JP7708575B2 - Immunochromatography Kit - Google Patents

Description

The present invention relates to an immunochromatography kit, and in particular to an immunochromatography kit that uses gold-coated silver nanoplates.

Silver nanoplates absorb light through interaction with light (localized surface plasmon resonance: LSPR), and it is known that suspensions of these nanoplates exhibit a color that depends on the shape of the nanoplates. It is also known that the amount of light absorbed, and therefore the color, can be changed by controlling the size and shape of the silver nanoplates. On the other hand, silver nanoplates dissolve and change shape through oxidation. This change in shape of the silver nanoplates due to oxidation can cause the intended color change.

Therefore, in order to stabilize the silver nanoplate against oxidation, the surface of the silver nanoplate is coated with gold. Patent Document 1 describes that the gold-coated silver nanoplate is stable and can be suitably used in immunochromatography tests. Patent Document 2 describes that by adjusting the concentration and pH of the water-soluble polymer, the gold-coated silver nanoplate is prepared in the form of a stable suspension, which can be used as a label for a detection reagent for a test substance (for example, a label for an antibody used to detect a target protein) and applied to detect various test substances with high sensitivity.

Known test strips used in immunochromatography tests include "half strips" consisting of a membrane and an absorbent pad, and "full strips" that also include a sample pad and a conjugate pad, with "full strips" being the most widely sold commercially. In half strips, the detection reagent for the test substance is used as the developing liquid, but in full strips, the detection reagent for the test substance is impregnated into the conjugate pad and dried, so that it can be used simply by applying a sample containing the test substance to the sample pad.

JP 2015-194495 A International Publication No. 2015/182770

Conventional gold-coated silver nanoplates can be used effectively in half strips, but in full strips, color development may decrease. The cause of this is thought to be that the gold-coated silver nanoplates are deteriorated by oxidation when the conjugate pad is impregnated with a detection reagent for the test substance containing the gold-coated silver nanoplates and then dried. Conventional gold-coated silver nanoplates can only form a thin coating relative to the size of the core silver nanoplate, and have insufficient resistance to oxidation, resulting in insufficient detection sensitivity in immunochromatography tests. Therefore, the present invention aims to provide an immunochromatography kit that uses gold-coated silver nanoplates that are highly resistant to oxidation.

As a result of intensive research aimed at solving the above problems, the present inventors discovered that high detection sensitivity can be obtained even in full stripping by using a gold-coated silver nanoplate having a thick gold layer, and thus completed the present invention. That is, the present invention provides the following immunochromatography kit.
[1] A test strip including a membrane;
A gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate;
An immunochromatography kit for detecting a test substance in a sample, comprising:
A specific binding substance for capturing the test substance is immobilized on the membrane,
An immunochromatography kit, wherein the average thickness of the gold layer is greater than 1.0 nm, and the gold-coated silver nanoplate carries a specific binding substance for detection of the test substance.
[2] The immunochromatography kit according to [1], wherein the average thickness of the gold layer is 1.5 nm to 10.0 nm.
[3] The immunochromatography kit according to [1] or [2], wherein the gold-coated silver nanoplate is contained in a dry state.
[4] The immunochromatography kit according to any one of [1] to [3], wherein the test strip comprises a conjugate pad containing the gold-coated silver nanoplate.
[5] The gold layer is formed on a main plane and an end plane of the silver nanoplate,
The immunochromatography kit according to any one of [1] to [4], wherein the ratio (T/D) of the average thickness (T) of the gold layer at the end surface to the average particle diameter (D) of the silver nanoplates is 0.05 or more.
[6] The immunochromatography kit according to any one of [1] to [5], wherein the average particle diameter of the silver nanoplate as the core is 55 nm or less.
[7] The immunochromatography kit according to any one of [1] to [6], wherein the gold-coated silver nanoplate has a maximum absorption wavelength in the visible light region, and the ratio (E _max /E ₉₀₀ ) of the extinction degree (E _max ) at the maximum absorption wavelength to the extinction degree (E ₉₀₀ ) at a wavelength of 900 nm of the gold-coated silver nanoplate is 5 or more.
[8] The immunochromatography kit according to any one of [1] to [7], wherein the capture specific binding substance and/or the detection specific binding substance comprises an antibody.
[9] The sample contains a plurality of test substances,
a capture specific binding substance for each of the plurality of types of test substances is immobilized on the membrane;
The immunochromatography kit according to any one of [1] to [8], wherein the gold-coated silver nanoplate carries a detection specific binding substance for each of the multiple types of test substances.
[10] The immunochromatography kit according to [9], wherein the capture specific binding substance is immobilized at a different site depending on the type of test substance to be bound.
[11] The immunochromatography kit according to [9] or [10], wherein the specific binding substance for detection is supported on a gold-coated silver nanoplate having a different maximum absorption wavelength for each type of test substance to be bound.
[12] The immunochromatography kit according to any one of [1] to [11], wherein the membrane further comprises a control site for determining the success or failure of the immunochromatography test.
[13] Further comprising a control specific binding substance that specifically binds to the control site;
The immunochromatography kit according to claim 12, wherein, when the control specific binding substance contains an antibody and the capture specific binding substance contains an antibody, the antibody of the control specific binding substance is derived from a host animal different from the antibody of the capture specific binding substance.
[14] The immunochromatography kit according to any one of [1] to [13], further comprising nanoparticles other than the gold-coated silver nanoplates.
[15] The immunochromatography kit according to any one of [1] to [14], wherein the test substance comprises at least one antigen selected from the group consisting of influenza virus antigens and coronavirus antigens.

According to the present invention, by using a gold-coated silver nanoplate with a thick gold layer, high detection sensitivity can be obtained even when performing immunochromatography tests using full strips that are at high risk of degradation due to oxidation.

1 shows the spectroscopic spectra of suspensions of gold-coated silver nanoplates (Ag@Au Suspensions 1 to 3). 1 shows a high angle annular dark field scanning transmission microscope (HAADF-STEM) image of gold-coated silver nanoplates in Ag@Au suspension 2. 1 shows the spectroscopic spectra of suspensions of gold-coated silver nanoplates (Ag@Au Suspensions 4 to 6).

The present invention will now be described in further detail.
The present invention relates to an immunochromatography kit for detecting a test substance in a sample, the immunochromatography kit comprising:
a test strip including a membrane on which a specific binding substance for capturing the test substance is immobilized;
A gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate, and carrying a specific binding substance for detecting the test substance;
Contains:

The term "silver nanoplate" as used herein refers to a plate-like particle made of silver and having a size on the order of nanometers (nm). It has upper and lower surfaces that are the main planes, and end surfaces (side surfaces) between the upper and lower surfaces. The end surfaces may be flat or curved, and may have rounded corners. The shape of the upper and lower surfaces of the silver nanoplate is not particularly limited, and may be, for example, a polygon such as a triangle, a square, a pentagon, or a hexagon (including shapes with rounded corners), or a circle. In the gold-coated silver nanoplate, the gold layer is formed on the main planes and end surfaces of the silver nanoplate, forming a so-called core-shell structure of the silver nanoplate and the gold layer.

The maximum long diameter of the main plane of the silver nanoplate is called the particle diameter, which corresponds to the diameter in the case of a circle, and corresponds to the length of the maximum side in the case of a triangle. If the maximum long diameter differs between the upper and lower surfaces of the silver nanoplate, the longer one is the maximum long diameter of the silver nanoplate. The upper limit of the average particle diameter of the silver nanoplate used in the suspension of the present invention is not particularly limited, but may be, for example, about 55 nm or less, and preferably about 50 nm or less, about 40 nm or less, or about 30 nm or less. The lower limit of the average particle diameter of the silver nanoplate is also not particularly limited, but may be, for example, about 1 nm or more, and preferably about 10 nm or more. The average thickness of the silver nanoplate is also not particularly limited, but may be, for example, about 20 nm or less, and preferably about 3 to about 15 nm. The particle size and thickness of the silver nanoplates may be measured, for example, by scanning electron microscope (SEM), scanning transmission electron microscope (STEM), or transmission electron microscope (TEM) observation, and the particle size may be measured by a dynamic light scattering particle size distribution analyzer (DLS). When measuring the particle size of the silver nanoplates using SEM observation photographs, STEM observation photographs, or TEM observation photographs, 80 points of data are prepared by excluding the top and bottom 10% from a total of 100 points of data obtained by measuring the particle size of 100 arbitrary silver nanoplates, and the average particle size may be calculated by averaging these data.

The ratio of the maximum major axis to the width of the axis perpendicular to the plane that contains it, i.e., the index defined as "maximum major axis/thickness", is called the "aspect ratio", and this can be used as one of the indices for expressing the shape of the nanoplate. When the shape of the silver nanoplate changes, the position of its maximum absorption wavelength also changes, so the aspect of the silver nanoplate can also be said to be an index of the maximum absorption wavelength. In the suspension of the present invention, the aspect ratio of the silver nanoplate is not particularly limited, but may be, for example, greater than about 1, and is preferably about 1.5 to about 10. When the aspect ratio is in this range, it is easy to adjust the maximum absorption wavelength due to surface plasmon resonance in the aqueous suspension to the visible light region.

In the immunochromatography kit of the present invention, the average thickness of the gold layer of the gold-coated silver nanoplate is thicker than that used in conventional immunochromatography kits, and is greater than about 1.0 nm, particularly at the end surface. A gold-coated silver nanoplate with a thick gold layer has high oxidation resistance and therefore high storage stability, and can detect test substances with good sensitivity even in the form of an immunochromatography kit that is not used immediately after preparation. In one embodiment, the average thickness of the gold layer of the gold-coated silver nanoplate is about 1.5 nm to about 10.0 nm. When the thickness of the gold layer is in this range, the oxidation of the silver nanoplate can be more effectively suppressed while maintaining the optical properties of the silver nanoplate.

The thickness of the gold layer at the end surface can be measured using a high-angle annular dark-field scanning transmission microscope (HAADF-STEM). Specifically, for 10 particles selected from the HAADF-STEM observation photograph, the gold thickness is measured at 10 points at any end of each particle, for a total of 100 points of data. Of these, 80 points of data are prepared by excluding the top and bottom 10%, and the average thickness (T) is calculated by averaging these data.

The thickness of the gold layer at the end face can also be calculated from the size of the silver nanoplate before and after gold coating measured using a transmission scanning microscope. For example, when the shape of the main plane of the silver nanoplate is circular, the average particle size (a) before gold coating and the average particle size (b) after gold coating are measured, and the thickness of the gold layer at the end face can be calculated using the following formula:
T = (b - a) / 2
When the shape of the main plane of the silver nanoplate is an equilateral triangle, the average height (c) of the equilateral triangle before and after gold coating is measured, and the average thickness (T) can be calculated by the following formula:
T = (d-c)/3
The average thickness (T) can be calculated by:

The color tone exhibited by the suspension of the present invention can be adjusted by adjusting the size and/or shape of the core silver nanoplate and the thickness of the gold layer to change the maximum absorption wavelength of the gold-coated silver nanoplate. In other words, the size and shape of the silver nanoplate are adjusted to take into account the change in maximum absorption wavelength due to subsequent gold coating, so that the suspension of the present invention has a maximum absorption wavelength in the visible light region. In one embodiment, the maximum absorption wavelength is between about 400 nm and about 680 nm.

In one embodiment, the gold-coated silver nanoplates are contained in the immunochromatography kit of the present invention in a dried state. The method for drying the gold-coated silver nanoplates is not particularly limited, but for example, the gold-coated silver nanoplates may be dried at room temperature under reduced pressure, or may be dried by freeze-drying. The gold-coated silver nanoplates used in the present invention can be in such a form because they have a thick gold layer and high resistance to oxidation.

In one embodiment, the ratio (T/D) of the average thickness (T) of the gold layer at the end surface to the average particle diameter (D) of the silver nanoplate is about 0.05 or more, preferably about 0.1 or more, and more preferably about 0.16 or more. That is, the gold-coated silver nanoplate may have a thick gold layer considering its small particle diameter. Since gold-coated silver nanoplates with small particle diameters have a maximum absorption wavelength in the visible light region, it is possible to multicolorize the detection method of a test substance that requires visual judgment, such as immunochromatography. In one embodiment, T/D may be about 0.5 or less, about 0.4 or less, or about 0.3 or less.

In one embodiment, the gold-coated silver nanoplate has a maximum absorption wavelength in the visible light region, and the ratio (E _max /E ₉₀₀ ) of the extinction degree (E _max ) at the maximum absorption wavelength to the extinction degree (E ₉₀₀ ) at a wavelength of 900 nm of the gold-coated silver nanoplate may be about 5 or more, preferably about 10 or more. That is, the gold-coated silver nanoplate exhibits a sharp spectrum, which is due to the high uniformity of the particle size. Therefore, the color of the gold-coated silver nanoplate is vivid and has high saturation.

As the silver nanoplate for preparing the gold-coated silver nanoplate in the immunochromatography kit of the present invention, those commonly used in the art can be used without any particular limitation, and for example, a commercially available product may be used, or one manufactured according to a known manufacturing method or a method described in the Examples below may be used. Note that when manufacturing a gold-coated silver nanoplate having a thick gold layer for a small particle size as mentioned in the specific embodiment above, attention should be paid to the elution of silver from the core silver nanoplate, the oxidation of the silver nanoplate, and the formation of agglomerates due to the grown gold layer. For example, in the coating mixture used to form the gold layer (containing the silver nanoplate, the gold-containing water-soluble compound, and the gold ion complexing agent), the silver concentration (C _Ag ) is about 0.25 mM or less, the gold concentration (C _Au ) is about 0.1 mM or more, and the ratio of C _Au to C _Ag (C _Au /C _Ag ) is about 0.50 or more. By adjusting the silver concentration and gold concentration and allowing the coating mixture to stand for a predetermined period of time, a gold-coated silver nanoplate having a thick gold layer despite its small particle size can be efficiently and stably produced. For more details, please refer to the specification of Japanese Patent Application No. 2020-149087.

The gold-coated silver nanoplate in the immunochromatography kit of the present invention carries a specific binding substance for detection of the test substance. The term "carrying" as used herein means that the gold-coated silver nanoplate and the specific binding substance for detection are bound to each other to form a complex, regardless of the mode of binding, such as covalent or non-covalent binding, or direct or indirect binding. As a method of carrying, any ordinary carrying method can be used without particular limitation. A method of directly binding the gold-coated silver nanoplate and the specific binding substance for detection using physical adsorption, chemical adsorption (covalent binding to the surface), chemical bonding (covalent bonding, coordinate bonding, ionic bonding, or metallic bonding), etc., or a method of binding a part of a water-soluble polymer to the surface of the gold-coated silver nanoplate and directly or indirectly binding the specific binding substance for detection to the end, main chain, or side chain of the water-soluble polymer can be adopted. For example, when the specific binding substance for detection is an antibody, the gold-coated silver nanoplate and the antibody solution are mixed, shaken, and centrifuged to obtain the gold-coated silver nanoplate carrying the antibody (labeled detection reagent) as a precipitate. In addition, when the gold-coated silver nanoplate and the antibody are supported by electrostatic adsorption, the efficiency of supporting the antibody can be improved by coating the surface of the gold-coated silver nanoplate with negatively charged polystyrene sulfonic acid.

The capture specific binding substance or the detection specific binding substance (sometimes collectively referred to as "specific binding substance for the test substance") can be any substance that can form a complex with the test substance to be detected and that can use the gold-coated silver nanoplate as a label, without any particular restrictions. Specific examples of combinations of a test substance and a substance that specifically binds to the test substance include an antigen and an antibody that binds to it, an antibody and an antigen that binds to it, a sugar chain or glycoconjugate and a lectin that binds to it, a lectin and a sugar chain or glycoconjugate that binds to it, a hormone or cytokine and a receptor that binds to it, a receptor and a hormone or cytokine that binds to it, a protein and a nucleic acid aptamer or peptide aptamer that binds to it, an enzyme and a substrate that binds to it, a substrate and an enzyme that binds to it, biotin and avidin or streptavidin, avidin or streptavidin and biotin, IgG and protein A or protein G, protein A or protein G and IgG, T cell immunoglobulin mucin domain-containing molecule 4 (Tim4) and phosphatidylserine (PS), PS and Tim4, or a first nucleic acid and a second nucleic acid that binds to it (hybridizes to it). The second nucleic acid may be a nucleic acid that includes a sequence complementary to the first nucleic acid.

When the test substance is an antigen, the substance that specifically binds to the antigen may be an antibody. The antibody may be a polyclonal antibody, a monoclonal antibody, a single chain antibody, or a fragment thereof that specifically binds to the antigen, and the fragment may be an F(ab) fragment, an F(ab') fragment, _an F(ab') fragment, or an F(v) fragment. The antigen as the test substance may be a lectin such as concanavalin A (ConA), malt agglutinin, or ricin, or may be a virus such as influenza virus, coronavirus, adenovirus, respiratory syncytial virus, rotavirus, human papillomavirus, human immunodeficiency virus, hepatitis B virus, Zika virus, or dengue virus, or a substance contained therein (e.g., hepatitis B virus antigen (HBs antigen) or influenza virus hemagglutinin), or may be an antigen such as Aspergillus flavus, Chlamydia, Treponema pallidum, Streptococcus, Bacillus anthracis, Staphylococcus aureus, Shigella, Escherichia coli, Salmonella enterica, Salmonella typhimurium, Salmonella paratyphi, Pseudomonas aeruginosa, or a virulent pathogen such as Aspergillus flavus. The pathogenic microorganism may be a pathogenic microorganism such as Vibrio parahaemolyticus or a substance contained therein (for example, aflatoxin (B1, B2, G1, G2, or M1, etc.) of Aspergillus flavus, verotoxin of enterohemorrhagic Escherichia coli, or streptolysin O of Streptococcus hemolyticus), a blood protein such as immunoglobulin G (IgG), rheumatoid factor, and C-reactive protein (CRP), a glycoprotein such as mucin, or a pituitary hormone (for example, growth hormone (GH), adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, or melanocyte stimulating hormone (MSH), etc.). The hormone may be a hormone such as thyrotropin releasing hormone (TH), thyrotropin releasing hormone (TRH), thyroid hormone (e.g., diiodothyronine or triiodothyronine), chorionic gonadotropin, calcium metabolism regulating hormone (e.g., calcitonin or parathormone), pancreatic hormone, gastrointestinal hormone, vasoactive intestinal peptide, follicular hormone (e.g., estrone), natural or synthetic luteal hormone (e.g., progesterone), male hormone (e.g., testosterone), and adrenal cortex hormone (e.g., cortisol), or may be other biological substances such as serotonin, urokinase, ferritin, substance P, prostaglandin, and cholesterol. The specific binding substance may be a tumor marker such as prostatic acid phosphatase (PAP), prostate specific antigen (PSA), alkaline phosphatase, transaminase, trypsin, pepsinogen, α-fetoprotein (AFP) and carcinoembryonic antigen (CEA), a carbohydrate antigen such as a blood group antigen, a marker used for detecting fecal occult blood such as hemoglobin and transferrin, a D-dimer which is a kind of fibrin degradation product in which stabilized fibrin cross-linked by the action of activated factor XIII in the blood coagulation/fibrinolysis system is decomposed by plasmin, or a protein or nucleic acid contained in extracellular vesicles. When the antigen as the test substance is an in vivo substance such as a hormone or cytokine, the specific binding substance for the in vivo substance may be not only an antibody but also a receptor, and when the antigen as the test substance is a sugar chain or a complex carbohydrate having a sugar chain, the specific binding substance for the sugar chain or complex carbohydrate having a sugar chain may be not only an antibody but also a lectin. Additionally, antigens as test substances may be haptens such as penicillin and cadmium.

When the test substance is an antibody, the substance that specifically binds to the antibody may be an antigen. The antigen may be the entire antigen or a fragment thereof that specifically binds to the antibody, or a fusion substance in which the antigen is bound to another carrier. The antibody as the test substance may be an autoantibody such as an anti-cyclic citrullinated peptide (CCP) antibody or an anti-phospholipid antibody, or an antibody against a foreign antigen such as an anti-chlamydia antibody, an anti-HIV antibody, or an anti-HCV antibody.

When the test substance is a glycan, the substance that specifically binds to the glycan may be a lectin. The lectin may be a galectin, a C-type lectin, a legume lectin, or a fragment thereof that specifically binds to the glycan. The glycan as the test substance may be a monosaccharide or polysaccharide, or may be a complex carbohydrate in which the monosaccharide or polysaccharide is bound to a protein or lipid. For example, when the test substance is a glycan containing mannose, the legume lectin concanavalin A (ConA) may be used as the substance that specifically binds to the glycan.

When the test substance is a lectin, the substance that specifically binds to the lectin may be a sugar chain. The sugar chain may be a monosaccharide, polysaccharide, or complex carbohydrate that specifically binds to the lectin, or a fusion substance in which they are bound to another carrier. The lectin as the test substance may be a galectin, a C-type lectin, or a legume lectin. For example, when the test substance is the legume lectin concanavalin A (ConA), a sugar chain containing mannose may be used as the substance that specifically binds to the lectin.

When the combination of the test substance and the substance that specifically binds to the test substance is a protein and a nucleic acid aptamer or peptide aptamer that binds to the protein, the nucleic acid aptamer may be, for example, an aptamer that binds to anthrax (Bacillus anthracis), Staphylococcus aureus, Shigella sonnei, Escherichia coli, Salmonella typhimurium, Streptococcus hemolyticus, Salmonella paratyphi A, Staphylococcal enterotoxin B, or a peptide aptamer that binds to anthrax (Bacillus anthracis). B), a DNA aptamer that binds to bacteria such as Pseudomonas aeruginosa or Vibrio parahaemolyticus, a tumor marker that appears on the surface of epithelial cells such as mucin 1, or an enzyme such as β-galactosidase or thrombin, or an RNA aptamer that binds to the Tat protein or Rev protein of human immunodeficiency virus, and the peptide aptamer may be, for example, a peptide aptamer that binds to the oncoprotein HPV16 E6 of human papillomavirus (HPV).

When the test substance is a vitamin, the specific binding substance for the vitamin may be a vitamin-binding protein such as transcalciferin, which binds to vitamin D, and transcobalamin, which binds to vitamin B12. When the test substance is an antibiotic, the specific binding substance for the antibiotic may be a penicillin-binding protein such as PBP1 and PBP2, which bind to penicillin.

The nucleic acid as the test substance or the substance that binds to the test substance may be, for example, DNA, RNA, an oligonucleotide, a polynucleotide, or an amplification product thereof.

In one embodiment, the sample contains multiple types of test substances,
a capture specific binding substance for each of the plurality of types of test substances is immobilized on the membrane;
The gold-coated silver nanoplate carries a specific binding substance for detection for each of the multiple types of test substances. That is, the immunochromatography kit of the present invention can simultaneously detect multiple types of test substances. In this case, the capture specific binding substance may be fixed to a different site for each type of test substance to be bound. In addition, the detection specific binding substance may be carried on a gold-coated silver nanoplate with a different maximum absorption wavelength for each type of test substance to be bound. In this way, coloring is observed at different locations or in different colors for each test substance to be detected, making it easier to detect the test substance.

In a specific embodiment, the test substance includes at least one antigen selected from the group consisting of influenza virus antigens and coronavirus antigens. The influenza virus antigen is not particularly limited, but may include, for example, influenza A virus antigen and/or influenza B virus antigen. The coronavirus antigen is not particularly limited, but may include, for example, SARS-CoV antigen and/or SARS-CoV-2 antigen.

In one embodiment, the test strip includes a conjugate pad containing the gold-coated silver nanoplates. The "conjugate pad" described in this specification is a portion impregnated with the gold-coated silver nanoplates, and is configured so that when the sample is added thereto, the gold-coated silver nanoplates are dissolved and begin to develop (flow) on the membrane. The conjugate pad can be prepared by any method commonly used in the art.

In one embodiment, the membrane further comprises a control site for determining whether the immunochromatography test is successful or not. The control site is not particularly limited, and may be, for example, a control line to which a substance such as an antibody capable of capturing the detection specific binding substance is immobilized. In a specific embodiment, the immunochromatography kit of the present invention further comprises a control specific binding substance that specifically binds to the control site. The control specific binding substance may be contained in the conjugate pad together with the gold-coated silver nanoplate. In addition, considering that the capture specific binding substance may flow out of the membrane and reach the control site during immersion in a blocking solution or a washing solution or during development of these solutions or during development of the sample, in order not to interfere with the determination at the control site, it is preferable that the control specific binding substance does not compete with not only the detection specific binding substance but also the capture specific binding substance at the control site. For example, when the control specific binding substance contains an antibody and the capture specific binding substance contains an antibody, the antibody of the control specific binding substance may be derived from a host animal different from that of the antibody of the capture specific binding substance.

In one embodiment, the immunochromatography kit of the present invention further includes nanoparticles other than the gold-coated silver nanoplates. In particular, by adopting nanoparticles that exhibit a color different from that of the gold-coated silver nanoplates, the options for colors that can be used to color the detection site and/or control site can be expanded. The nanoparticles are not particularly limited as long as they can be used in immunochromatography tests, and may be, for example, gold nanoparticles and palladium-coated gold nanoparticles.

The immunochromatography kit of the present invention may further include any configuration that is commonly used in the art, as long as it does not impair the object of the present invention. For example, the immunochromatography kit may further include a standard sample of the test substance, a buffer solution, or an instruction manual that describes how to use the kit.

The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples.

1. Preparation Example 1
(1) Preparation of silver nanoplate seed particles 10 mL of 0.5 g/L polystyrene sulfonic acid (molecular weight 70,000) aqueous solution and 11.5 mL of 10 mM sodium borohydride aqueous solution were added to 200 mL of 2.5 mM trisodium citrate aqueous solution with stirring, and then 200 mL of 0.74 mM silver nitrate aqueous solution was added at 86 mL/min. After stopping the stirring, the obtained solution was left to stand in an incubator (30°C) for 60 minutes to prepare an aqueous dispersion (seed suspension) of silver nanoplate seed particles.

(2) Preparation of silver nanoplate aqueous dispersion 45 mL of 10 mM ascorbic acid aqueous solution and 300 mL of the above seed suspension were added to 2000 mL of distilled water while stirring. Then, 120 mL of 7.4 mM silver nitrate aqueous solution was added at 26 mL/min. 20 mL of 250 mM trisodium citrate aqueous solution was added to the obtained solution, and stirring was stopped. The obtained solution was left to stand in an incubator (23 ° C.) under an air atmosphere for 20 hours to prepare silver nanoplate aqueous dispersion 1 (AgPL suspension 1). In addition, AgPL suspensions 2 and 3 were prepared in the same manner as AgPL suspension 1, except that the amount of the above seed suspension was changed to 42 mL or 100 mL.

The silver nanoplates in each AgPL suspension were observed with a scanning transmission electron microscope (STEM), and the maximum long diameter of 100 random silver nanoplates was measured. The average particle size was calculated by averaging 80 data points, excluding the top and bottom 10%, out of a total of 100 data points. The average particle sizes of the silver nanoplates in AgPL suspensions 1, 2, and 3 were 13.2 nm, 32.8 nm, and 20.2 nm, respectively.

(3) Gold Coating Treatment of Silver Nanoplates 150 mL of distilled water was mixed with 14 mL of 24 mM chloroauric acid solution, 8 mL of 200 mM sodium hydroxide solution, and 103 mL of 10 mM sodium sulfite solution to prepare growth solution 1. Then, 312 mL of AgPL suspension 1, 69 mL of 5 mass% polyvinylpyrrolidone solution, 14 mL of 500 mM ascorbic acid solution, 14 mL of 500 mM sodium hydroxide solution, 3 mL of 100 mM sodium sulfite solution, and 275 mL of growth solution 1 diluted 2.9 times were mixed with 312 mL of distilled water and left at room temperature for 24 hours to prepare an aqueous dispersion 1 of gold-coated silver nanoplates (Ag@Au suspension 1) (yellowish). Then, in the same manner as above, Ag@Au suspension 2 (blue tone) was prepared by diluting growth solution 1 1.4 times into AgPL suspension 2, and Ag@Au suspension 3 (red tone) was prepared by diluting growth solution 1 1.7 times into AgPL suspension 3. Ag@Au suspensions 1 to 3 were appropriately diluted, and their spectra were measured using a spectrophotometer V-770 (manufactured by JASCO Corporation) as shown in FIG. 1. Also, a photograph of the gold-coated silver nanoplates in Ag@Au suspension 2 observed with a high-angle annular dark-field scanning transmission microscope (HAADF-STEM) is shown in FIG. 2.

(4) Characteristics of Gold-Coated Silver Nanoplates The Ag@Au suspensions 1 to 3 were observed with an STEM, and the average thickness of the gold layer at the end face of the gold-coated silver nanoplate in each suspension was calculated. Specifically, for the convenience of calculation, a gold-coated silver nanoplate with a circular shape was selected, the average particle diameter was measured, and the difference from the average particle diameter measured before gold coating was divided by 2 to calculate the average thickness of the gold layer at the end face. Table 1 below shows the silver concentration and gold concentration of the mixed solution during the gold coating operation, the average particle diameter of the core silver nanoplate and the average thickness of the gold layer at the end face, the maximum absorption wavelength of the gold-coated silver nanoplate, and the extinction degree at the maximum absorption wavelength and the extinction degree at 900 nm.

(5) Preparation of conventional gold-coated silver nanoplates According to the preparation method of metal fine particles A, B and C described in the examples of Patent Document 1 (Patent No. 6440166), suspensions of gold-coated silver nanoplates, that is, Ag@Au suspensions 4 (yellow tone), 5 (red tone), and 6 (blue tone) were prepared. Ag@Au suspensions 4 to 6 were appropriately diluted, and the spectroscopic spectra measured with a spectrophotometer V-770 (manufactured by JASCO Corporation) are shown in FIG. 3. In addition, the average thicknesses of the gold layers at the end faces of the gold-coated silver nanoplates in Ag@Au suspensions 4 to 6 measured according to the method described in (4) above were 0.14 nm, 0.21 nm, and 0.31 nm, respectively, all of which were thinner than 1.0 nm.

(6) Supporting antibodies on gold-coated silver nanoplates The gold-coated silver nanoplates in the Ag@Au suspension 1 were precipitated by centrifugation, and after removing the supernatant, 0.5 mM trisodium citrate aqueous solution was added to disperse the precipitated gold-coated silver nanoplates. The same operation was repeated twice, and the concentration of the Ag@Au suspension was adjusted so that the extinction degree at the maximum absorption wavelength was finally 2.0. 1 mL of this suspension was mixed with 0.1 mL of a solution of anti-human chorionic gonadotropin (hCG) antibody (host animal: mouse) diluted to a concentration of 50 μg/mL with 5 mM borate buffer (pH 7.5), and the resulting mixture was allowed to stand at room temperature for 60 minutes. Next, 0.100 mL of 0.5 mM bovine serum albumin solution (in 5 mM borate buffer) was added, and the resulting suspension was allowed to stand at room temperature for 60 minutes. This was centrifuged to precipitate the complex of the antibody and the gold-coated silver nanoplate, and the supernatant was removed. Then, 50 μL of 20 mM Tris-HCl buffer (containing 0.05% polyethylene glycol (PEG) (Mw: 20,000), 0.25 mM BSA, and 150 mM NaCl) was added to the complex to redisperse it, and Ag@Au suspension 1 carrying anti-hCG antibody (anti-hCG antibody/Ag@Au suspension 1) was prepared. Similarly, Ag@Au suspensions 2 to 6 carrying anti-hCG antibody were prepared from AgAu suspensions 2 to 6, respectively.

(7) Preparation of Conjugate Pad 900 μL of 20 mM Tris-HCl buffer (pH 8.2; containing 0.05% polyethylene glycol (Mw: 20,000) and 4% sucrose) was mixed with 300 μL of anti-hCG antibody/Ag@Au suspension 1. This mixture was uniformly applied to the entire surface of a glass fiber pad (GRADE 8964, manufactured by AHLSTROM) measuring 8 mm in length and 300 mm in width at an amount of 40 μL/cm, and then placed in a vacuum desiccator (manufactured by AS ONE Corporation) and dried overnight with a vacuum pump (belt-driven oil rotary vacuum pump TSW-50N, manufactured by Sato Vacuum Co., Ltd.) to prepare a conjugate pad 1 containing Ag@Au carrying anti-hCG antibody. Similarly, conjugate pads 2 to 6 containing Ag@Au carrying anti-hCG antibody were prepared from anti-hCG antibody/Ag@Au suspensions 2 to 6, respectively.

(8) Preparation of immunochromatographic test strips 50 mM phosphate buffer containing 0.5 mg/mL anti-hCG antibody (host animal: mouse) was applied uniformly and linearly at 1 μL/cm to the detection site of a nitrocellulose membrane, and 50 mM phosphate buffer containing 0.5 mg/mL anti-mouse IgG antibody (host animal: rabbit) was applied uniformly and linearly at 1 μL/cm to the control site on the upper end side of the detection site, and these antibodies were immobilized on the membrane. This membrane was blocked with 50 mM borate buffer (pH 8.5) (containing 0.5% casein). The blocked membrane was immersed in 50 mM Tris-HCl buffer (pH 7.6) (containing 0.5% sucrose, 0.01% sodium dodecylbenzenesulfonate), washed, and dried.

The dried membrane was attached to the adhesive tape of a 40 mm x 60 mm backing sheet (GL-57888, manufactured by Lohmann), and one of the conjugate pads 1 to 6 containing Ag@Au carrying anti-hCG antibodies was attached to the lower end of the membrane so as to overlap by 2 mm. Next, a 17 mm x 60 mm sample pad (GRADE0319, manufactured by Ahlstrom) impregnated with 25 mM Tris-HCl buffer (pH 8.2) was attached to the opposite side of the conjugate pad from the membrane so as to overlap by 4 mm. Then, a 20 mm x 60 mm absorbent pad (GRADE0270, manufactured by Ahlstrom) was attached to the upper end of the membrane so as to overlap by 5 mm. Finally, the backing sheet equipped with the membrane, conjugate pad, sample pad, and water-absorbent pad was cut into strips measuring 60 mm in length and 5 mm in width using a roll cutter, and these were placed in a housing case (Type-T housing case, manufactured by Trust Medical) to produce a test strip [hCG+M].

2. Immunochromatography Test 1
100 μL of 10 mM phosphate buffered saline (containing 0.25 mM BSA) containing hCG at a concentration of 250 mIU/mL or the same phosphate buffered saline (blank) not containing hCG was dropped onto the sample pad of the test strip [hCG+M] prepared in the above item 1 (8), and developed on the membrane. Then, the coloring of the detection site and the control site was visually evaluated according to the following criteria, and a luminance analysis was also performed when the test sample containing hCG was developed.
(Visual inspection criteria)
++: Vivid coloring +: Coloring -: No coloring

In the luminance analysis, the test strip after the test sample was spread was scanned with a scanner (device name: CanoScan LiDE500F, manufacturer: Canon Inc.), and the minimum luminance (which decreases when colored) of the detection site, control site, and other sites (control area) was quantified using the image analysis software Image-J (open source, publicly owned image processing software developed by Wayne Rasband at the National Institutes of Health, USA; http://imagej.nih.gov/ij/). More specifically, the minimum luminance of each area was measured five times, and the median of the obtained values was used as the minimum luminance of the detection site, control site, or control area. The luminance difference was then calculated by subtracting the minimum luminance of the detection site or control site from the minimum luminance of the control area. The results are shown in Tables 2 and 3.

In immunochromatography tests using gold-coated silver nanoplates with an average gold layer thickness of more than 1.0 nm (Ag@Au suspensions 1-3), only coloring was observed at the control site when a test sample not containing hCG was developed, and it was confirmed that the test system was valid. However, in immunochromatography tests using gold-coated silver nanoplates with an average gold layer thickness of less than 1.0 nm (Ag@Au suspensions 4-6), no coloring was observed at the control site, and the test system did not function (Table 2). This is thought to be because the gold-coated silver nanoplates were deteriorated by oxidation. In addition, in immunochromatography tests using Ag@Au suspensions 1-3, when a test sample containing hCG was developed, hCG could be detected well at the detection site (Table 3).

3. Preparation Example 2
(1) Supporting of antibodies on gold-coated silver nanoplates As the antibody solutions, anti-influenza virus type B (Flu-B) antibody (host animal: mouse) diluted to a concentration of 50 μg/mL with 5 mM borate buffer (pH 8.3) was used for Ag@Au suspension 1, anti-influenza virus type A (Flu-A) antibody (host animal: mouse) similarly diluted for Ag@Au suspension 2, and anti-SARS-CoV-2 (CoV2) antibody (host animal: mouse) similarly diluted for Ag@Au suspension 3. In addition, 0.05 wt % Lipidure in 20 mM Tris-HCl buffer was used. A suspension of Ag@Au carrying anti-Flu-B antibody (Suspension A), a suspension of Ag@Au carrying anti-Flu-A antibody (Suspension B), and a suspension of Ag@Au carrying anti-CoV2 antibody (Suspension C) were prepared in the same manner as in Item 1(6) above, except that BL802 (NOF Corporation) was contained and PEG was not contained.

(2) Preparation of control antibody A black aqueous dispersion containing surface-treated palladium-coated gold nanorods (Au@Pd suspension) was prepared according to the preparation method of "Suspension E" described in the examples of Japanese Patent No. 6717732. Then, a suspension of Au@Pd carrying rabbit IgG (Suspension R) was prepared in the same manner as in item 1(6) above, except that the Au@Pd suspension was used as the suspension, rabbit IgG diluted with 5 mM borate buffer (pH 8.0) to a concentration of 50 μg/mL was used as the antibody solution, and 20 mM Tris-HCl buffer contained 0.05 wt% Lipidure BL802 (NOF Corporation).

(3) Preparation of Conjugate Pads Conjugate pad [ABC] and conjugate pad [ABC+R] were prepared in the same manner as in item 1(7) above, except that a mixture of suspensions A to C or a mixture of suspensions A to C and R was used instead of suspension 1 of Ag@Au carrying anti-hCG antibody, that 20 mM Tris-HCl buffer contained 0.05 wt % Lipidure BL802 (NOF Corporation), and that no PEG was contained.

(4) Preparation of immunochromatographic test strips Test strips [ABC+M] were prepared in the same manner as in item 1(8) above, except that 50 mM borate buffer (containing 10% sucrose) containing 0.25 mg/mL anti-CoV2 antibody, 50 mM borate buffer (containing 10% sucrose) containing 0.25 mg/mL anti-Flu-A antibody, and 50 mM borate buffer (containing 10% sucrose) containing 0.25 mg/mL anti-Flu-B antibody were linearly applied to the detection site of a nitrocellulose membrane from the bottom to the top of the membrane, and 2.1 mg/mL anti-mouse IgG antibody (host animal: rabbit) was applied to the control site on the top side of the detection site, and conjugate pad [ABC] was used as the conjugate pad. Test strip [ABC+R] was prepared in the same manner as test strip [ABC+M], except that the concentration of the antibody solution applied to the detection site was changed to 1.0 mg/mL, the antibody solution applied to the control site was changed to 0.3 mg/mL anti-rabbit IgG antibody (host animal: goat), and conjugate pad [ABC+R] was used as the conjugate pad.

4. Immunochromatography Test Example 2
100 μL of 10 mM phosphate buffered saline (containing 0.25 mM BSA) containing the following test samples (a) to (e) was dropped onto the sample pad of the test strip [ABC+M] or test strip [ABC+R] prepared in item 3 (4) above, and allowed to spread on the membrane. Then, the coloring of the detection site and the control site was evaluated according to the method described in item 2 above. The results are shown in Tables 4 and 5.
(a) Blank (no antigen)
(b) Flu-A antigen (1.5 μg/mL)
(c) Flu-B antigen (5.0 μg/mL)
(d) CoV2 antigen (3.0 μg/mL)
(e) Flu-A antigen (1.5 μg/mL) + Flu-B antigen (5.0 μg/mL) +
CoV2 antigen (3.0 μg/mL)

Whether test strip [ABC+M] or test strip [ABC+R] was used, Flu-A was detected in blue, Flu-B in yellow, and CoV2 in red, but the difference in brightness was greater when test strip [ABC+R] was used. In addition, the color of the control site was black in test strip [ABC+M], a mixture of blue, yellow, and red gold-coated silver nanoplates, while in test strip [ABC+R], the color was black as a tone of palladium-coated gold nanorods.

In the test strip [ABC+M], when the concentration of the antibody fixed to the detection site was 1.0 mg/mL, the black color of the control site disappeared. This is thought to be because the fixed antibody flowed out to the upper end of the membrane during immersion in the blocking solution or washing solution, or during the development of these solutions, or during the development of the test sample, and competitively bound to the antibody supported on the gold-coated silver nanoplate with the anti-mouse IgG antibody at the control site. In other words, if the capture specific binding substance of the test substance competes with the substance that binds to the control site, there is a risk that the control cannot be judged if the amount of the capture specific substance immobilized is increased to increase the detection sensitivity. On the other hand, as in the test strip [ABC+R], if the capture specific binding substance of the test substance does not compete with the substance that binds to the control site, it is possible to increase the detection sensitivity without adversely affecting the control judgment by increasing the amount of the capture specific substance immobilized.

From the above, it was found that the use of gold-coated silver nanoplates with a thick gold layer can increase the detection sensitivity of immunochromatographic tests and also enables multicolor analysis. Therefore, it is possible to easily detect even trace amounts of test substances using immunochromatographic tests.

Claims (14)

a test strip including a membrane;
A gold-coated silver nanoplate having a silver nanoplate as a core and a gold layer covering the silver nanoplate;
An immunochromatography kit for detecting a test substance in a sample, comprising:
A specific binding substance for capturing the test substance is immobilized on the membrane,
The average thickness of the gold layer is more than 1.0 nm, and the gold-coated silver nanoplate carries a specific binding substance for detecting the test substance ;
An immunochromatography kit , wherein the average particle size of the silver nanoplate as the core is 55 nm or less . The immunochromatography kit according to claim 1, wherein the average thickness of the gold layer is 1.5 nm to 10.0 nm. The immunochromatography kit according to claim 1 or 2, wherein the gold-coated silver nanoplates are contained in a dry state. The immunochromatography kit according to any one of claims 1 to 3, wherein the test strip includes a conjugate pad containing the gold-coated silver nanoplate. The gold layer is formed on a main surface and an end surface of the silver nanoplate,
The immunochromatography kit according to any one of claims 1 to 4, wherein the ratio (T/D) of the average thickness (T) of the gold layer at the end surface to the average particle diameter (D) of the silver nanoplates is 0.05 or more. The immunochromatography kit according to any one of claims 1 to 5, wherein the gold-coated silver nanoplate has a maximum absorption wavelength in the visible light region, and the ratio (E _max /E ₉₀₀ ) of the extinction degree (E _max ) at the maximum absorption wavelength to the extinction degree (E ₉₀₀ ) at a wavelength of 900 nm of the gold-coated silver nanoplate is 5 or more . The immunochromatography kit according to any one of claims 1 to 6 , wherein the capture specific binding substance and/or the detection specific binding substance comprises an antibody. The sample contains a plurality of test substances,
a capture specific binding substance for each of the plurality of types of test substances is immobilized on the membrane;
The immunochromatography kit according to any one of claims 1 to 7 , wherein the gold-coated silver nanoplate carries a detection specific binding substance for each of the multiple types of test substances. The immunochromatography kit according to claim 8 , wherein the capture specific binding substances are immobilized at different sites depending on the type of test substance to be bound. The immunochromatography kit according to claim 8 or 9 , wherein the specific binding substance for detection is supported on a gold-coated silver nanoplate having a different maximum absorption wavelength for each type of test substance to be bound. The immunochromatography kit according to any one of claims 1 to 10 , wherein the membrane further comprises a control site for determining whether the immunochromatography test is successful or not. further comprising a control specific binding substance that specifically binds to the control site;
The immunochromatography kit described in claim 11, wherein when the control specific binding substance contains an antibody and the capture specific binding substance contains an antibody, the antibody of the control specific binding substance is derived from a host animal different from the antibody of the capture specific binding substance. The immunochromatography kit according to any one of claims 1 to 12 , further comprising nanoparticles other than the gold-coated silver nanoplates. The immunochromatography kit according to any one of claims 1 to 13 , wherein the test substance comprises at least one antigen selected from the group consisting of an influenza virus antigen and a coronavirus antigen.