immunochromatographic method

Abstract

Provided is an immunochromatographic method with high detection sensitivity. An immunochromatographic method includes: a mixing step of obtaining a mixture containing a magnetic particle complex by mixing a sample that can contain an antigen and a magnetic particle modified with a substance having specific affinity for the antigen; a capturing step of capturing the magnetic particle; a dissociation step of obtaining an antigen concentrate by mixing the captured magnetic particle and a dissociation solution in an amount smaller than the sample that can contain the antigen, and dissociating the modified magnetic particle; a neutralization step of obtaining a neutralized antigen concentrate; a development step of developing on an insoluble carrier having a reaction site on which a second binding substance is immobilized, in a state in which a gold particle complex is formed as a complex of the antigen in the neutralized antigen concentrate and a gold particle modified with a first binding substance; a capturing step of capturing the gold particle complex on the reaction site; and a silver amplification step of performing silver amplification on the gold particle complex.

Description

TECHNICAL FIELD

[0001] The present application relates to an immunoassay. BACKGROUND

[0002] The immunoassay has been frequently used recently because of its simple operation and ability to perform measurement in a short time.

[0003] For example, in the case of detecting an antigen such as an influenza virus using the immunoassay, the following operation is performed.

[0004] First, a label modified with an antibody (labeled antibody) is prepared and mixed with a sample containing an antigen. The labeled antibody binds to the antigen to form a complex. In this state, when it is developed on an insoluble support having a detection line on which an antibody specifically reacting with the antigen is coated, the complex is captured on the detection line (test line) by reacting with the antibody, and the detection is confirmed by the naked eye or the like.

[0005] As such an immunoassay, for example, the method disclosed in Patent Literature 1 can be cited.

[0006] PRIOR ART DOCUMENTS

[0007] PATENT LITERATURE

[0008] Patent Literature 1: Japanese Patent No. 5728453 SUMMARY

[0009] PROBLEMS TO BE SOLVED BY THE INVENTION

[0010] Recently, in the case where an immunoassay is expected to be applicable also to a subject fluid in which the concentration of an antigen is extremely low, a method having higher sensitivity than the conventional method (for example, the method disclosed in Patent Literature 1) is required for the immunoassay.

[0011] Therefore, in view of the above circumstances, an object of the present application is to provide an immunoassay having high detection sensitivity.

[0012] MEANS FOR SOLVING THE PROBLEMS

[0013] The present inventors and others have intensively studied the above problems, and as a result, have found that the above problems can be solved by using a subject fluid concentrated by a prescribed method, thereby completing the present application.

[0014] That is, the present inventors and others have found that the above problems can be solved by the following structure.

[0015] (1) An immunoassay comprising:

[0016] a mixing step of obtaining a mixture containing a magnetic particle complex of a complex of the antigen and the modified magnetic particle by mixing the sample that can contain the antigen and the magnetic particle modified with the substance having specific affinity for the antigen, i.e., the modified magnetic particle;

[0017] a capturing step of capturing the magnetic particle in the mixture containing the magnetic particle complex using magnetism;

[0018] a dissociation step of obtaining an antigen concentrate by dissociating the modified magnetic particle from the magnetic particle complex by mixing the magnetic particle captured in the capturing step and a dissociation liquid that is an alkaline or acidic liquid in a smaller amount than the sample that can contain the antigen;

[0019] a neutralization step of obtaining a neutralized antigen concentrate by neutralizing the antigen concentrate using a neutralization liquid;

[0020] a development step of developing on an insoluble carrier having a reaction site at which a second binding substance capable of binding to the antigen is immobilized, in a state in which a gold particle complex of a complex of the antigen in the neutralized antigen concentrate and a gold particle modified with a first binding substance capable of binding to the antigen, i.e., a modified gold particle, is formed;

[0021] a capturing step of capturing the gold particle complex at the reaction site of the insoluble carrier; and

[0022] a silver amplification step of performing silver amplification on the gold particle complex captured in the capturing step.

[0023] (2) The immunochromatography method according to the above (1), wherein the ratio of the dissociation liquid to the sample that can contain the antigen is 1 / 5 or less in terms of mass ratio.

[0024] (3) The immunochromatography method according to the above (1) or (2), wherein the sample that can contain the antigen is urine.

[0025] (4) The immunochromatography method according to any one of the above (1) to (3), wherein the antigen is a sugar chain.

[0026] (5) The immunochromatography method according to any one of the above (1) to (4), wherein the dissociation liquid contains NaOH or HC1.

[0027] (6) The immunochromatography method according to any one of the above (1) to (5), wherein the neutralizing solution contains HC1 and at least one selected from the group consisting of tris(hydroxymethyl)methylglycine, TRIS, HEPES, acetamidoglycine, glycylamide and N,N-dihydroxyethylglycine, or contains NaOH and at least one selected from the group consisting of tris(hydroxymethyl)methylglycine, TRIS, HEPES, acetamidoglycine, glycylamide and N,N-dihydroxyethylglycine.

[0028] (7) The immunochromatography method according to any one of the above (1) to (6), wherein the antigen is lipoarabinomannan.

[0029] (8) The immunochromatography method according to any one of the above (1) to (7), wherein the particle diameter of the magnetic particle before modification is 0.05 μm to 10 μm.

[0030] Effects of the Invention

[0031] According to the present application, as described below, it is possible to provide an immunochromatography method with high detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a schematic view of one mode of the insoluble carrier used in the method of the present application. DETAILED DESCRIPTION

[0033] Hereinafter, the immunochromatography method of the present application will be described.

[0034] In addition, in the present specification, the numerical range represented by "~" means a range including the numerical values recited before and after "~" as the lower limit value and the upper limit value.

[0035] Also, in the present specification, each component can be used singly or two or more kinds can be used at the same time. Here, in the case where two or more kinds of each component are used at the same time, the content of the component means the total content unless otherwise specified.

[0036] Also, in the present specification, "further improvement in detection sensitivity and S / N ratio (signal / noise ratio)" is also referred to as "more excellent effects of the present application and the like".

[0037] The immunochromatography method of the present application (hereinafter, also referred to as "the method of the present application") is an immunochromatography method which includes:

[0038] a mixing step of obtaining a mixture containing a magnetic particle complex of a complex of the antigen and the modified magnetic particle by mixing a sample which can contain the antigen and a magnetic particle modified with a substance having specific affinity with the antigen, i.e., a modified magnetic particle;

[0039] a capturing step of capturing the magnetic particles in the mixture containing the magnetic particle complex using a magnet;

[0040] a dissociating step of dissociating the modified magnetic particles from the magnetic particle complex by mixing the magnetic particles captured in the capturing step and a dissociating liquid which is an alkaline or acidic liquid and is smaller in amount than the sample which can contain the antigen, to obtain an antigen concentrated liquid;

[0041] a neutralizing step of neutralizing the antigen concentrated liquid by using a neutralizing liquid, to obtain a neutralized antigen concentrated liquid;

[0042] a developing step of developing, in a state where a gold particle complex which is a complex of the antigen in the neutralized antigen concentrated liquid and a modified gold particle which is a gold particle modified with a first binding material capable of binding to the antigen, is formed, on an insoluble carrier having a reaction site where a second binding material capable of binding to the antigen is immobilized;

[0043] a capturing step of capturing the gold particle complex at the reaction site of the insoluble carrier; and

[0044] a silver amplification step of performing silver amplification on the gold particle complex captured in the capturing step.

[0045] Hereinafter, each step will be described. In addition, the mixing step to the neutralizing step will be collectively referred to as "magnetic particle process".

[0046] [The mixing step]

[0047] The mixing step is a step of obtaining a mixture containing a magnetic particle complex which is a complex of the antigen and the modified magnetic particle, by mixing a sample which can contain the antigen and a modified magnetic particle which is a magnetic particle modified with a material having specific affinity to the antigen.

[0048] [The sample]

[0049] The sample used in the mixing step is not particularly limited as long as it is a sample which can contain the antigen. As such a sample, for example, biological samples, particularly, body fluids (e.g., blood, serum, plasma, cerebrospinal fluid, tears, sweat, urine, pus, nasal discharge, or sputum) or excreta (e.g., feces) of animals (particularly, humans), organs, tissues, mucous membranes, or skin, scraping specimens (swabs) thought to contain them, gargle, or plants or animals themselves or their dried bodies can be given.

[0050] The sample is preferably a liquid, and more preferably urine, from the viewpoint of more excellent effects and the like of the present application.

[0051] [The antigen]

[0052] As the antigen, for example, bacteria, a bacterium (e.g., Mycobacterium tuberculosis, lipid arabinomannan (LAM) contained in Mycobacterium tuberculosis), a virus (e.g., influenza virus), or a nucleoprotein thereof, and the like can be mentioned. In addition, LAM is a major antigen in tuberculosis and is a major constituent of the cell membrane and cell wall, i.e., a glycolipid.

[0053] From the viewpoint of more excellent reasons of the effects of the present application, the antigen is preferably an antigen that is a sugar chain (particularly, a glycolipid), and more preferably LAM.

[0054] <Pre-treatment of sample>

[0055] The above sample can be used directly as the sample, or in the form of an extract obtained by extracting the antigen using a suitable extraction solvent, and further in the form of a dilution obtained by diluting the extract with a suitable diluent, or in the form of a concentrate obtained by concentrating the extract with a suitable method.

[0056] As the above extraction solvent, a solvent used in a general immunological analysis method (e.g., water, physiological saline, or a buffer, and the like) or a water-miscible organic solvent capable of directly performing an antigen-antibody reaction by dilution with the solvent can be used.

[0057] [Modified magnetic particle]

[0058] The above modified magnetic particle is a magnetic particle modified with a substance having specific affinity with the above antigen.

[0059] <Magnetic particle>

[0060] The material of the above magnetic particle is not particularly limited as long as it is a material having magnetism, and as specific examples, iron, cobalt, nickel, oxides thereof, ferrite, and alloys thereof, and the like can be mentioned. Among them, from the viewpoint of more excellent reasons of the effects of the present application, iron oxide is preferred.

[0061] The magnetic particle can be a particle in which a material having magnetism is formed into a particle shape alone, or can be a particle in which a material having magnetism is used as a core and the surface thereof is coated with a high molecule (e.g., polystyrene, silica gel, and the like) or the like, or a particle in which a material having magnetism is used and a high molecule or the like is used as a core to coat the surface thereof.

[0062] (Particle diameter)

[0063] The particle diameter of the magnetic particle is not particularly limited, but from the viewpoint of more excellent reasons of the effects of the present application, it is preferably 0.05 μm to 10 μm, and more preferably 0.1 μm to 5 μm.

[0064] In addition, the particle diameter can be measured using a commercially available particle size distribution meter or the like. As a method for measuring the particle size distribution, optical microscopy, confocal laser microscopy, electron microscopy, atomic force microscopy, static light scattering, laser diffraction, dynamic light scattering, centrifugal sedimentation, electric pulse measurement, chromatography, ultrasonic attenuation, and the like are known, and devices corresponding to each principle are commercially available. As a method for measuring the particle diameter, dynamic light scattering can be preferably used in terms of the range of the particle diameter and ease of measurement. As a commercially available measuring device using dynamic light scattering, NANOTRAC UPA (Nikkiso Co., Ltd.), dynamic light scattering particle size distribution measuring device LB-550 (HORIBA, Ltd.), and dense particle analyzer FPAR-1000 (OTSUKA ELECTRONICS Co., Ltd.) can be mentioned. In the present application, the value of the median particle diameter (d=50) measured at a measurement temperature of 25°C is obtained as the particle diameter.

[0065] <Substance having specific affinity to antigen>

[0066] The substance having specific affinity to antigen is not particularly limited, but from the viewpoint of more excellent effects of the present application and the like, it is preferably a protein, more preferably an antibody (e.g., polyclonal antibody, or monoclonal antibody), and further preferably a monoclonal antibody from the viewpoint of achieving higher detection sensitivity.

[0067] The above antibody is not particularly limited, and for example, an antiserum prepared from the serum of an animal immunized with an antigen or an immunoglobulin fraction purified from the antiserum can be used. Also, a monoclonal antibody obtained by cell fusion using spleen cells of an animal immunized with an antigen, or a fragment thereof [e.g., F(ab')2, Fab, Fab', or Fv] can be used. The preparation of these antibodies can be performed by a conventional method.

[0068] As an example of the substance having specific affinity to antigen when the antigen is LAM, the A194-01 antibody described in International Publication No. 2017 / 139153 can be mentioned. The contents described in International Publication No. 2017 / 139153 regarding the A194-01 antibody are all incorporated into the present specification as a part of the disclosure of the present specification.

[0069] As another example of the substance having specific affinity to antigen when the antigen is LAM, an antibody having the sequence described as MoAb1 in paragraph

[0080] of International Publication No. 2013 / 129634 can be mentioned. The contents described in International Publication No. 2013 / 129634 regarding the MoAb1 antibody are all incorporated into the present specification as a part of the disclosure of the present specification.

[0070] Method for manufacturing modified magnetic particle

[0071] The method for manufacturing the above-described modified magnetic particle is not particularly limited, and a publicly known method can be used. For example, a method in which a magnetic particle is activated with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and an antibody is supported on the magnetic particle, or the like can be mentioned.

[0072] [Mixing]

[0073] In the above-described mixing step, the above-described sample and the above-described modified magnetic particle are mixed.

[0074] Thus, in the case where the above-described sample contains an antigen, the antigen in the sample reacts with the substance of the above-described modified magnetic particle having specific affinity with the above-described antigen, and a complex of the antigen and the above-described modified magnetic particle is formed in the above-described sample. On the other hand, in the case where the above-described sample does not contain an antigen, no complex is formed.

[0075] [Trapping step]

[0076] The trapping step is a step of magnetically trapping the magnetic particle in the mixture containing the magnetic particle complex after the above-described mixing step.

[0077] Here, "trapping the magnetic particle in the mixture" means "trapping the magnetic particle complex in the mixture and the magnetic particle (unmodified magnetic particle) and the modified magnetic particle remaining in the mixture".

[0078] The method for magnetically trapping the magnetic particle in the mixture after the above-described mixing step is not particularly limited, and for example, a method in which the mixture after the above-described mixing step is added to a conical tube provided on a magnetic stand, the magnetic particle is collected with a magnet, and then the remaining sample is extracted, or the like can be mentioned.

[0079] [Disaggregating step]

[0080] The disaggregating step is a step of causing the above-described modified magnetic particle to be disaggregated from the above-described magnetic particle complex by mixing the magnetic particle trapped in the above-described trapping step and a disaggregating liquid which is an alkaline or acidic liquid and is less than the above-described sample which can contain an antigen, and obtaining an antigen concentrated liquid.

[0081] In the dissociation step, the modified magnetic particles are dissociated from the magnetic particle complex (a complex of the antigen and the modified magnetic particles) by the dissociation liquid, and are separated into the antigen and the modified magnetic particles. As a result, a dissociation liquid containing the antigen is obtained. Here, since the amount of the dissociation liquid is less than the amount of the "antigen-possibly-containing sample" used in the above mixing step, the concentration of the antigen in the dissociation liquid is higher than the concentration of the antigen in the "antigen-possibly-containing sample" used in the above mixing step. That is, by the dissociation step, a liquid in which the concentration of the antigen is concentrated (antigen-concentrated liquid) is obtained.

[0082] [The dissociation liquid]

[0083] The dissociation liquid is not particularly limited as long as it is a basic or acidic liquid.

[0084] The above basic liquid is not particularly limited, and examples thereof include an aqueous NaOH solution, an aqueous KOH solution, and the like. Among them, from the viewpoint of more excellent effects and the like of the present application, an aqueous NaOH solution is preferred.

[0085] The above acidic liquid is not particularly limited, and examples thereof include an aqueous HCl solution, an aqueous H2SO4 solution, an aqueous HNO3 solution, and the like. Among them, from the viewpoint of more excellent effects and the like of the present application, an aqueous HCl solution is preferred.

[0086] From the viewpoint of more excellent effects and the like of the present application, the dissociation liquid is preferably a basic liquid.

[0087] From the viewpoint of more excellent effects and the like of the present application, the dissociation liquid preferably contains NaOH or HCl, more preferably contains NaOH, and further preferably is an aqueous NaOH solution.

[0088] [Amount]

[0089] As described above, the amount of the dissociation liquid is less than the amount of the "antigen-possibly-containing sample" used in the above mixing step.

[0090] From the viewpoint of more excellent effects and the like of the present application, the ratio of the dissociation liquid to the "antigen-possibly-containing sample" used in the above mixing step is preferably 1 / 5 or less in terms of mass ratio.

[0091] [Neutralization step]

[0092] The neutralization step is a step of obtaining a neutralized antigen-concentrated liquid by neutralizing the antigen-concentrated liquid obtained in the above dissociation step using a neutralization liquid.

[0093] In the above dissociation step, since dissociation is performed using a dissociation liquid that is an alkaline or acidic liquid, the antigen concentrated solution obtained in the dissociation step is generally alkaline or acidic. On the other hand, in the case where an alkaline or acidic liquid is used in the development step described later, sometimes the first binding substance or the second binding substance described later is modified, resulting in a decrease in detection sensitivity. Therefore, in the neutralization step, the antigen concentrated solution is neutralized using a neutralization liquid.

[0094] [Neutralization liquid]

[0095] The neutralization liquid is not particularly limited, and for example, a well-known buffer solution can be used.

[0096] For the reason that the effects of the present application and the like are more excellent, in the case where the dissociation liquid used in the above dissociation step is an alkaline liquid, the neutralization liquid preferably contains HC1 (particularly, 1 M HC1) and at least one selected from the group consisting of tris(hydroxymethyl)methylglycine, TRIS, HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), acetamidoglycine, glycinamide, and N,N-dihydroxyethylglycine, and in the case where the dissociation liquid used in the above dissociation step is an acidic liquid, preferably contains NaOH (particularly, 1 M NaOH) and at least one selected from the group consisting of tris(hydroxymethyl)methylglycine, TRIS, HEPES, acetamidoglycine, glycinamide, and N,N-dihydroxyethylglycine.

[0097] [Amount]

[0098] For the reason that the effects of the present application and the like are more excellent, the amount of the neutralization liquid is preferably less than the amount of the "sample that can contain an antigen" used in the above mixing step.

[0099] For the reason that the effects of the present application and the like are more excellent, the ratio of the neutralization liquid to the "sample that can contain an antigen" used in the above mixing step is preferably 1 / 10 or less, more preferably 1 / 100 or less, in terms of mass ratio.

[0100] For the reason that the effects of the present application and the like are more excellent, the total amount of the above dissociation liquid and the above neutralization liquid is preferably less than the amount of the "sample that can contain an antigen" used in the above mixing step.

[0101] For the reason that the effects of the present application and the like are more excellent, the ratio of the total amount of the above dissociation liquid and the above neutralization liquid to the "sample that can contain an antigen" used in the above mixing step is preferably 1 / 2 or less, more preferably 1 / 3 or less, further preferably 1 / 4 or less, particularly preferably 1 / 5 or less.

[0102] [Development step]

[0103] The development step is a step in which a gold particle complex of a complex of an antigen in the neutralized antigen concentrate obtained in the neutralization step and a gold particle modified with a first binding substance capable of binding to the antigen, i.e., a modified gold particle, is developed on an insoluble carrier having a reaction site on which a second binding substance capable of binding to the antigen is immobilized.

[0104] (Gold particle complex)

[0105] As described above, in the development step, first, a gold particle complex of a complex of an antigen in the neutralized antigen concentrate obtained in the neutralization step and a gold particle modified with a first binding substance capable of binding to the antigen, i.e., a modified gold particle, is formed.

[0106] (Modified gold particle)

[0107] The modified gold particle is a gold particle modified with a first binding substance capable of binding to the antigen.

[0108] (Gold particle)

[0109] The gold particle is not particularly limited.

[0110] The gold particle functions as a catalyst for reducing silver ions in the silver amplification step described later.

[0111] The particle diameter of the gold particle is preferably 100 nm or less, more preferably 50 nm or less, further preferably 30 nm or less, and particularly preferably 15 nm or less, from the viewpoint of more excellent effects of the present application and the like.

[0112] The lower limit of the particle diameter of the gold particle is not particularly limited, but is preferably 1 nm or more, more preferably 2 nm or more, and further preferably 5 nm or more, from the viewpoint of more excellent effects of the present application and the like.

[0113] In addition, the particle diameter of the gold particle can be found by the same method as that for the magnetic particle.

[0114] (First binding substance)

[0115] The first binding substance is not particularly limited as long as it can bind to the antigen.

[0116] Specific examples and preferable modes of the first binding substance are the same as those of the "substance having specific affinity to an antigen" used in the mixing step.

[0117] (Method for producing modified gold particle)

[0118] The method for producing the above-described modified gold particles is not particularly limited, and a publicly known method can be used. For example, a chemical bonding method in which an SH group is introduced to an antibody by chemical bonding of gold to the SH group, and then the antibody is fixed using an Au-S bond generated on the Au surface when the antibody is brought into proximity with the gold particles, can be mentioned.

[0119] <Insoluble carrier>

[0120] The above-described insoluble carrier is an insoluble carrier having a reaction site (test line) on which a second binding substance capable of binding to the above-described antigen is fixed. The insoluble carrier can have a plurality of test lines depending on the type of antigen (for example, a test line for influenza A virus and a test line for influenza B virus). Also, in order to confirm the development of the above-described gold particle complex, the insoluble carrier can have a control line on the more downstream side than the test line. Also, in the case where a reducing agent solution is used in the silver amplification process described later, in order to detect the reducing agent solution, a color developing reagent immobilization line can be provided on the more downstream side than the test line.

[0121] As a specific example of the above-described insoluble carrier, for example, as shown in Figure 1 Fig. 1, a nitrocellulose membrane 100 having, from the upstream side, a gold colloid holding pad 10, a test line 20, a control line 30, and a color developing reagent immobilization line 40 can be mentioned. Among these, the gold colloid holding pad 10 is a pad that holds gold particles modified with a first binding substance (modified gold particles), and the test line 20 is a line on which a second binding substance is fixed. The control line 30 is a line for confirming development, and the color developing reagent immobilization line 40 is a line for detecting a reducing agent solution described later. Here, the upstream side and the downstream side refer to the direction of development from the upstream side to the downstream side when the gold particle complex develops.

[0122] As a more specific example of the above-described insoluble carrier (or an immunochromatography kit having the same), for example, the insoluble carrier and the immunochromatography kit described in Japanese Patent No. 5728453 can be mentioned. The contents described in Japanese Patent No. 5728453 regarding the insoluble carrier and the immunochromatography kit are all incorporated into the present specification as a part of the disclosure of the present specification.

[0123] <Insoluble carrier>

[0124] The insoluble carrier is preferably a porous carrier. In particular, for the reason that the effects of the present application are more excellent, a nitrocellulose membrane (nitrocellulose membrane), a cellulose membrane, an acetyl cellulose membrane, a polysulfone membrane, a polyethersulfone membrane, a nylon membrane, glass fiber, nonwoven fabric, cloth, or silk, and the like are preferable, and a nitrocellulose membrane is more preferable.

[0125] <Second binding substance>

[0126] The second binding substance is not particularly limited as long as it can bind to the antigen described above.

[0127] Specific examples of the second binding substance and the preferred mode are the same as the "substance having specific affinity to the antigen" used in the mixing step described above.

[0128] In addition, the second binding substance described above can be the same as or different from the first binding substance described above, but from the viewpoint of more excellent effects of the present application and the like, it is preferred that they be different substances.

[0129] Also, in the case where the first binding substance and the second binding substance are antibodies, from the viewpoint of more excellent effects of the present application and the like, it is preferred that the antibody as the first binding substance and the antibody as the second binding substance be different.

[0130] Also, in the case where the first binding substance and the second binding substance are antibodies, from the viewpoint of more excellent effects of the present application and the like, it is preferred that the epitope of the first binding substance (a part of the antigen recognized by the first binding substance) and the epitope of the second binding substance (a part of the antigen recognized by the second binding substance) be different. The difference in the epitope of the antibody can be confirmed by, for example, ELISA (Enzyme-Linked Immuno Sorbent Assay).

[0131] [Development]

[0132] The method of developing on the insoluble support having the test line in the state where the gold particle complex is formed is not particularly limited. For example, a method in which a nitrocellulose membrane 100 (or an immunochromatography kit having the nitrocellulose membrane 100) shown in FIG. 1 is prepared, the neutralized antigen concentrate solution obtained in the neutralization step described above is dropped onto the gold colloid holding pad, and it is moved from the upstream side to the downstream side using the capillary phenomenon as shown in FIG. 2, and the like can be given. Figure 1 Figure 1

[0133] [Capture Step]

[0134] The capture step is a step of capturing the gold particle complex described above at the reaction site of the insoluble support.

[0135] As described above, since the second binding substance capable of binding to the antigen is fixed at the reaction site of the insoluble support, the gold particle complex (the complex of the antigen and the modified gold particle) developed on the insoluble support in the development step described above is captured at the reaction site (the test line) of the insoluble support.

[0136] In addition, in the case where the sample does not contain the antigen, the gold particle complex described above is not formed, and thus the gold particle complex is not captured at the reaction site of the insoluble support.​​

[0137] [Silver amplification process]

[0138] The silver amplification process is a process of amplifying silver to the gold particle complex captured in the above-mentioned capturing process.

[0139] The silver amplification process is a process of forming large silver particles in the gold particle complex captured at the reaction site of the insoluble carrier after the above-mentioned capturing process by imparting silver ions to the insoluble carrier. More specifically, it is a process of reducing silver ions using the gold particles of the above-mentioned gold particle complex as a catalyst to form silver particles (for example, 10 μm or more in diameter).

[0140] Thus, the detection sensitivity of the captured gold particle complex is significantly improved.

[0141] [Preferred mode]

[0142] The method of imparting silver ions to the insoluble carrier after the above-mentioned capturing process is not particularly limited, but from the viewpoint of more excellent effects of the present application, a method using the following reducing agent solution and the following silver amplification solution is preferred.

[0143] Further, in addition to the reducing agent solution and the silver amplification solution, a washing solution can be used in order to wash the complex remaining in the insoluble carrier by a specific binding reaction. The above-mentioned reducing agent solution can also function as a washing solution.

[0144] <Reducing agent solution>

[0145] The above-mentioned reducing agent solution contains a reducing agent capable of reducing silver ions. The reducing agent capable of reducing silver ions can use any material of inorganic or organic or a mixture thereof as long as it can reduce silver ions to silver. As the inorganic reducing agent, a reducing metal salt capable of changing the valence by metal ions such as Fe 2+ , V 2+ , and Ti 3+ , and a reducing metal complex salt can be preferably selected. In the case of using an inorganic reducing agent, it is necessary to complex the oxidized ions or to remove or make harmless by reduction. For example, in the system using Fe 2+ as a reducing agent, citric acid or ethylenediaminetetraacetic acid (EDTA) can be used to form a complex of Fe 3+ as an oxide to make it harmless. In the present application, it is preferred to use such an inorganic reducing agent, and as a more preferred mode of the present application, it is preferred to use a metal salt of Fe 2+ as a reducing agent.

[0146] Further, developing main agents (e.g., methyl gallate salts, hydroquinone, substituted hydroquinones, 3-pyrazolidinones, p-aminophenols, p-phenylenediamines, hindered phenols, amidoximes, azines, catechols, pyrogallols, ascorbic acid (or derivatives thereof), and leuco dyes) used in wet silver halide photographic light-sensitive materials and other materials obvious to those skilled in the art, such as the materials described in U.S. Patent No. 6,020,117, and the like can be used as reducing agents.

[0147] As the reducing agent, an ascorbic acid reducing agent is also preferable. Useful ascorbic acid reducing agents include ascorbic acid and its analogs, isomers, and derivatives, and examples of the useful ascorbic acid reducing agents include D- or L-ascorbic acid and its sugar derivatives (e.g., γ-lactobionic acid ascorbic acid, gluconic acid ascorbic acid, alginic acid ascorbic acid, glucoheptonic acid ascorbic acid, maltobionic acid ascorbic acid), sodium salts of ascorbic acid, potassium salts of ascorbic acid, erythorbic acid (or L-erythromycin ascorbic acid) and its salts (e.g., alkali metal salts, ammonium salts, or salts known in the art), enediol-type ascorbic acid, enaminol-type ascorbic acid, thioenol-type ascorbic acid, and the like, with D, L, or D, L-ascorbic acid (and alkali metal salts thereof) or erythorbic acid (or alkali metal salts thereof) being particularly preferable, with sodium salts being preferable. If desired, mixtures of these reducing agents can be used.

[0148] For the reason that the effects of the present application are more excellent, the reducing agent solution preferably flows in a manner such that the angle between the development direction in the development step and the development direction of the reducing agent solution becomes 0 degrees to 150 degrees, and more preferably flows in a manner such that the angle between the development direction in the development step and the development direction of the reducing agent solution becomes 0 degrees to 135 degrees.

[0149] Further, as a method of adjusting the angle between the development direction in the development step and the development direction of the reducing agent solution, for example, the method described in the examples of Japanese Patent Application Publication No. 2009-150869, and the like can be mentioned.

[0150] < Silver Amplification Solution >

[0151] The silver amplification solution described above is a liquid containing a compound containing a silver ion. As the compound containing silver, for example, an organic silver salt, an inorganic silver salt, or a silver complex can be used. Preferred examples include silver nitrate, silver acetate, silver lactate, silver butyrate, and silver thiosulfate, which are compounds containing a silver ion having high solubility in a solvent such as water. Silver nitrate is particularly preferable. As the silver complex, a silver complex coordinated to a ligand having a water-soluble group such as a hydroxyl group and a sulfone group is preferable, and examples include hydroxyl sulfide silver and the like.

[0152] The organic silver salt, the inorganic silver salt, or the silver complex is contained in the silver amplification solution as silver at a concentration of 0.001 mol / L to 5 mol / L, preferably at a concentration of 0.005 mol / L to 3 mol / L, and more preferably at a concentration of 0.01 mol / L to 1 mol / L.

[0153] As the adjuvant of the silver amplification solution, a buffer, a preservative such as an antioxidant or an organic stabilizer, a speed modifier, and the like can be given. As the buffer, for example, acetic acid, citric acid, sodium hydroxide, or one of these salts, or a buffer using tris(hydroxymethyl)aminomethane, other buffers generally used in chemical experiments can be used. By appropriately using these buffers, it is possible to adjust the pH to the most suitable one for the amplification solution thereof. Also, as the antifogging agent, an alkylamine can be used as the adjuvant, and particularly, dodecylamine is preferable. Also, in order to improve the solubility of these adjuvants, a surfactant can be used, and particularly, C9H 19 -C6H4-O-(CH2CH2O) 50 H.

[0154] For the reason that the effect of the present application is more excellent, the silver amplification solution is preferably flowed from the direction opposite to the development direction in the development process, and more preferably flowed in a manner that the angle between the development direction in the development process and the development direction of the silver amplification solution becomes 45 degrees to 180 degrees.

[0155] Further, as the method of adjusting the angle between the development direction in the development process and the development direction of the silver amplification solution, for example, the method described in the example of Japanese Patent Application Laid-Open No. 2009-150869, and the like can be given.

[0156] Example

[0157] Hereinafter, the present application will be further described in detail by examples, but the present application is not limited thereto.

[0158] [A] Example in which the antigen is LAM

[0159] [Preparation of the test liquid]

[0160] A liparabinomannan (02249-61, Nacalai Tesque Inc.) extracted from Mycobacterium tuberculosis was added to a urine test liquid (Bioreclamation IVT Inc.) in which the urine of a healthy person was stored, and a test liquid (a sample that can contain an antigen) having the LAM concentration described in Table 1 was prepared.

[0161] [Example A]

[0162] The immunochromatography method of Example A was performed as follows.

[0163] [Development process]

[0164] The magnetic particles (Dynabeads MyOne-COOH, particle size: 1 pm, manufactured by Thermo Fisher Scientific Inc.) were activated with EDC (1-ethyl-3-(3- dimethylaminopropyl) carbodiimide), and an anti-LAM monoclonal antibody (a substance having specific affinity to an antigen) was supported on the magnetic particles, to obtain modified magnetic particles, i.e., magnetic particles modified with the anti-LAM monoclonal antibody. In addition, the anti-LAM monoclonal antibody used was antibody A194-01, which was produced according to the production method of anti-lipid arabino mannan monoclonal antibody (A194-01 described in International Publication No. 2017 / 139153 (DETAILED DESCRIPTION D. Anti-LAM and Anti-PIM6 / LAM Antibodies 1. A194-01)).

[0165] To a 15-mL conical tube, 6 mL of the above-described test body fluid was added, 1 mg of the above-described modified magnetic particles was added thereto, and the mixture was stirred for 40 minutes and allowed to react. In this way, a mixture containing magnetic particle complexes of LAM and the above-described modified magnetic particles was obtained.

[0166] [Trapping Step]

[0167] After the reaction, the conical tube was placed on a magnet stand, and the magnetic particles (unmodified magnetic particles, modified magnetic particles, magnetic particle complexes) were magnetically collected for 10 minutes. The above-described magnetic particles (unmodified magnetic particles, modified magnetic particles, magnetic particle complexes) were trapped by extracting the remaining test body fluid from the conical tube with a pipette (manufactured by Eppendorf).

[0168] [Trapping Step]

[0169] Immediately, 200 pL of an aqueous 50 mM NaOH solution (alkaline liquid) was added. After the addition, 5-minute ultrasonic treatment was performed, and the mixture was allowed to stand for 60 minutes. In this way, the modified magnetic particles were dissociated from the magnetic particle complexes. After the standing, the conical tube was again placed on a magnet stand, the dissociated modified magnetic particles were magnetically collected for 10 minutes, and the supernatant (LAM concentrate) was recovered.

[0170] In addition, the ratio of the dissociation liquid to the test body fluid was 1 / 30 in terms of mass ratio.

[0171] [Neutralization Step]

[0172] The above LAM concentrate was neutralized using 10 μL of 10 mM TRIS-HCl as a neutralizing solution to obtain a neutralized LAM concentrate (neutralized antigen concentrate).

[0173] [Development process]

[0174] As shown in Figure 1 FIG. 1, a nitrocellulose membrane 100 having a gold colloid holding pad 10, a test line 20, a control line 30, and a color developing reagent immobilized line 40 from the upstream side was prepared. In addition, the gold colloid holding pad 10 is a pad that holds gold colloids modified with anti-LAM monoclonal antibodies (modified gold particles), the test line 20 is a line on which anti-LAM monoclonal antibodies are immobilized, the control line 30 is a line for confirming development, and the color developing reagent immobilized line 40 is a line for detecting a reducing agent solution described later.

[0175] The above neutralized LAM concentrate was added dropwise to the gold colloid holding pad. Thereby, gold particle complexes as complexes of LAM in a liquid and gold colloid particles modified with anti-LAM monoclonal antibodies (modified gold particles) in the gold colloid holding pad were formed. In this state, development was performed toward the downstream side of the above nitrocellulose membrane.

[0176] [Capture process]

[0177] The gold particle complexes developed in the development process were captured on the test line.

[0178] [Silver amplification process]

[0179] The silver amplification process was performed as follows.

[0180] <Preparation of reducing agent solution>

[0181] In 290 g of water, 23.6 mL of an aqueous iron nitrate (III) solution at 1 mol / L, which was prepared by dissolving iron nitrate (III) nine hydrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) in water, and 13.1 g of citric acid (manufactured by FUJIFILM Wako Pure Chemical Corporation) were dissolved. After all of the components were dissolved, 36 mL of nitric acid (10 mass%) was added while stirring with a stirrer, and 60.8 g of iron (II) sulfate ammonium hexahydrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) was added as a reducing agent solution.

[0182] <Preparation of silver amplification solution>

[0183] To 66 g of water, 8 mL of a silver nitrate solution (containing 10 g of silver nitrate) and 24 mL of a 1 mol / L aqueous ferric nitrate solution were added. In addition, this solution was mixed with a solution prepared by dissolving nitric acid (10 mass%) 5.9 mL, dodecylamine (manufactured by FUJIFILM Wako Pure Chemical Corporation) 0.1 g, and surfactant C 12 H 25 -C6H4-O-(CH2CH2O) 50 H 0.1 g, and this was used as a silver amplification solution.

[0184] <Development of the reductant solution>

[0185] The reductant solution prepared as described above was made to flow in the nitrocellulose membrane from the same direction (from the more upstream side) as in the above development process.

[0186] <Development of the silver amplification solution>

[0187] After the color reagent immobilized line changed color, the silver amplification solution prepared as described above was made to flow from the direction opposite to the development direction in the development process (from the downstream side). Thus, silver amplification was performed on the gold particle complex captured on the test line.

[0188] [Evaluation]

[0189] The coloring of the test line was confirmed by the naked eye, and evaluation was performed according to the following criteria.

[0190] ++: coloring was dense

[0191] +: there was coloring

[0192] -: there was no coloring

[0193] The results are shown in Table 1. The smaller the minimum LAM concentration (minimum detection sensitivity) among the LAM concentrations of the test body fluids evaluated as ++ or +, the higher the detection sensitivity was indicated.

[0194] [Comparative Example A]

[0195] The immunoassay was performed according to the same procedure as in Example A, except that the development process was performed using the test body fluid itself instead of the neutralized LAM concentrate solution, and the mixing process to the neutralization process were not performed. The results are shown in Table 1.

[0196] [Table 1]

[0197]

[0198] As shown in Table 1, Example A in which the magnetic particle process was performed showed higher detection sensitivity compared to Comparative Example A in which the magnetic particle process was not performed (from the mixing step to the neutralization step).

[0199] Examples of the antigen being an influenza virus

[0200] [Preparation of the test body fluid]

[0201] A test body fluid (a liquid that can contain an antigen) having an influenza virus concentration (plaque forming unit (PFU) concentration) described in Table 2 was prepared by diluting a simulated positive test body (BD Flu Exam Control A+B- (Becton, Dickinson and Company)) with an extraction solution (1 mass% BIGCHAP (N, N-Bis(3-D-gluconamidopropyl) cholamide) 1 mass% BSA (Bovine Serum Albumin)-PBS (Phosphate buffered salts)).

[0202] [Example B1]

[0203] The immunochromatography method of Example B1 was performed as follows.

[0204] [The mixing step]

[0205] The above test body fluid 5.2 mL and magnetic particles (Dynabeads MyOne Carboxylic acid, particle diameter: 1 pm, Thermo Fisher Scientific Inc.) modified with an anti-influenza A monoclonal antibody (Anti-Influenza A SPT N-57307, Medix Biochemica Corporation) were mixed, stirred, and allowed to react. In this way, a mixture of magnetic particle complexes containing complexes of influenza viruses and magnetic particles modified with an anti-influenza A monoclonal antibody, i.e., modified magnetic particles, was obtained.

[0206] [The capturing step]

[0207] After the reaction, the magnetic particles (unmodified magnetic particles, modified magnetic particles, magnetic particle complexes) were collected using a magnet, and the supernatant was removed, thereby capturing the above magnetic particles (unmodified magnetic particles, modified magnetic particles, magnetic particle complexes).

[0208] [The dissociation step]

[0209] Then, 200 μL of a 50 mM NaOH aqueous solution (alkaline liquid) was added dropwise, and the mixture was stirred. This dissociated the modified magnetic particles from the magnetic particle complex. The dissociated modified magnetic particles were then collected again using the same magnet, and all the supernatant (influenza virus concentrate) (antigen concentrate) was recovered.

[0210] In addition, the ratio of the dissociation fluid to the tested body fluid is 1 / 26 by mass.

[0211] [Neutralization process]

[0212] The recovered supernatant was neutralized by adding 9.1 μL of 1M HCl tris(hydroxymethyl)methylglycine, yielding a neutralized influenza virus concentrate (neutralized antigen concentrate). This concentrates the influenza virus concentration in the tested body fluids by 25 times.

[0213] [Development Process]

[0214] like Figure 1 As shown, a nitrocellulose membrane 100 is prepared, comprising a gold colloid holding pad 10, a test line 20, a control line 30, and a chromogenic reagent immobilization line 40, starting from the upstream side. The gold colloid holding pad 10 is a pad holding gold colloid (modified gold particles) modified with anti-influenza A monoclonal antibody; the test line 20 is a line immobilized with anti-influenza A monoclonal antibody; the control line 30 is a line used to confirm the development; and the chromogenic reagent immobilization line 40 is a line used to detect the reducing agent solution described later.

[0215] The above-mentioned neutralized influenza virus concentrate was dropped onto a gold colloidal holding pad. This formed a gold particle complex, which was a complex of the influenza virus in liquid and gold colloidal particles modified with anti-influenza A monoclonal antibodies (modified gold particles). In this state, it was spread downstream of the aforementioned nitrocellulose membrane.

[0216] [Capture Process]

[0217] The gold particle complex that unfolds during the unfolding process is captured on the test line.

[0218] [Silver Amplification Process]

[0219] The silver amplification process shall be performed in accordance with the same steps as in Example A.

[0220] Thus, the gold particle complexes captured on the test line were amplified with silver.

[0221] 〔evaluate〕

[0222] The coloration of the test lines was confirmed by visual inspection, and the evaluation was conducted according to the following criteria.

[0223] Positive: Confirmed staining.

[0224] Negative: No coloring was confirmed.

[0225] The results are shown in Table 2. The smaller the minimum concentration of influenza virus among the concentrations of the influenza virus of the test body fluid evaluated as positive (minimum detection sensitivity) is, the higher the detection sensitivity is.

[0226] [Example B2]

[0227] In the dissociation process, 200 μL of 50 mM aqueous HC1 (acidic liquid) was used instead of 200 μL of 50 mM aqueous NaOH, and in the neutralization process, 9.1 μL of 1 M NaOH tris(hydroxymethyl)methylglycine was used instead of 9.1 μL of 1 M HC1 tris(hydroxymethyl)methylglycine, and otherwise, the immunoassay was performed according to the same procedure as Example Bl, and was evaluated. The results are shown in Table 2.

[0228] [Comparative Example B]

[0229] The immunoassay was performed according to the same procedure as Example Bl, except that the process from the mixing process to the neutralization process was not performed, and in the development process, the above test body fluid was used instead of the neutralized influenza virus concentrate, and was evaluated. The results are shown in Table 2.

[0230] [Table 2]

[0231]

[0232] In addition, in Table 2, "E+" indicates an exponent, for example, "5.00E+03" indicates "5.00 x 10 3 ".

[0233] As is clear from Table 2, Examples Bl to B2 in which the magnetic particle process was performed showed higher detection sensitivity than Comparative Example B in which the magnetic particle process (from the mixing process to the neutralization process) was not performed. Among them, Example Bl in which the dissociation liquid contained NaOH showed even higher detection sensitivity.

[0234] [C] Reference

[0235] As a reference, as a simulated test body for detecting a bacterial test body, a 500 nm particulate analyte (reference example) was compared with the molecular analyte used in Example Bl.

[0236] We plan to prepare 500nm silica beads (manufactured by Polysciences, Inc.: product name Uniform Silica Microspheres #24759) with streptavidin adsorbed on their surface. Furthermore, we plan to replace the antibody-modified gold colloid with gold colloids (manufactured by Cosmo Bio Co., Ltd.: Biotin Gold Nanoparticles, 50nm) with biotin adsorbed on their surface by reacting with the silica beads.

[0237] To achieve the same concentration as the magnetic concentration in Example B1, silica beads were separated by centrifugation. After discarding the supernatant, the concentration of the test sample was prepared to be the same as in Example B1. The determination was performed using the above-described developing process with a kit containing biotin-adsorbed gold colloids. The process was the same as in Example B1. The concentration of the background before and after silver development was used as noise, and the results were evaluated visually according to the following criteria. The results are shown in Table 3.

[0238] A: No concentration was observed at all.

[0239] B: Very low concentrations were observed.

[0240] C: A slight increase in concentration was observed.

[0241] D: Capable of recognizing increases in concentration, but within permissible levels.

[0242] [Table 3]

[0243] Example B1 Reference Example Antigen Influenza virus 500 nm silica beads (mock fungus) Noise before silver amplification A A Noise after silver amplification B C

[0244] As shown in Table 3, regarding the noise before silver amplification, the noise levels for particulate and molecular specimens similar to bacterial specimens are the same. However, regarding the noise after silver amplification for high sensitivity, the noise of molecular specimens is slightly lower than that of particulate specimens similar to bacterial specimens.

[0245] Symbol Explanation

[0246] 10 Gold Colloidal Retention Pad

[0247] 20 test lines

[0248] 30 control lines

[0249] 40 Immobilization lines for colorimetric reagents

[0250] 100 Nitrocellulose Membrane

Claims

1. An immunochromatography method comprising: a mixing step of obtaining a mixture containing magnetic particle complexes as complexes of an antigen and modified magnetic particles by mixing a sample that can contain the antigen and the modified magnetic particles modified with a substance having specific affinity to the antigen; a capturing step of capturing magnetic particles in the mixture containing the magnetic particle complexes using magnetism; a dissociation step of obtaining an antigen concentrate by dissociating the modified magnetic particles from the magnetic particle complexes by mixing the magnetic particles captured in the capturing step and a dissociation liquid that is a basic or acidic liquid and is less than the sample that can contain the antigen, the dissociation liquid containing NaOH or HC1; a neutralization step of obtaining a neutralized antigen concentrate by neutralizing the antigen concentrate using a neutralization liquid, the neutralization liquid containing HC1 and at least one selected from the group consisting of tris(hydroxymethyl)methylglycine, TRIS, HEPES, acetamidoglycine, glycylamide, and N,N-dihydroxyethylglycine or containing NaOH and at least one selected from the group consisting of tris(hydroxymethyl)methylglycine, TRIS, HEPES, acetamidoglycine, glycylamide, and N,N-dihydroxyethylglycine; a development step of developing gold particle complexes as complexes of the antigen in the neutralized antigen concentrate and gold particles modified with a first binding substance capable of binding to the antigen, the gold particles being modified gold particles, on an insoluble carrier having a reaction site at which a second binding substance capable of binding to the antigen is immobilized; a capturing step of capturing the gold particle complexes at the reaction site of the insoluble carrier; and a silver amplification step of performing silver amplification on the gold particle complexes captured in the capturing step.

2. The immunochromatography method according to claim 1, wherein the ratio of the dissociation liquid to the sample that can contain the antigen is 1 / 5 or less in terms of mass ratio.

3. The immunochromatography method according to claim 1 or 2, wherein the sample that can contain the antigen is urine.

4. The immunochromatography method according to claim 1, wherein the antigen is a sugar chain.

5. The immunochromatography method according to claim 1, wherein the antigen is a lipoarabinomannan.

6. The immunochromatography method according to claim 1, wherein the particle diameter of the magnetic particles before modification is 0.05 μm to 10 μm.

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