivative; or ceramic. Specific examples of the membrane include glass fiber filter papers and nitrocellulose membranes commercially available from Sartorius, Millipore, Toyo Roshi Kaisha, Ltd., Whatman, and the like.
[0058] The immunochromatographic test strip that may be used in the invention is preferably provided with an absorption pad at the downstream end of the insoluble membrane. The absorption pad is a portion having liquid absorbability. The absorption pad controls the development of the sample by absorption of the sample that has migrated/passed through the insoluble membrane. As the absorption pad, a known absorption pad that has been conventionally used for immunochromatographic test strips may be used. For example, a filter paper may be used.
[0059] The non-specific reaction in the description means that a signal derived from the conjugate is detected, and hence that the biological sample is judged as positive, in spite of the fact that the analyte is absent or substantially absent in the biological sample.
(Immunochromatographic Test Strip)
[0060] In the description, the “immunochromatography” means an immunoassay utilizing the nature of a porous material such as a cellulose membrane that allows a test specimen to flow slowly therethrough (capillary phenomenon) while dissolving a reagent. In the description, “using a test strip” means that the analyte contained in the biological sample is detected by bringing the test strip into contact with the biological sample. The immunochromatographic test strip is preferably placed on a solid support such as a plastic adhesive sheet. The solid support is constituted with a substance that does not prevent the capillary flow of the sample and the conjugate. The immunochromatographic test strip may be immobilized on the solid support using an adhesive or the like. In this case, the component of the adhesive or the like is also constituted with a substance that does not prevent the capillary flow of the sample and the conjugate. For the purpose of increasing the mechanical strength of the insoluble membrane, and preventing evaporation of water (drying) during the assay, the test strip may be laminated with a polyester film or the like. The immunochromatographic test strip may be used in a state where the test strip is stored or mounted in an appropriate container (housing) taking into account the size of the immunochromatographic test strip, the method of addition of the sample, the position where the sample is added, the position where each detection region is formed in the insoluble membrane, the detection method for the signal, and/or the like. The test strip stored or mounted in this state is referred to as “device”.
[0061] The immunochromatographic assay method of the invention may comprise the following steps (A) to (D):
[0062] (A) bringing a biological sample that may contain an analyte into contact with a sample application region;
[0063] (B) bringing the biological sample into contact with a conjugate to form a first complex;
[0064] (C) bringing, in a detection region containing a cationic substance, the first complex into contact with a binding partner immunologically reactive with the biological sample, to form a second complex; and
[0065] (D) measuring a signal derived from the conjugate.
[0066] In the description, the immunochromatographic assay method means an assay method utilizing immunochromatography.
[0067] When appropriate, the immunochromatographic test strip may be prepared with modification or alteration of the methods described in the Examples.
[0068] The reason why the invention can produce the effect that suppresses the non-specific reaction has not been fully elucidated, but the reason can be inferred as follows. The invention is, however, not limited by the following explanation.
[0069] The inventors discovered that the immunochromatographic test strip shows an increase in the non-specific reaction in the presence of an anionic substance in the detection region, but that the test strip shows suppression of the non-specific reaction in the presence of a cationic substance in the detection region. It can thus be thought that the causative non-specific substance is positively charged, and hence that the non-specific reaction can be suppressed by repelling of the causative non-specific substance by the cationic substance (
EXAMPLES
[0070] The invention is described below concretely by way of Examples. However, these do not limit the scope of the invention. In the description, “%” represents “% by mass” unless otherwise specified.
[Preparation of Anti-Influenza Virus Monoclonal Antibodies]
[0071] The anti-influenza A virus monoclonal antibodies and the anti-influenza B virus monoclonal antibodies used in the following tests were obtained by immunization of mice using recombinant influenza nucleoproteins as antigens, by a method conventionally used by those skilled in the art for the production of monoclonal antibodies.
[Test Strip Preparation Example 1] Preparation of Immunochromatographic Test Strip
1) Preparation of Colloidal Gold Particle-Labeled Anti-Influenza Virus Monoclonal Antibodies (Conjugates)
[0072] PBS containing an anti-influenza A virus monoclonal antibody at a concentration of 100 μg/mL and PBS containing an anti-influenza B virus monoclonal antibody at a concentration of 100 μg/mL were prepared. To 20 mL of 1 OD/mL colloidal gold particle solution, 1 mL of each antibody solution was added, and the resulting mixture was stirred at room temperature for minutes. To each of the colloidal gold particle/anti-influenza A virus monoclonal antibody mixture and the gold colloid particle/anti-influenza B virus monoclonal antibody mixture, 2 mL of 10% aqueous bovine serum albumin (BSA) solution was added, and each resulting mixture was stirred for 5 minutes, followed by centrifugation at 10° C. at 10,000 rpm for 45 minutes to obtain a precipitate (conjugate). To each conjugate obtained, Conjugate Dilution Buffer (manufactured by Scripps) was added, and the conjugate was suspended therein.
2) Preparation of Sample Pad
[0073] Each conjugate prepared in 1) as described above was mixed with a solution containing 1.33% casein and 4% sucrose (pH 7.5) to a concentration of 8 to 20 OD/mL, to prepare a conjugate solution. A glass fiber pad having a total length of 28 mm was soaked with the conjugate solution such that a line having a line width of 4 mm was formed. The pad was dried by heating at 70° C. for 30 minutes, to provide a sample pad containing a conjugate region.
3) Preparation of Insoluble Membrane to which Anti-Influenza Virus Monoclonal Antibody is Immobilized (Antibody-Immobilized Membrane)
[0074] An anti-influenza A virus monoclonal antibody, an anti-influenza B virus monoclonal antibody, and an anti-mouse IgG antibody, whose concentrations were adjusted to 0.75 mg/mL, 1.0 mg/mL, and 0.75 mg/mL, respectively, with phosphate buffer containing various cationic substances at predetermined concentrations were provided as solid-phase antibody application solutions, and the solid-phase antibody application solutions were applied on a short-side end of a nitrocellulose membrane (pore size, 8 μm) to form lines in the direction perpendicular to the longitudinal direction of the insoluble membrane. Thereafter, the membrane was dried at 70° C. for 45 minutes to provide an antibody-immobilized membrane. The lines were applied such that the anti-influenza A virus monoclonal antibody (c1), the anti-influenza B virus monoclonal antibody (c2), and the control antibody (c3) are arranged in this order from the upstream side when the test strip is assembled.
4) Preparation of Immunochromatographic Test Strip
[0075] The sample pad prepared in 2) was layered on the insoluble membrane obtained in 3), to prepare an immunochromatographic test strip.
Example 1
[0076] Using the immunochromatographic test strip prepared in Test Strip Preparation Example 1, a detection test for influenza A and B viruses was carried out.
1. Test Method
(1) Samples
[0077] Nasal mucus from a healthy subject, who was not infected with influenza, was diluted by mixing with a sample diluent, to provide each sample for use.
(2) Procedure
[0078] After dispensing 135 μL of each sample prepared in (1) into a test tube, the immunochromatographic test strip was inserted into the tube, and the presence or absence of color development of the test lines A and B on the antibody-immobilized membrane in the detection regions was visually judged 5, 15, 30, and 45 minutes later.
2. Test Results
[0079] The results of the judgment of the presence/absence of color development of the test line B are shown in Tables 1 and 2. The test line B indicates detection of influenza B virus. The “additive concentration” represents the concentration in the solid-phase antibody application solution.
TABLE-US-00001 TABLE 1 Number 1 2 3 4 5 6 Additive name No additive SILK-1000 WJ WS WS-HQ WS-HCAQ Type of additive — Peptide Peptide Peptide Cationic peptide Cationic peptide Additive concentration (%) — 0.25% 0.25% 0.25% 0.25% 0.25% Time 5 − − − − − − (minutes) 15 − − − − − − 30 + − + + − − 45 + + + + − −
TABLE-US-00002 TABLE 2 Number 7 8 9 10 11 Additive WR WK-H WK-HCAQ WG L-Lysine name hydrochloride Type of Peptide Peptide Cationic Peptide Cationic additive peptide amino acid Additive 0.25% 0.25% 0.25% 0.25% 0.25% concentration (%) Time 5 − − − − − (minutes) 15 − − − − − 30 + − − + − 45 + + − + −
[0080] In Table 1 and Table 2, “−” represents the absence of color development of the test line B. The test line B indicates detection of influenza B virus. Thus, “−” indicates the absence of a false-positive result. “+” represents color development of the line, indicating occurrence of a false positive result. All of the cases where a non-cationic peptide was added, and the case of the non-addition control, showed non-specific reaction after 30 minutes and/or 45 minutes of the reaction. In contrast, none of the samples to which a cationic peptide or a cationic amino acid was added showed non-specific reaction. None of the tested samples showed non-specific reaction in the test line A. The test line A indicates detection of influenza A virus. It was thus shown that addition of a cationic surfactant to the detection region enables prevention of non-specific reaction that occurs in the detection of influenza B virus.
Example 2
1. Test Method
[0081] A detection test for influenza A and B viruses was carried out in the same manner as in Example 1 except that different types of cationic peptide or cationic amino acid were used at different concentrations.
2. Test Results
[0082] The results of the judgment of the presence/absence of color development of the test line B are shown in Tables 3 and 4. The test line B indicates detection of influenza B virus.
TABLE-US-00003 TABLE 3 Number 12 13 14 15 Additive name No WK-HCAQ additive Type of cationic − Cationic peptide substance Cationic substance − 0.10% 0.15% 0.20% concentration (%) Time 5 − − − − (minutes) 15 − − − − 30 + − − − 45 + − − −
TABLE-US-00004 TABLE 4 Number 16 17 18 Additive name L-Lysine hydrochloride Type of cationic Cationic amino acid substance Cationic substance 0.10% 0.50% 1% concentration (%) Time 5 − − − (minutes) 15 − − − 30 − − − 45 − − −
[0083] In Table 3 and Table 4, “−” represents the absence of color development of the test line B. The test line B indicates detection of influenza B virus. Thus, “−” indicates the absence of a false-positive result. “+” represents color development of the line, indicating occurrence of a false positive result. In the measurement systems in which the cationic peptide was added, a sufficient effect for suppression of the non-specific reaction could be found in the detection of influenza B virus even in the case where the concentration of the cationic peptide was decreased to 0.10%. In the measurement systems in which the cationic amino acid was added, a sufficient effect for suppression of the non-specific reaction could be found even in the case where the concentration of the cationic peptide was increased to 1%. None of the tested samples showed non-specific reaction in the detection of influenza A virus.
INDUSTRIAL APPLICABILITY
[0084] By the invention, non-specific reaction in an immunochromatographic assay for an analyte can be effectively suppressed.
REFERENCE SIGNS LIST
[0085] (a) Plastic adhesive sheet [0086] (b) Insoluble membrane [0087] (c1) Test line A [0088] (c2) Test line B [0089] (c3) Control line [0090] (d) Conjugate pad [0091] (e) Sample pad [0092] (f) Absorption pad [0093] (g) Third pad [0094] (h) Conjugate region [0095] (i) Film