Immunochromatography kit

Abstract

To provide an immunochromato kit using a gold-coated silver nanoplate with high oxidation resistance.SOLUTION: An immunochromato kit of the present invention for detecting a test substance in a sample includes: a test strip including a membrane; a silver nanoplate as a core; and a gold-coated silver nanoplate having a gold layer coated on the silver nanoplate. A specific binding substance for trapping of the test substance is fixed on the membrane. The average thickness of the gold layer is more than 1.0 nm. The gold-coated silver nanoplate carries a specific binding substance for detection of the test substance.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an immunochromatography kit, and particularly to an immunochromatography kit using gold-coated silver nanoplatelets.

Background Art

[0002] Since silver nanoplatelets absorb light by interacting with light (localized surface plasmon resonance: LSPR), it is known that their suspension exhibits a color depending on the shape of the nanoplatelets. It is also known that by controlling the size and shape of silver nanoplatelets, the absorbed light can be changed, that is, the color can be changed. On the other hand, silver nanoplatelets dissolve due to oxidation and their shape changes. This shape change of silver nanoplatelets due to oxidation can cause an unintended color change.

[0003] Therefore, in order to stabilize silver nanoplatelets against oxidation, the surface of silver nanoplatelets is coated with gold. Patent Document 1 describes that gold-coated silver nanoplatelets are stable and can be suitably used in immunochromatography tests. Further, Patent Document 2 describes that by adjusting the concentration and pH of a water-soluble polymer, gold-coated silver nanoplatelets can be prepared in the form of a stable suspension and used as a label for a detection reagent for a test substance (for example, a label for an antibody used for detecting a target protein), and applied to detect various test substances with high sensitivity.

[0004] As a test strip used in an immunochromatography test, a "half strip" composed of a membrane and an absorption pad, and a "full strip" having a sample pad and a conjugate pad in addition thereto are known, and the "full strip" is widely sold as a commercial product. In the half strip, a detection reagent for a test substance is used as a developing solution, but in the full strip, since the detection reagent for a test substance is impregnated in the conjugate pad and dried, it can be used simply by applying a sample containing the test substance to the sample pad.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] In conventional gold-coated silver nanoplatelets, they can be suitably used in half-strips, but in full-strips, the color development property may decrease. As a cause, it was considered that the gold-coated silver nanoplatelets were deteriorated by oxidation when the detection reagent of the test substance containing the gold-coated silver nanoplatelets was impregnated into the conjugate pad and dried. In conventional gold-coated silver nanoplatelets, only a thin film could be formed with respect to the size of the core silver nanoplatelets, and the oxidation resistance was insufficient, so the detection sensitivity of the immunochromatography test was not sufficient. Therefore, an object of the present invention is to provide an immunochromatography kit using a gold-coated silver nanoplatelet having high oxidation resistance.

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that high detection sensitivity can be obtained even in full-strips by using a gold-coated silver nanoplatelet having a thick gold layer, and have completed the present invention. That is, the present invention provides an immunochromatography kit shown below. 〔1〕A test strip including a membrane, A gold-coated silver nanoplatelet having a silver nanoplatelet as a core and a gold layer covering the silver nanoplatelet, An immunochromatography kit for detecting a test substance in a sample, comprising: On the membrane, a specific binding substance for capturing the test substance is immobilized. An immunochromatography kit, wherein 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. 〔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 includes a conjugate pad containing the gold-coated silver nanoplate. 〔5〕The gold layer is formed on the main plane and the end face 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 face to the average particle diameter (D) of the silver nanoplate 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 gold-coated silver nanoplate has a maximum absorption wavelength in the visible light region, and the ratio (E 900 / E max ) of the extinction coefficient (E max ) at the maximum absorption wavelength to the extinction coefficient (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 〔1〕 to 〔6〕. 〔8〕The immunochromatography kit according to any one of 〔1〕 to 〔7〕, wherein the specific binding substance for capture and / or the specific binding substance for detection contains an antibody. 〔9〕The sample contains a plurality of types of test substances, On the membrane, specific binding substances for capturing each of the plurality of types of test substances are immobilized. The immunochromatography kit according to any one of [1] to [8] above, wherein the gold-coated silver nanoplate carries a detection-specific binding substance for each of the plurality of test substances. 〔10〕The immunochromatography kit according to [9] above, wherein the capture-specific binding substance is fixed at a different site for each type of test substance to which it binds. 〔11〕The immunochromatography kit according to [9] or

[10] above, wherein the detection-specific binding substance is carried on gold-coated silver nanoplates having different maximum absorption wavelengths for each type of test substance to which it binds. 〔12〕The immunochromatography kit according to any one of [1] to

[11] above, wherein the membrane further includes a control site for determining the success or failure of an immunochromatography test. 〔13〕Further comprising a control-specific binding substance that specifically binds to the control site, 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

[12] above. 〔14〕The immunochromatography kit according to any one of [1] to

[13] above, further comprising nanoparticles other than the gold-coated silver nanoplate. 〔15〕The immunochromatography kit according to any one of [1] to

[14] above, wherein the test substance includes at least one selected from the group consisting of influenza virus antigen and coronavirus antigen.

Advantages of the Invention

[0008] According to the present invention, by using a gold-coated silver nanoplate having a thick gold layer, a high detection sensitivity can be obtained even when an immunochromatography test is performed using a full strip with a high risk of deterioration due to oxidation.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Embodiments for Carrying Out the Invention

[0010] Hereinafter, the present invention will be described in more detail. The present invention relates to an immunochromatography kit for detecting a test substance in a sample, and the immunochromatography kit includes a test strip including a membrane on which a specific binding substance for capturing the test substance is immobilized, a silver nanoplate as a core, a gold layer covering the silver nanoplate, and a gold-coated silver nanoplate carrying a specific binding substance for detecting the test substance, and includes.

[0011] The "silver nanoplate" described in this specification refers to plate-shaped particles having a size on the order of nanometers (nm) manufactured from silver, and includes an upper surface and a lower surface that are the main planes, and end faces (side faces) between the upper surface and the lower surface. The end face may be a plane or a curved surface, and the corners may be rounded. The shapes of the upper surface and the lower surface of the silver nanoplate are not particularly limited, and may be, for example, polygons such as triangles, quadrilaterals, pentagons, and hexagons (including shapes with rounded corners) or circles. In the gold-coated silver nanoplate, the gold layer is formed on the main plane and the end face of the silver nanoplate, and a so-called core-shell structure of the silver nanoplate and the gold layer is formed.

[0012] The maximum major axis length of the main plane of the silver nanoplate is referred to as the particle size, which corresponds to the diameter in the case of a circular shape, for example, and to the length of the maximum side in the case of a triangular shape. When the maximum major axis lengths are different between the upper and lower surfaces of the silver nanoplate, the longer one is taken as the maximum major axis length of the silver nanoplate. The upper limit value of the average particle size 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, preferably about 50 nm or less, about 40 nm or less, or about 30 nm or less. The lower limit value of the average particle size of the silver nanoplate is also not particularly limited, but may be, for example, about 1 nm or more, preferably about 10 nm or more. Further, the average thickness of the silver nanoplate is not particularly limited, but may be, for example, about 20 nm or less, preferably about 3 to about 15 nm. The particle size and thickness of the silver nanoplate may be measured, for example, by observation with a scanning electron microscope (SEM), a scanning transmission electron microscope (STEM), or a transmission electron microscope (TEM), and the particle size may also be measured with a dynamic light scattering particle size distribution measuring device (DLS). When measuring the particle size of the silver nanoplate using an SEM observation photograph, a STEM observation photograph, or a TEM observation photograph, 80 data points excluding the upper and lower 10% may be prepared from a total of 100 data points obtained by measuring the particle sizes of 100 arbitrary silver nanoplate, and the average particle size may be calculated by obtaining their average value.

[0013] The ratio of the maximum major axis length to the width of the axis perpendicular to the plane containing it, that is, the index defined by "maximum major axis length / thickness", is called the "aspect ratio", and this can be used as one of the indicators for representing 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 ratio of the silver nanoplate can also be said to be an indicator 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, preferably about 1.5 to about 10. When the aspect ratio is in such a range, it is easy to adjust the maximum absorption wavelength due to surface plasmon resonance in the aqueous suspension to the visible light region.

[0014] 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, especially exceeding about 1.0 nm at the end face. The gold-coated silver nanoplate with a thick gold layer has high oxidation resistance and thus high storage stability, and can detect the test substance with good sensitivity even in the form of an immunochromatography kit that is not used immediately after preparation. In one aspect, 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 within such a range, oxidation of the silver nanoplate can be more effectively suppressed while maintaining the optical properties of the silver nanoplate.

[0015] The thickness of the gold layer at the end face can be measured using a high-angle annular dark-field scanning transmission microscope (HAADF-STEM). Specifically, for 10 arbitrary particles selected from the HAADF-STEM observation photograph, 80 data points excluding the upper and lower 10% can be prepared from a total of 100 data points obtained by measuring the gold thickness at 10 arbitrary end points of each particle, and the average thickness (T) can be calculated by obtaining the average value thereof.

[0016] Also, the thickness of the gold layer at the end face can be calculated from the sizes 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 diameter (a) before gold coating and the average particle diameter (b) after gold coating are measured, and the following formula: T = (b - a) / 2 can be used to calculate the average thickness (T). When the shape of the main plane of the silver nanoplate is equilateral triangular, the average height (c) of the equilateral triangle before gold coating and the average height (d) of the equilateral triangle after gold coating are measured, and the following formula: T = (d - c) / 3 can be used to calculate the average thickness (T).

[0017] The color tone exhibited by the suspension of the present invention can be adjusted by adjusting the size and / or shape of the silver nanoplate serving as the core and the thickness of the gold layer, and changing the maximum absorption wavelength of the gold-coated silver nanoplate. That is, the size and shape of the silver nanoplate are adjusted in consideration of the change in the maximum absorption wavelength due to the subsequent gold coating, so that the suspension of the present invention has a maximum absorption wavelength in the visible light region. In one aspect, the maximum absorption wavelength is present at about 400 nm to about 680 nm.

[0018] In one aspect, in the immunochromatography kit of the present invention, the gold-coated silver nanoplate is included in a dry state. The method for drying the gold-coated silver nanoplate is not particularly limited. For example, the gold-coated silver nanoplate may be dried under reduced pressure at room temperature, or may be dried by freeze-drying. Since the gold-coated silver nanoplate used in the present invention has a thick gold layer and high oxidation resistance, it can be in such a form.

[0019] In one aspect, the ratio (T / D) of the average thickness (T) of the gold layer on the end face 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 relative to its small particle size. Since the gold-coated silver nanoplate with a small particle size has a maximum absorption wavelength in the visible light region, it is possible to achieve multi-colorization of a detection method for a test substance that requires visual determination such as immunochromatography. In one aspect, T / D may be about 0.5 or less, about 0.4 or less, or about 0.3 or less.

[0020] In one aspect, the gold-coated silver nanoplate has a maximum absorption wavelength in the visible light region, and the ratio (E 900 ) of the extinction coefficient (E max ) at the maximum absorption wavelength to the extinction coefficient (E max ) at a wavelength of 900 nm of the gold-coated silver nanoplate (E 900) may be about 5 or more, preferably about 10 or more. That is, the gold-coated silver nanoplate shows a sharp spectral spectrum because of the high uniformity of the particle size. Therefore, the color of the gold-coated silver nanoplate is vivid and has a high chroma.

[0021] 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 adopted without particular limitation. For example, commercially available products may be used, or those manufactured according to known production methods or the methods described in the examples below may be used. When manufacturing a gold-coated silver nanoplate having a thick gold layer despite a small particle size as mentioned in the above specific embodiment, attention should be paid to elution of silver from the core silver nanoplate, oxidation of the silver nanoplate, and formation of agglomerates due to the grown gold layer. For example, in the coating mixture used for forming the gold layer (including the silver nanoplate, a water-soluble gold-containing compound, and a complexing agent for gold ions), 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 Ag to C Au (C Au / C Ag ) is adjusted to be about 0.50 or more, and if the coating mixture is allowed to stand for a predetermined time, a gold-coated silver nanoplate having a thick gold layer despite a small particle size can be efficiently and stably produced. For more details, refer to the specification of Japanese Patent Application No. 2020-149087.

[0022] The gold-coated silver nanoplate in the immunochromatography kit of the present invention carries a specific binding substance for detecting the test substance. The "carrying" described in this specification means that the gold-coated silver nanoplate and the specific binding substance for detection are combined to form a complex regardless of the mode such as covalent bond, non-covalent bond, or direct or indirect bond. As the method of carrying, ordinary carrying methods can be used without particular limitation, and physical adsorption, chemical adsorption (covalent bond to the surface), chemical bond (covalent bond, coordination bond, ionic bond or metal bond), etc. are utilized to directly bond the gold-coated silver nanoplate and the specific binding substance for detection, or a part of a water-soluble polymer is bonded to the surface of the gold-coated silver nanoplate, and the specific binding substance for detection is directly or indirectly bonded to the end, main chain or side chain of the water-soluble polymer. For example, when the specific binding substance for detection is an antibody, the gold-coated silver nanoplate carrying the antibody (labeled detection reagent) can be obtained as a precipitate by mixing the gold-coated silver nanoplate and the antibody solution, shaking, and centrifuging. Further, when the gold-coated silver nanoplate and the antibody are carried by electrostatic adsorption, coating the surface of the gold-coated silver nanoplate with polystyrene sulfonic acid having a negative charge can improve the carrying efficiency of the antibody.

[0023] As the capture-specific binding substance or the detection-specific binding substance (sometimes collectively referred to as "specific binding substance for the test substance"), those that can form a complex with the test substance to be detected and can utilize the gold-coated silver nanoplate as a label can be used without particular limitation as long as they meet the requirements. Specific examples of combinations of a test substance and a specific binding substance for 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 and 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 (hybridizes with) it, etc. The second nucleic acid may be a nucleic acid containing a sequence complementary to the first nucleic acid.

[0024] When the test substance is an antigen, the specific binding substance to the antigen may be an antibody. The antibody may be a polyclonal antibody, monoclonal antibody, single-chain antibody or a fragment thereof that specifically binds to the antigen, and the fragment may be an F(ab) fragment, F(ab’) fragment, F(ab’)2 fragment or F(v) fragment. The antigen as the test substance may be a lectin such as concanavalin A (ConA), malt agglutinin and lysine, a virus such as influenza virus, coronavirus, adenovirus, RS virus, rotavirus, human papillomavirus, human immunodeficiency virus and hepatitis B virus, Zika virus, dengue virus or a substance they possess (for example, hepatitis B virus antigen (HBs antigen) or hemagglutinin of influenza virus), a pathogenic microorganism such as Aspergillus flavus, Chlamydia, Treponema pallidum, Streptococcus, Bacillus anthracis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coli, Salmonella, Salmonella typhi, Salmonella paratyphi, Pseudomonas aeruginosa and Vibrio parahaemolyticus or a substance they possess (for example, aflatoxin (such as B1, B2, G1, G2 or M1) of Aspergillus flavus, verotoxin of enterohemorrhagic Escherichia coli or streptolysin O of Streptococcus), a blood protein such as immunoglobulin G (IgG), rheumatoid factor and C-reactive protein (CRP), a glycoprotein such as mucin, a hormone such as insulin, pituitary hormones (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)), thyrotropin-releasing hormone (TRH), thyroid hormones (for example, diiodothyronine or triiodothyronine), human chorionic gonadotropin, calcium metabolism-regulating hormones (for example, calcitonin or parathyroid hormone), pancreatic hormones, gastrointestinal hormones, vasoactive intestinal peptide, follicular hormones (for example, estrone), natural or synthetic luteal hormones (for example, progesterone), male hormones (for example, testosterone), and adrenal cortical hormones (for example, cortisol), serotonin, urokinase,It may also be other in-vivo substances such as ferritin, substance P, prostaglandin, and cholesterol, and may also be tumor markers such as prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), alkaline phosphatase, transaminase, trypsin, pepsinogen, α-fetoprotein (AFP), and carcinoembryonic antigen (CEA), may also be sugar chain antigens such as blood group antigens, may also be markers used for detecting occult blood in feces such as hemoglobin and transferrin, may also be D-dimer which is a kind of fibrin degradation product obtained by degradation of stabilized fibrin cross-linked by the action of activated factor XIII in the blood coagulation and fibrinolysis systems, and may also be proteins and nucleic acids possessed by extracellular vesicles. When the antigen as the test substance is an in-vivo substance such as a hormone or cytokine, the specific binding substance to the in-vivo substance may be not only an antibody but also a receptor. When the antigen as the test substance is a sugar chain or a complex carbohydrate having a sugar chain, the specific binding substance to the sugar chain or the complex carbohydrate having a sugar chain may be not only an antibody but also a lectin. Further, the antigen as the test substance may be a hapten such as penicillin and cadmium.,

[0025] When the test substance is an antibody, the specific binding substance to the antibody may be an antigen. The antigen may be the whole antigen that specifically binds to the antibody or a fragment thereof, or may be a fusion substance obtained by binding them 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 may be an antibody against a foreign antigen such as an anti-chlamydia antibody, an anti-HIV antibody, or an anti-HCV antibody.

[0026] When the test substance is a sugar chain, the specific binding substance to the sugar chain may be a lectin. The lectin may be a galectin, C-type lectin, leguminous lectin or a fragment thereof that specifically binds to the sugar chain. The sugar chain as the test substance may be a monosaccharide or a polysaccharide, or it may be a glycoconjugate in which they are bound to a protein or a lipid. For example, when the test substance is a sugar chain containing mannose, concanavalin A (ConA), a leguminous lectin, can be used as the specific binding substance to the sugar chain.

[0027] When the test substance is a lectin, the specific binding substance to the lectin may be a sugar chain. The sugar chain may be a monosaccharide, polysaccharide, glycoconjugate or a fusion substance in which they are bound to another carrier that specifically binds to the lectin. The lectin as the test substance may be a galectin, C-type lectin or leguminous lectin. For example, when the test substance is concanavalin A (ConA), a leguminous lectin, a sugar chain containing mannose can be used as the specific binding substance to the lectin.

[0028] When the combination of a test substance and a specific binding substance for the test substance is a protein and a nucleic acid aptamer or peptide aptamer that binds thereto, the nucleic acid aptamer may be, for example, a DNA aptamer that binds to bacteria such as Bacillus anthracis, Staphylococcus aureus, Shigella sonnei, Escherichia coli, Salmonella typhimurium, Streptococcus hemolyticus, Salmonella paratyphi A, Staphylococcal enterotoxin B, Pseudomonas aeruginosa, Vibrio parahaemolyticus, tumor markers that appear on the cell surface of epithelium such as mucin 1, or enzymes such as β-galactosidase and thrombin, or an RNA aptamer that binds to the Tat protein or Rev protein of the human immunodeficiency virus. The peptide aptamer may be, for example, a peptide aptamer that binds to the oncogenic protein HPV16 E6 of the human papillomavirus (HPV).

[0029] When the test substance is vitamins, the specific binding substance for the vitamins may be a vitamin-binding protein such as transcalsiferin that binds to vitamin D and transcobalamin that 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 that binds to penicillin.

[0030] The nucleic acid as the test substance or a substance that binds to the test substance may be, for example, DNA, RNA, oligonucleotide, polynucleotide, or an amplification product thereof.

[0031] In one embodiment, the sample contains a plurality of types of test substances. On the membrane, specific binding substances for capturing each of the plurality of types of test substances are immobilized. The gold-coated silver nanoplate carries a specific binding substance for detection for each of the plurality of types of test substances. That is, with the immunochromatography kit of the present invention, it is possible to simultaneously detect a plurality of types of test substances. In this case, the specific binding substance for capture may be immobilized at different sites for each type of test substance to which it binds. Further, the specific binding substance for detection may be carried on gold-coated silver nanoplates having different maximum absorption wavelengths for each type of test substance to which it binds. By doing so, coloring is observed at different locations or in different colors for each test substance to be detected, facilitating the detection of the test substance.

[0032] In a specific embodiment, the test substance includes at least one selected from the group consisting of influenza virus antigens and coronavirus antigens. The influenza virus antigen is not particularly limited, and may include, for example, an influenza A virus antigen and / or an influenza B virus antigen. The coronavirus antigen is not particularly limited, and may include, for example, a SARS-CoV antigen and / or a SARS-CoV-2 antigen.

[0033] In one embodiment, the test strip includes a conjugate pad containing the gold-coated silver nanoplate. As used herein, the "conjugate pad" is a site in which the gold-coated silver nanoplate is incorporated. When the sample is added here, the gold-coated silver nanoplate elutes and is configured to start spreading (flowing) on the membrane. The conjugate pad can be produced by any method commonly used in the art.

[0034] In one aspect, the membrane further comprises a control site for determining the success or failure of an immunochromatography test. The control site is not particularly limited, and for example, it may be a control line on which a substance such as an antibody capable of capturing the detection-specific binding substance is immobilized. In a specific aspect, 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 included in the conjugate pad together with the gold-coated silver nanoplate. Also, considering that the capture-specific binding substance may flow out of the membrane and reach the control site during immersion in the blocking solution or washing solution, during the development of these solutions, or during the 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 the detection-specific binding substance and 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, an antibody derived from a host animal different from that of the antibody of the capture-specific binding substance may be employed as the antibody of the control-specific binding substance.

[0035] In one aspect, the immunochromatography kit of the present invention further comprises nanoparticles other than the gold-coated silver nanoplate. In particular, by employing nanoparticles that exhibit a color different from that of the gold-coated silver nanoplate, the options for colors that can be colored at the detection site and / or the control site can be expanded. The nanoparticles are not particularly limited as long as they can be used in an immunochromatography test, and for example, gold nanoparticles and palladium-coated gold nanoparticles may be used.

[0036] The immunochromatography kit of the present invention may further include any configuration commonly used in the art, as long as the object of the present invention is not impaired. For example, the immunochromatography kit may further include a standard product of the test substance, a buffer solution, an instruction manual describing the usage method, and the like.

[0037] Hereinafter, the present invention will be specifically described by way of examples, but the scope of the present invention is not limited to these examples.

Examples

[0038] 1. Preparation Example 1 (1) Preparation of seed particles of silver nanoplate To 200 mL of a 2.5 mM aqueous solution of trisodium citrate, 10 mL of a 0.5 g / L aqueous solution of polystyrene sulfonic acid (molecular weight 70,000) and 11.5 mL of a 10 mM aqueous solution of sodium borohydride were added with stirring. Then, 200 mL of a 0.74 mM aqueous solution of silver nitrate was added at a rate of 86 mL / min. After stopping the stirring, the obtained solution was allowed to stand in an incubator (30 °C) for 60 minutes to prepare an aqueous dispersion of silver nanoplate seed particles (seed suspension).

[0039] (2) Preparation of silver nanoplate aqueous dispersion To 2000 mL of distilled water, 45 mL of a 10 mM aqueous solution of ascorbic acid and 300 mL of the above seed suspension were added with stirring. Then, 120 mL of a 7.4 mM aqueous solution of silver nitrate was added at a rate of 26 mL / min. To the obtained solution, 20 mL of a 250 mM aqueous solution of trisodium citrate was added, and the stirring was stopped. The obtained solution was allowed to stand in an incubator (23 °C) under an air atmosphere for 20 hours to prepare a 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 used was changed to 42 mL or 100 mL.

[0040] The silver nanoplates in each AgPL suspension were observed with a scanning transmission electron microscope (STEM), and the average particle size was calculated by obtaining the average value of 80 data points excluding the top and bottom 10% from a total of 100 data points obtained by measuring the maximum major axis of any 100 silver nanoplates. 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.

[0041] (3) Gold coating treatment of silver nanoplates 14 mL of 24 mM chloroauric acid aqueous solution, 8 mL of 200 mM sodium hydroxide aqueous solution, and 103 mL of 10 mM sodium sulfite aqueous solution were mixed in 150 mL of distilled water to prepare Growth Solution 1. Then, 312 mL of AgPL suspension 1, 69 mL of 5 mass% polyvinylpyrrolidone aqueous solution, 14 mL of 500 mM ascorbic acid aqueous solution, 14 mL of 500 mM sodium hydroxide aqueous solution, 3 mL of 100 mM sodium sulfite aqueous solution, and 275 mL of a solution obtained by diluting Growth Solution 1 by 2.9 times were mixed and allowed to stand at room temperature for 24 hours to prepare an aqueous dispersion 1 of gold-coated silver nanoplates (Ag@Au suspension 1) (yellowish tint). Then, in the same manner as above, Ag@Au suspension 2 (blueish tint) was prepared using a solution obtained by diluting Growth Solution 1 by 1.4 times with AgPL suspension 2, and Ag@Au suspension 3 (reddish tint) was prepared using a solution obtained by diluting Growth Solution 1 by 1.7 times with AgPL suspension 3. The spectral spectra of Ag@Au suspensions 1 to 3 diluted as appropriate and measured with a spectrophotometer V-770 (manufactured by JASCO Corporation) are shown in Fig. 1. Also, a high-angle annular dark-field scanning transmission microscope (HAADF-STEM) observation photograph of the gold-coated silver nanoplates in Ag@Au suspension 2 is shown in Fig. 2.

[0042] (4) Characteristics of gold-coated silver nanoplates Ag@Au suspensions 1 to 3 were observed by STEM, and the average thickness of the gold layer on the end face of the gold-coated silver nanoplate in each suspension was calculated. Specifically, for the sake of calculation convenience, gold-coated silver nanoplates with a circular shape were selected to measure the average particle diameter, 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 on the end face. Table 1 below summarizes the silver concentration and gold concentration of the mixed solution during the gold coating operation, the average particle diameter of the silver nanoplate of the core, the average thickness of the gold layer on the end face, the maximum absorption wavelength of the gold-coated silver nanoplate, and the extinction coefficient at the maximum absorption wavelength and the extinction coefficient at 900 nm.

[0043]

Table 1

[0044] (5) Preparation of conventional gold-coated silver nanoplates According to the preparation methods of metal fine particles A, B, and C described in the examples of Patent Document 1 (Japanese Patent No. 6440166), suspensions of gold-coated silver nanoplates, namely 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 spectral spectra measured by a spectrophotometer V-770 (manufactured by JASCO Corporation) are shown in Figure 3. Also, the average thicknesses of the gold layers on 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.

[0045] (6) Loading of antibodies onto gold-coated silver nanoplates The gold-coated silver nanoplatelets in Ag@Au suspension 1 were precipitated by centrifugation. After removing the supernatant, an aqueous solution of 0.5 mM trisodium citrate was added to disperse the precipitated gold-coated silver nanoplatelets. The same operation was repeated twice, and finally the concentration of the Ag@Au suspension was adjusted so that the absorbance at the maximum absorption wavelength became 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. Then, 0.100 mL of a 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 nanoplatelets, and the supernatant was removed. Thereafter, 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 and redispersed to prepare Ag@Au suspension 1 carrying anti-hCG antibody (anti-hCG antibody / Ag@Au suspension 1). Similarly, Ag@Au suspensions 2 to 6 carrying anti-hCG antibody (anti-hCG antibody / Ag@Au suspensions 2 to 6) were prepared from AgAu suspensions 2 to 6, respectively.

[0046] (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 a rate of 40 μL / cm, and then placed in a vacuum desiccator (manufactured by AS ONE) and dried overnight with a vacuum pump (belt-driven oil rotary vacuum pump TSW-50N, manufactured by Sato Vacuum Co., Ltd.) to prepare 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.

[0047] (8) Preparation of Test Strip for Immunochromatography On the detection site of a nitrocellulose membrane, a 50 mM phosphate buffer containing 0.5 mg / mL anti-hCG antibody (host animal: mouse) was uniformly and linearly applied in an amount of 1 μL / cm. On the control site on the upper end side of the detection site, a 50 mM phosphate buffer containing 0.5 mg / mL anti-mouse IgG antibody (host animal: rabbit) was uniformly and linearly applied in an amount of 1 μL / cm to fix these antibodies 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 and 0.01% sodium dodecylbenzenesulfonate for washing and then dried.

[0048] The dried membrane was attached to the adhesive tape part of a backing sheet (GL-57888, manufactured by Lohmann) with a size of 40 mm in length and 60 mm in width. Any one of the conjugate pads 1 to 6 containing Ag@Au carrying anti-hCG antibody was attached so as to overlap 2 mm on the lower end side of the membrane. Next, a sample pad (GRADE0319, manufactured by Ahlstrom) with a size of 17 mm in length and 60 mm in width impregnated with 25 mM Tris-HCl buffer (pH 8.2) was attached on the side opposite to the membrane of the conjugate pad so as to overlap it by 4 mm. Then, a water-absorbing pad (GRADE0270, manufactured by Ahlstrom) with a size of 20 mm in length and 60 mm in width was attached so as to overlap 5 mm on the upper end side of the membrane. Finally, the backing sheet equipped with the membrane, conjugate pad, sample pad, and water-absorbing pad was cut into a strip shape with a length of 60 mm and a width of 5 mm using a roll cutter and installed in a housing case (Type-T housing case, manufactured by Trust Medical) to prepare a test strip [hCG+M].

[0049] 2. Immunochromatography Test 1 To the sample pad of the test strip [hCG+M] prepared in the above item 1(8), 100 μL of 10 mM phosphate buffered saline containing hCG at a concentration of 250 mIU / mL (containing 0.25 mM BSA) or the same phosphate buffered saline without hCG (blank) was dropped 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 luminance analysis was also performed when a test sample containing hCG was developed. (Criteria for visual determination) ++: Clear coloring + : Coloring - : No coloring

[0050] In luminance analysis, the test strip after developing the test sample was scanned by a scanner (device name: CanoScan LiDE500F, manufacturer: Canon Inc.), and the minimum luminance (which becomes lower when colored) of the detection site, the control site, and the other sites (control region) was quantified using the image analysis software Image-J (open-source and publicly available 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 part was measured 5 times each, and the median of the obtained numerical values was adopted as the minimum luminance of the detection site, the control site, or the control region. Then, the minimum luminance of the detection site or the control site was subtracted from the minimum luminance of the control region to obtain the luminance difference. The results are shown in Tables 2 and 3.

[0051]

Table 2

[0052]

Table 3

[0053] In the immunochromatography test using gold-coated silver nanoplatelets (Ag@Au suspensions 1 to 3) with an average gold layer thickness greater than 1.0 nm, only the coloring at the control site was observed when the test sample not containing hCG was developed, and it was confirmed that the test system was established. However, in the immunochromatography test using gold-coated silver nanoplatelets (Ag@Au suspensions 4 to 6) with an average gold layer thickness less than 1.0 nm, no coloring at the control site was observed, and the test system did not function (Table 2). This is considered to be because the gold-coated silver nanoplatelets deteriorated due to oxidation. Also, in the immunochromatography test using Ag@Au suspensions 1 to 3, when the test sample containing hCG was developed, hCG could be detected well at the detection site (Table 3).

[0054] 3. Preparation Example 2 (1) Loading of antibody onto gold-coated silver nanoplatelets As the antibody solution, for Ag@Au suspension 1, an anti-influenza B virus (Flu-B) antibody (host animal: mouse) diluted to a concentration of 50 μg / mL with 5 mM borate buffer (pH 8.3), for Ag@Au suspension 2, an anti-influenza A virus (Flu-A) antibody (host animal: mouse) diluted in the same manner, and for Ag@Au suspension 3, an anti-SARS-CoV-2 (CoV2) antibody (host animal: mouse) diluted in the same manner were used. Except for containing 0.05 wt% of Lipidure BL802 (NOF Corporation) in 20 mM Tris-HCl buffer and not containing PEG, in the same manner as in item 1(6) above, a suspension of Ag@Au loaded with anti-Flu-B antibody (suspension A), a suspension of Ag@Au loaded with anti-Flu-A antibody (suspension B), and a suspension of Ag@Au loaded with anti-CoV2 antibody (suspension C) were prepared respectively.

[0055] (2) Preparation of control antibody According to the preparation method of "suspension E" described in the examples of Patent No. 6717732, a black aqueous dispersion (Au@Pd suspension) containing surface-treated palladium-coated gold nanorods was prepared. Then, the Au@Pd suspension was used as the suspension, and rabbit IgG diluted to a concentration of 50 μg / mL with 5 mM borate buffer (pH 8.0) was used as the antibody solution. A suspension of Au@Pd carrying rabbit IgG (suspension R) was prepared in the same manner as in item 1(6) above, except that 0.05 wt% of Lipidure BL802 (NOF Corporation) was included in 20 mM Tris-HCl buffer.

[0056] (3) Preparation of conjugate pad The conjugate pads [ABC] and [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 the suspension 1 of Ag@Au carrying the anti-hCG antibody, 0.05 wt% of Lipidure BL802 (NOF Corporation) was included in 20 mM Tris-HCl buffer, and no PEG was included.

[0057] (4) Preparation of test strip for immunochromatography At the detection site of the nitrocellulose membrane, 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 in sequence from the lower end to the upper end of the membrane. And at the control site on the upper end side of the detection site, 2.1 mg / mL anti-mouse IgG antibody (host animal: rabbit) was applied. Except for using conjugate pad [ABC] as the conjugate pad, a test strip [ABC+M] was prepared in the same manner as in item 1(8) above. Also, the concentration of the antibody solution applied to the detection site was changed to 1.0 mg / mL, and the antibody solution applied to the control site was changed to 0.3 mg / mL anti-rabbit IgG antibody (host animal: goat). Except for using conjugate pad [ABC+R] as the conjugate pad, a test strip [ABC+R] was prepared in the same manner as the test strip [ABC+M].

[0058] 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 spread on the membrane. Then, according to the method described in item 2 above, the coloring of the detection site and the control site was evaluated. The results are shown in Tables 4 and 5. (a) Blank (without 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)

[0059]

Table 4

[0060]

Table 5

[0061] In both cases of using the test strip [ABC+M] and the test strip [ABC+R], Flu-A was detected in blue, Flu-B in yellow, and CoV2 in red, but the detection was with a greater luminance difference when using the test strip [ABC+R]. Also, regarding the color of the control site, in the test strip [ABC+M], it showed black as a mixed color of blue, yellow, and red gold-coated silver nanoplate mixtures, and in the test strip [ABC+R], it showed black as the color tone of palladium-coated gold nanorods.

[0062] Note that in the test strip [ABC+M], when the concentration of the antibody immobilized on the detection site was set to 1.0 mg / mL, the black color of the control site disappeared. This is presumably because the immobilized antibody flowed out to the upper end side of the membrane during immersion in the blocking solution or washing solution, during the development of these solutions, or during the development of the test sample, and competitively bound to the anti-mouse IgG antibody at the control site with the antibody carried on the gold-coated silver nanoplate. That is, when the specific binding substance for capturing the test substance competes with the substance that binds to the control site, if an attempt is made to increase the detection sensitivity by increasing the fixed amount of the specific capturing substance, there is a risk that the control cannot be judged. On the other hand, when the specific binding substance for capturing the test substance, like in the test strip [ABC+R], does not compete with the substance that binds to the control site, it becomes possible to increase the detection sensitivity without adversely affecting the control judgment by increasing the fixed amount of the specific capturing substance.

[0063] From the above, it was found that by using a gold-coated silver nanoplate having a thick gold layer, the detection sensitivity of an immunochromatography test can be enhanced and multi-color analysis becomes possible. Therefore, even a trace amount of a test substance can be easily detected by an immunochromatography test.

Claims

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; 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 diameter of the silver nanoplate as the core is 55 nm or less.

2. The immunochromatography kit according to claim 1, wherein the average thickness of the gold layer is 1.5 nm to 10.0 nm.

3. The immunochromatography kit according to claim 1 or 2, wherein the gold-coated silver nanoplate is included in a dry state.

4. 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.

5. the gold layer is formed on the main plane and end faces 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 on the end face to the average particle diameter (D) of the silver nanoplate is 0.05 or more.

6. The gold-coated silver nanoplate has a maximum absorption wavelength in the visible light region, and the absorbance (E 900 at a wavelength of 900 nm of the gold-coated silver nanoplate) with respect to the absorbance (E max at the maximum absorption wavelength) of the ratio (E max / E 900 ) is 5 or more. The immunochromatographic kit according to any one of claims 1 to 5.

7. The immunochromatography kit according to any one of claims 1 to 6, wherein the specific binding substance for capture and / or the specific binding substance for detection includes an antibody.

8. the sample contains a plurality of types of test substances; specific binding substances for capturing each of the plurality of types of test substances are immobilized on the membrane; The immunochromatography kit according to any one of claims 1 to 7, wherein the gold-coated silver nanoplate carries specific binding substances for detecting each of the plurality of types of test substances.

9. The immunochromatography kit according to claim 8, wherein the specific binding substance for capture is immobilized at different sites for each type of test substance to which it binds.

10. The immunochromatography kit according to claim 8 or 9, wherein the specific binding substance for detection is carried on a gold-coated silver nanoplate having a maximum absorption wavelength different for each type of test substance to which it binds.

11. The immunochromatography kit according to any one of claims 1 to 10, wherein the membrane further comprises a control site for determining the success or failure of an immunochromatography test.

12. further comprising a control-specific binding substance that specifically binds to the control site, 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 claim 11.

13. The immunochromatography kit according to any one of claims 1 to 12, further comprising nanoparticles other than the gold-coated silver nanoplates.

14. The immunochromatography kit according to any one of claims 1 to 13, wherein the test substance contains at least one selected from the group consisting of influenza virus antigen and coronavirus antigen.

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