JP6440166B2 - Immunochromatography kit - Google Patents

Description

  The present invention relates to an immunochromatography kit, and more particularly to an immunochromatography kit excellent in detection sensitivity of a test substance. The immunochromatography kit is a test kit that uses an immunochromatographic method when detecting a test substance.

  Noble metal colloids hardly change chemically and constitute so-called nanoparticles having a particle size of about several nanometers to several tens of nanometers. In addition, each colloid has a unique color and is expected to be applied to various uses. For example, application to a detection technique using a molecular recognition function has been proposed.

  Japanese Patent No. 4995882 (Patent Document 1) discloses a localized surface plasmon resonance (LSPR) sensor composed of metal nanoparticles in which the absorption spectrum of particles is sharply modified. 1) The particle size is 5 to 100 nm, 2) flat and triangular particles having a side of about 40 nm, and 3) a sharp particle size distribution in the vicinity of the apex 40 nm as measured by the photon correlation method. To do.

  Further, Japanese Patent No. 4778738 (Patent Document 2) focuses on plate-like silver nanoparticles in the field of devices capable of recognizing target species of interest such as peptides, metabolites, molecules or ions, and interacts with target analytes. Disclosed is a sensor in which plate-like silver nanoparticles having a functioning receptor attached to the surface are dispersed.

Japanese Patent No. 4995882 Japanese Patent No. 4778738

  However, when plate-like silver nanoparticles are applied to detection technology using molecular recognition function, there are issues regarding the stability of plate-like silver nanoparticles to body fluids and biochemical buffer solutions, and improvements to application to detection technology remain. There is room for. Also, spherical nanoparticles of other metals such as gold are not suitable for multicolor design such as changing the detection color for each test substance because the absorption wavelength control region of localized surface plasmon resonance (LSPR) is limited. First, when nanoparticles are applied to a detection technique, there is also a problem that it is difficult to determine a detection line when measuring a plurality of test substances at the same time.

  Accordingly, an object of the present invention is to solve the above-mentioned problems of the prior art, and to use an immunochromatography kit which is one of detection techniques using a molecular recognition function, which is excellent in detection of a test substance and uses a body fluid or a biochemical buffer. An object of the present invention is to provide an immunochromatography kit capable of

  As a result of intensive studies to achieve the above object, the present inventors have found that when metal fine particles formed by coating the surface of plate-like silver nanoparticles with gold are used in an immunochromatography kit, body fluids or biochemical buffer solutions of metal fine particles The inventors have found that it is possible to improve the detection sensitivity of a test substance while ensuring the stability to the above, and have completed the present invention.

  That is, the immunochromatography kit of the present invention has plate-shaped silver nanoparticles and metal fine particles (a) made of gold covering the surface of the plate-shaped silver nanoparticles, and has a specific binding property to a test substance and And a molecule (b) having a binding property to the metal fine particles (a).

In a preferred example of the immunochromatography kit of the present invention, an immunochromatography kit comprising a chromatographic carrier that exhibits one or more detection lines after an immunochromatographic test,
The color of the detection line is based on the color of one type of metal fine particles (a) or a mixed color of a plurality of types of metal fine particles (a) exhibiting different colors.

In another preferred embodiment of the immunochromatography kit of the present invention, an immunochromatography kit comprising a chromatographic carrier that exhibits a plurality of types of detection lines after an immunochromatography test,
In the chromatographic carrier, plural kinds of molecules (b) having binding properties to different test substances are fixed at different positions.

  In a preferred example of the immunochromatography kit of the present invention, a water-soluble polymer (c) having no binding property to the test substance and having binding properties to the metal fine particles (a) is further provided.

  ADVANTAGE OF THE INVENTION According to this invention, the immunochromatography kit which can improve the detection sensitivity of a test substance can be provided, ensuring the stability with respect to the bodily fluid and biochemical buffer of a metal microparticle.

1 shows a schematic diagram of an immunochromatographic test using one embodiment of the immunochromatographic kit of the present invention. FIG. The schematic of the immunochromatography test using other embodiment of the immunochromatography kit of this invention is shown. The schematic of the immunochromatography test using other embodiment of the immunochromatography kit of this invention is shown. The schematic of the immunochromatography test using other embodiment of the immunochromatography kit of this invention is shown. The schematic of the immunochromatography test using other embodiment of the immunochromatography kit of this invention is shown. The schematic of the immunochromatography test using other embodiment of the immunochromatography kit of this invention is shown. It is an example of the 2nd developing solution which can be used for the immunochromatography kit shown in FIG. It is a figure which shows the optical characteristic of the seed particle of a plate-shaped silver nanoparticle. It is a figure which shows the optical characteristic of plate-shaped silver nanoparticle AD. It is a figure which shows the optical characteristic of metal microparticles A-D. It is a figure of the measurement result of the spectral characteristic of the plate-like silver nanoparticle in a buffer solution. It is a figure of the measurement result of the spectral characteristic of the metal microparticles B in a buffer solution. It is a figure which shows the result of the visual determination of [immunochromatography test Example 1] and [immunochromatography test comparative example 1]. It is a figure which shows the result of the brightness | luminance analysis at the time of using the developing solution A prepared using the metal microparticle A of [immunochromatography test Example 1]. It is a figure which shows the result of the brightness | luminance analysis at the time of using the developing solution B prepared using the metal microparticle B of [immunochromatography test Example 1]. It is a figure which shows the result of the brightness | luminance analysis at the time of using the developing solution C prepared using the metal microparticle C of [immunochromatography test Example 1]. It is a figure which shows the result of the brightness | luminance analysis at the time of using the developing solution D prepared using the metal microparticle D of [immunochromatography test Example 1]. It is a figure which shows the result of the brightness | luminance analysis at the time of using the antibody-gold colloid developing solution I prepared using the spherical gold colloid of [Immunochromatography test comparative example 1]. Comparison of the results of luminance analysis of the developing liquids A to D and the antibody-gold colloid developing liquid I prepared using the metal fine particles A to D and the spherical gold colloid of [Immunochromatographic Test Example 1] and [Immunochromatographic Test Comparative Example 1] FIG. It is a figure which shows the result of the visual determination of [immunochromatography test Example 2] and [immunochromatography test comparative example 2]. It is a figure which shows the result of the brightness | luminance analysis at the time of using the developing solution E prepared using the metal microparticles B of [Immunochromatography test Example 2]. It is a figure which shows the result of the brightness | luminance analysis at the time of using the antibody-gold colloid developing solution II prepared using the spherical gold colloid of [Immunochromatography test comparative example 2]. It is the figure which compared the result of the brightness | luminance analysis of the developing solution E and the antibody-gold colloid developing solution II which were prepared using the metal microparticles B of [Immunochromatographic test Example 2] and [Immunochromatographic test Comparative Example 2] and spherical gold colloid. . It is a figure which shows the preparation ratio of the developing liquid IL of [immunochromatography test Example 3]. It is a figure which shows the spectral characteristics of the developing solution FH of [Immunochromatography test Example 3] and the antibody-gold colloid developing solution III of [Immunochromatography test comparative example 2]. It is a figure which shows the spectral characteristics of the developing liquid IL of [immunochromatography test Example 3]. It is a figure which shows the result of the color measurement of [immunochromatography test Example 3] and [immunochromatography test comparative example 2]. It is a figure which shows the SEM observation photograph of a seed particle. It is a figure which shows the SEM observation photograph of the metal microparticle A. It is a figure which shows the SEM observation photograph of the metal microparticles B. It is a figure which shows the SEM observation photograph of the metal microparticle C. FIG. It is a figure which shows the SEM observation photograph of the metal microparticle D. FIG. It is a figure which shows the chromaticity coordinate of yellow, magenta, and cyan, and the chromaticity coordinate of the aqueous dispersion of metal microparticles A, B, and C in a CIE1931xy chromaticity diagram. It is a figure which shows the chromaticity coordinate of red, blue, and green and the chromaticity coordinate of liquid mixture A, B, and C in a CIE1931xy chromaticity diagram. It is a figure which shows the result of the visual determination of [immunochromatography test Example 4]. It is a figure which shows the result of the color measurement of [immunochromatography test Example 4]. It is a figure which shows the result of the brightness | luminance analysis at the time of using the developing solution M prepared using the metal microparticles B and C of [immunochromatography test Example 4]. It is a figure which shows the preparation ratio of the developing liquid QT of [immunochromatography test Example 5]. It is a figure which shows the result of the colorimetry of [immunochromatography test Example 5].

  The present invention is described in detail below. The immunochromatography kit of the present invention comprises a plate-like silver nanoparticle and metal fine particles (a) made of gold covering the surface of the plate-like silver nanoparticle, a specific binding property to a test substance, and the metal And a molecule (b) having a binding property to the fine particles (a).

The metal fine particles (a) used in the immunochromatography kit of the present invention are composed of plate-like silver nanoparticles and gold covering the surface of the plate-like silver nanoparticles. Since the metal fine particles (a) have a plate shape, plasmon absorption is strong in the visible light region to the near infrared light region other than the region where the spherical fine particles absorb (J. Phys. Chem. B, 107, 668 (2003)). ) Because the color of the test substance detection line during the immunochromatography test can be set in a wider wavelength range than when spherical fine particles are used, the test substance detection line can be multicolored and the metal fine particles accumulated in the test substance detection line The luminance is lowered by the plasmon absorption of (a), and the contrast difference with the background is increased, so that the visibility is increased. In addition, when the plate-like silver nanoparticles are concentrated by centrifugation or oxidized in various buffers essential for biochemical tests, the aspect ratio and absorbance change. Moreover, although the reason is unknown, when the spherical fine particles are concentrated by centrifugation, aggregation or adsorption to the centrifuge tube occurs and the absorbance changes. In contrast, since the metal fine particles (a) are coated with gold, they have high dispersion stability and oxidation resistance in various buffer solutions. Further, when the metal fine particles (a) are concentrated by centrifugation, aggregation and adsorption to the centrifuge tube do not occur, so the change in absorbance is small. In other words, it has strong durability against centrifugation and buffer exposure, which are pretreatment operations when using metal microparticles for immunochromatography, and is less susceptible to oxidation and aggregation than other metal microparticles, and can maintain a high absorbance. Therefore, when this is used as a labeling substance, the detection sensitivity for the test substance can be improved.
The detection sensitivity in the present invention can be quantified by visual determination and luminance difference analysis. In the visual determination, the presence or absence of the detection line after the immunochromatographic test is visually confirmed, and the lowest test substance concentration at which the detection line can be confirmed can be set as the detection sensitivity. In the luminance analysis, the minimum test substance concentration when the absolute value of the value obtained by subtracting 1 from 2 below is 2 (detection limit luminance difference) or more can be used as the detection sensitivity.
1. 1. Difference in luminance between a detection line and an area other than the detection line in an immunochromatographic test using a developing solution not containing a test substance. Luminance difference between the detection line on the immunochromatographic carrier and the part other than the detection line during the immunochromatographic test using a developing solution containing the test substance

  In the immunochromatography kit of the present invention, the metal fine particles (a) can be used in the same form as a normal labeling substance used in an immunochromatography method. For example, the metal fine particles (a) are dispersed in a developing solution or the molecules ( It is carried on a chromatographic carrier integrally with b) or on an absorbent paper joined to the development start part of the chromatograph together with the molecule (b).

  The plate-like silver nanoparticles constituting the metal fine particles (a) are flat particles having two main surfaces, and the shape of the main surfaces is a polygonal shape such as a triangle, pentagon, hexagon, etc., and the corners are curved. For example, it is known that the shape of particles can be controlled by adjusting the rate of reducing silver ions and selecting the type of dispersant adsorbed on the nanoparticle surface. . In addition, the shape of the main surface is preferably a circular shape with curved corners because it is easy to flow with little physical catching with the substances constituting the chromatographic test paper when the chromatograph is developed. In addition, the thickness of the plate-like silver nanoparticles is usually 40 nm or less, preferably 5 to 20 nm, and the particle diameter that is the maximum length of the main surface of the particles is usually 10 to 1000 nm, preferably 10 to 100 nm. Furthermore, the aspect ratio (particle diameter / thickness) of the plate-like silver nanoparticles is usually 2 or more, and preferably 2 to 10 in which the absorption wavelength of LSPR is expressed in the visible light region and multicolor design is possible. In the case of detecting with near-infrared light, plate-like silver nanoparticles having an aspect ratio that allows LSPR to appear at 800 to 2000 nm (for example, LSPR appears near 900 nm with an aspect ratio of 11) may be used. As an example of multicolor design, plate-like silver nanoparticles that express a single LSPR by adjusting the aspect ratio of the plate-like silver nanoparticles may be used. The Munsell color system, which is one of the color systems that represent the color quantitatively, has a Munsell value (hereinafter also simply referred to as a Munsell value) of 5Y 8.5 / 14, and the coordinates of the CIE 1931xy chromaticity diagram ( Hereinafter, yellow (yellowish color, plate-like silver nanoparticles expressing LSPR in the vicinity of 400 to 500 nm) having x: 0.4498, y: 0.4811, and Munsell value of 5RP 5 / 14, magenta with chromaticity coordinates of x: 0.4142, y: 0.2428 (magenta color, plate-like silver nanoparticles expressing LSPR in the vicinity of 500 to 600 nm), Munsell value of 7.5B 6 / 10, chromaticity coordinates x: 0.1934, y: 0.2374 cyan (cyan color, plate-like silver nanoparticles expressing LSPR in the vicinity of 600 to 750 nm Etc., can be selected absorption wavelength of any LSPR by adjusting the aspect ratio of the plate-shaped silver nanoparticles. FIG. 33 shows chromaticity coordinates of yellow, magenta, and cyan in the CIE 1931xy chromaticity diagram. Two or more kinds of plate-like silver nanoparticles having different aspect ratios may be mixed to design a color. For example, yellow and magenta are mixed and red (Munsell value: 5R 4/14, chromaticity coordinates x: 0.5734, y: 0.3057), magenta and cyan are mixed and blue (Munsell value: 10B 4/14, Chromaticity coordinates x: 0.1310, y: 0.1580), yellow and cyan mixed and green (Munsell value: 2.5G 6.5 / 10, chromaticity coordinates x: 0.3000, y: 0.6000 ) Etc. can be designed. FIG. 34 shows chromaticity coordinates of red, blue and green in the CIE 1931xy chromaticity diagram. Furthermore, it is possible to use a multi-color design to which subtractive mixing using three or more kinds of plate-like silver nanoparticles with different aspect ratios that express yellow, magenta, and cyan colors as three primary colors. For example, when a black color is designed by mixing plate-type silver nanoparticles of yellow, magenta, and cyan colors in an arbitrary ratio, the test substance detection line during the immunochromatography test has a contrast difference from the background (white) Increases the visibility (detection sensitivity). In addition, when designing a color by mixing a plurality of plate-like silver nanoparticles with different aspect ratios, the thickness of the plate-like silver nanoparticles is small so that the difference in the flow rate of the particles during the development of the chromatograph is small. 5-15 nm is preferable, and the particle diameter which becomes the maximum length of the main surface of the particle is preferably 10-80 nm.

  The method for preparing the plate-like silver nanoparticles is not particularly limited, and can be appropriately selected depending on the purpose such as adjusting the particle size. For example, it is known that silver particles can be synthesized by adding silver ions to an aqueous solution in which an appropriate dispersant is dissolved and then reducing the silver ions by an appropriate reduction method. , A method of reducing silver ions with light (SCIENCE, 294, 1901 (2001)), a method of reducing with heat (Nano Lett., 2, 903 (2002)), a method of reducing with a reducing agent (Adv. Mater., 14, 1084 (2002)). Furthermore, a method of reducing silver ions in the presence of copper ions, which are different metals (Japanese Patent No. 5059317), can be mentioned. In addition, when synthesizing plate-like silver nanoparticles, spherical silver nanoparticles are usually synthesized as impurities. For example, it is described in “Adv. Funct. Mater., 18, 2005 (2008)”. In such a technique, a seed liquid dispersion of plate-like silver nanoparticles having stacking faults is prepared in advance in order to grow the shape of the particles into a plate shape, and silver ions and a reducing agent are added thereto to add silver. The shape of the nanoparticles is grown into a plate shape. This method using seed particles is preferable because the generation of spherical particles can be greatly reduced. These seed particles are formed when silver ions are reduced in an aqueous solution of trisodium citrate in which a polyanionic polymer such as polystyrene sulfonic acid is dissolved. As a result of the relatively strong interaction between the polyanionic polymer and the surface of the silver nanoparticles, defects are generated. A structure is believed to arise. The seed particles having stacking faults grow in the <111> direction and grow into plate-shaped silver nanoparticles having a large {111} plane. Plate-like silver nanoparticles can be obtained by adding silver ions and a reducing agent to an aqueous dispersion containing seed particles. At this time, plate-like silver nanoparticles with a wide aspect ratio can be obtained by adjusting the amount of seed particles added. When the number of seed particles is set to be large, the ratio of the amount of silver ions consumed by individual seed particles decreases, so that the particle diameter of the obtained plate-like silver nanoparticles tends to be small. Further, when the number of seed particles is set to be small, the ratio of the amount of silver ions consumed by each seed particle increases, and therefore the particle diameter of the obtained plate-like silver nanoparticles tends to increase. As a reducing agent, ascorbic acid can be used. In addition, when plate-like silver nanoparticles are prepared by these techniques, they are usually prepared in the form of an aqueous dispersion. In addition, in order to separate particles having shapes other than the target shape such as spherical silver nanoparticles and excess organic substances used in the synthesis from the product, the dispersion of silver nanoparticles is subjected to a centrifuge or filtered through a filter. Or you may. The prepared plate-like silver nanoparticles have a distribution in particle diameter in the dispersion, and the absorption spectrum changes according to the distribution. Narrowing the particle size distribution is suitable for multicolor design because the half-value width of the absorption spectrum is narrowed and the color becomes vivid.

  The metal fine particles (a) used in the immunochromatography kit of the present invention require that the surface of the plate-like silver nanoparticles is coated with gold. For example, when the plate-like silver nanoparticles are not coated with gold and are supported on a chromatographic carrier or are supported on an absorbent paper at the development start portion, the silver nanoparticles are dried on the chromatographic carrier during storage. Since the silver nanoparticles are in contact with a developing solution having a wide pH during use, the plate-like silver nanoparticles are often oxidized. When the silver nanoparticles are oxidized, the shape of the silver nanoparticles also changes, which causes a problem that a color change accompanying a change in the plasmon absorption wavelength occurs. In particular, when plate-like silver nanoparticles are applied to an immunochromatography kit, there is room for improvement in the application of plate-like silver nanoparticles because precise shape control of the plate-like silver nanoparticles is required. Even when the present inventor is used in an immunochromatography kit by coating a plate-like silver nanoparticle with a metal thin film that is less oxidizable than silver and forming a metal shell on the surface of the plate-like silver nanoparticle. It has been found that oxidation of plate-like silver nanoparticles can be prevented. Examples of the metal that can be used as the metal shell of the plate-like silver nanoparticles include gold, which is a noble metal than silver.

  The method for coating the surface of the plate-like silver nanoparticles with metal is not particularly limited, and can be appropriately selected depending on the purpose such as adjusting the film thickness of the metal. For example, when coating with gold, gold ions are added to an aqueous dispersion of plate-like silver nanoparticles, and the gold ions are chemically reduced, so that the surface of the plate-like silver nanoparticles is made of a gold thin film (gold (Materials Chemistry and Physics, 90, 361 (2005), Angew. Chem. Int. Ed., 51, 5629 (2012)). For example, in order to prevent aggregation of the plate-like silver nanoparticles and etching of the surface, first, citric acid, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol ( PVA), polyacrylic acid (PA), gelatin, amine and the like are added to treat the surface of the plate-like silver nanoparticles, and then gold ions and a reducing agent are added to the dispersion. Then, metal fine particles in which the surface of the plate-like silver nanoparticles is coated with gold can be obtained. In the present invention, “gold covering the surface of the plate-like silver nanoparticles” refers to gold existing on the surface of the plate-like silver nanoparticles, but in addition to the gold existing alone. In addition, those existing in the state of an alloy with silver are also included.

  The molecule (b) used in the immunochromatography kit of the present invention is a molecule (for example, antibody or antigen) having specific binding property to a test substance (for example, antigen or antibody). It is necessary to have binding properties. As a result, a complex is formed in which the test substance and the metal fine particles (a) are bonded via the molecule (b), so that the test substance can be detected based on the color tone of the metal fine particles (a).

  The molecule (b) preferably has a functional group such as an amino group, a carboxyl group, or a hydroxyl group because it has binding properties to the metal fine particles (a), and in particular, a mercapto group, a dithiol group, or a sulfide group. It is preferable to have. However, even if these functional groups are not present, they may be physically adsorbed by interfacial energy or electrostatic adsorption of the compound.

The molecule (b) is appropriately selected depending on the type of the test substance, but is an immunoreactive substance, lectin and sugar chain, avidin and biotin, nucleic acid and nucleic acid that hybridizes with the nucleic acid, nucleic acid It can be applied to any substance as long as it can interact with small organic molecules, proteins, nucleic acids, cells, cell tissues, microorganisms and the like that specifically bind to aptamers such as aptamers and peptide aptamers. Examples of immunoreactive substances include various antibodies and antigens. Here, as antibodies (IgG, IgM, IgE, IgD, IgA, etc.), autoantibodies such as anti-DNA antibodies, anti-ENA antibodies, anti-cardioribine antibodies, anti-mitochondrial antibodies, anti-smooth muscle antibodies, various immunoglobulins, etc. For example, the present invention can be applied to analysis of specific immunoglobulins (specific IgE, etc.) that specifically recognize allergens. Examples of the antigen include a glycoprotein, a specific protein composed of a plurality of subunits, and a protein complex such as prostate specific antigen (PSA).
Examples of the molecule (b) include anti-concanavalin A antibody, mannose and its derivatives (test substance: concanavalin A), anti-verotoxin antibody, globotrisaccharide and its derivative (test substance: verotoxin), sialyl lactose and its Derivatives, anti-influenza antibody (test substance: influenza virus and its antigen), anti-hepatitis B virus antibody (test substance: hepatitis B virus and its antigen), anti-insulin antibody (test substance: insulin), anti-aflatoxin antibody (test) Substance: aflatoxin), anti-human chorionic gonadotropin antibody (test substance: human chorionic gonadotropin), anti-human immunodeficiency virus antibody (test substance: human immunodeficiency virus and its antigen), anti-lysine antibody, galactose and its derivatives (test) Substance: ricin), anti-Salmonella anti (Test substance: Salmonella), etc., Bacillus anthracis, Staphylococcus aureus, D group Shigella sonnei, Escherichia sumirum, Escherichia smut ), Streptococcus hemoliticus, Paratyphi (Salmonella paratyphi A), Staphylococcal enterotoxin B (Staphylococcal enterotoxin B), Pseudomonas aeruginosa, etc. Cell surface DNA aptamers that bind to cancer markers, etc. appearing, peptide aptamers, etc. that bind with a tumor protein HPV16 E6 of human papillomavirus (HPV) and the like.

  Examples of test substances include concanavalin A (Con A), malt agglutinin, lectins such as ricin, serum protein components such as immunoglobulin G (IgG), glycoproteins such as mucin, prostatic acid phosphatase (PAP), prostate specific antigen (PSA), alkaline phosphatase, transaminase, trypsin, pepsinogen, α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and other cancer specific substances, peptide hormones (growth hormone (GH), adrenocorticotropic hormone (ACTH), Melamine cell stimulating hormone (MSH), prolactin, thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), pituitary hormone, calcium metabolism regulating hormone, pancreatic hormone, gastrointestinal hormone, vasoactive hormone Mon, placental hormones such as human chorionic gonadotropin (hCG), fecal occult blood, influenza virus, adenovirus, RS virus, rotavirus, human papilloma virus, hepatitis B virus and other antigens, antibodies, metabolites, etc. , Chlamydia, Syphilis treponema, Streptococcus, Bacillus anthracis, Staphylococcus aureus, Shigella, Escherichia coli, Salmonella typhimurium, Paratyphi, Pseudomonas aeruginosa, Vibrio parahaemolyticus and other antigens, antibodies and metabolites, rheumatoid factor, serotonin , Follicular hormones such as ferritin, substance P and estrone, natural or synthetic luteinizing hormones such as progesterone, male hormones such as testosterone, adrenocortical hormones such as cortisol, cholesterol, bile acids, cardiotonic steroids, sapogenins and other STEROY , Epinephrine, dopamine, bioactive alkaloids, amino group-containing psychotropic drugs, low molecular peptides such as TRH, thyroid hormones such as diiodothyronine, antibiotics such as prostaglandins, vitamins, penicillin , DNA, RNA, oligonucleotides, polynucleotides, amplified products thereof, and the like.

  In the immunochromatography kit of the present invention, the molecule (b) can be used in the same form as a normal antibody used in an immunochromatography method. For example, the molecule (b) is dispersed in a developing solution and fixed alone on a chromatographic carrier. In a form integrated with the fine metal particles (a) and dispersed in the developing solution and fixed alone on the chromatographic carrier, a chromatograph integrated with the fine metal particles (a) The form supported by the support | carrier and being independently fixed to the position different from the said metal microparticle of a chromatographic support | carrier is mentioned.

  The immunochromatography kit of the present invention preferably further comprises a water-soluble polymer (c) that does not have binding properties to the test substance and has binding properties to the metal fine particles (a). When the metal fine particles (a) are dispersed in the developing solution, the dispersion stability of the metal fine particles (a) in the developing solution is improved by adding a water-soluble polymer (c) to the developing solution. Can be made. In addition, the feature of “having no binding property to the test substance” means that the water-soluble polymer (c) is a substance different from the molecule (b).

  The water-soluble polymer (c) preferably has a functional group such as an amino group, a carboxyl group, or a hydroxyl group because it has binding properties to the metal fine particles (a), and in particular, a mercapto group, a dithiol group, It preferably has a sulfide group. However, even if these functional groups are not present, they may be physically adsorbed by interfacial energy or electrostatic adsorption of the compound. Specific examples include polyethylene glycol, polyacrylic acid, polymethacrylic acid, polyvinyl pyrrolidone, polyallylamine, dextran, polyacrylamide, polymethacrylamide, polyvinyl phenol, polyvinyl benzoate, and polyvinyl alcohol. In addition, these water-soluble polymers may be used individually by 1 type, and may be used in combination of 2 or more type.

  The immunochromatography kit of the present invention usually comprises a chromatographic carrier. A chromatographic carrier exhibits one or more detection lines after an immunochromatographic test. As the chromatographic carrier, a porous substance exhibiting a capillary phenomenon is preferable, and a nitrocellulose membrane, a cellulose membrane, an acetylcellulose membrane, a polysulfone membrane, a polyethersulfone membrane, a nylon membrane, a glass fiber, a nonwoven fabric, a cloth, a thread, and the like are preferable. Can be mentioned.

On the chromatographic carrier, the molecule (b) is immobilized alone, or the same molecule (b) is immobilized in plural or different kinds of molecules (b) are immobilized in plural. The part functions as a part (detection line) for determining the presence or absence of the test substance.
When the chromatographic carrier exhibits a plurality of types of detection lines after the immunochromatographic test, a plurality of types of molecules (b) having binding properties to different test substances are fixed to the chromatographic carrier at different positions. Is preferred. In this case, it becomes possible to detect different test substances at once.
Further, the color of the detection line presented after the immunochromatographic test is based on the color of one type of metal fine particles (a) or a mixed color of a plurality of types of metal fine particles (a) exhibiting different colors.
For this reason, when the chromatographic carrier is provided with a plurality of immobilized portions by a plurality of types of molecules (b) having binding properties to different test substances, the difference in color tone of the metal fine particles (a) is used. It is also possible to cause each fixed portion to function as a detection line exhibiting a different color (multiple detection lines). Thereby, a plurality of test substances can be detected at once.

  The chromatographic carrier may be used on a support such as paper or plastic. Further, the chromatogram carrier may be provided with an absorbing portion (for example, paper or nonwoven fabric) for absorbing and removing the developing solution at an end portion located on the downstream side when viewed from the developing direction of the developing solution. In addition, an absorption part (for absorbing a solution containing a test substance) capable of carrying a complex of metal fine particles (a) and an antibody (b) at an end located upstream from the direction in which the development liquid is developed. For example, glass fiber) may be provided.

  A commercially available product can be suitably used as the chromatographic carrier on which the molecule (b) is immobilized alone. A chromatographic carrier carrying a complex composed of the molecule (b) and the metal fine particles (a) and having the molecule (b) immobilized alone is prepared using the above-mentioned commercially available chromatographic carrier. it can. When the absorption part for removing the developing solution of the commercially available chromatographic carrier is the upper part, the molecule (b) and the metal fine particles (a) are located at the lower part of the part where the molecule (b) is immobilized. The solution can be prepared by dropping a solution in which the above are integrated linearly and drying.

  The immunochromatography kit of the present invention usually comprises a developing solution. Examples of the developing solution include a mixture of a sample that may contain a test substance and an appropriate solvent (for example, water, physiological saline, or a buffer solution), and the sample itself is placed on a chromatographic carrier. If it can be developed, the sample itself can be used as a developing solution.

  The sample that may contain the test substance is not particularly limited. For example, biological samples, particularly body fluids of animals (particularly humans) (for example, blood, serum, plasma, spinal fluid, tears) Fluid, sweat, urine, pus, runny nose or sputum) or excrement (eg, feces), organs, tissues, mucous membranes and skin, swabs suspected of containing them, gargle, or animals and plants themselves Or those dry bodies can be mentioned.

  In the immunochromatography kit of the present invention, when the molecule (b) is immobilized alone on the chromatographic carrier, a developing solution further containing metal fine particles (a) and molecules (b) can be used.

  When the molecule (b) is immobilized on the chromatographic carrier alone, the above-described developing solution contains a sample that may contain the test substance, the metal fine particles (a), and the molecules (b). Although mixed, the developing solution may be divided into two types. For example, a developing solution containing a sample that may contain a test substance may be used as the first developing solution, and a developing solution containing metal fine particles (a) or molecules (b) may be used as the second developing solution. . Further, when the molecule (b) is integrated with the metal fine particles and supported on the chromatographic carrier, only the developing solution containing the sample that may contain the test substance may be developed. Similarly, a sample that may contain a test substance when the molecule (b) is integrated with the metal fine particles and is supported on the absorption part at the upstream side when viewed from the direction in which the developing solution is developed. It is sufficient to develop only the developing solution containing

  The immunochromatography kit of the present invention can be used for various detection methods using an immunochromatography method. Hereinafter, a part of the detection method (immunochromatography test) using the embodiment of the immunochromatography kit of the present invention will be described in detail with reference to the drawings. The immunochromatographic test described below is described on the assumption that the test substance is present in a sample that may contain the test substance. Of course, the test substance may not exist.

  FIG. 1 shows a schematic diagram of an immunochromatographic test using one embodiment of the immunochromatography kit of the present invention. The chromatographic carrier shown in FIG. 1 is installed on a plastic, and an absorbent paper is provided on the upper end thereof (this is an immunochromatographic test paper). Further, in this chromatographic carrier, the molecule (b) is fixed in a straight line, and a determination portion is formed. The developing solution shown in FIG. 1 includes a first developing solution containing a test substance and a buffer solution (actually, the above-described biological sample or the like), metal fine particles (a), molecules (b), and a buffer solution. In the second developing solution, metal fine particles (a) and molecules (b) are bonded to form a complex. According to the immunochromatographic test shown in FIG. 1, first, a first developing solution is developed on an immunochromatographic test paper (FIG. 1 (a)). Here, when the test substance is contained in the first developing solution as shown in FIG. 1, the test substance binds to the molecule (b) immobilized on the chromatographic carrier (FIG. 1 (b)). Next, in order to remove the unbound test substance remaining on the chromatographic carrier, the chromatographic carrier is washed by developing a buffer solution (FIG. 1 (b)). The removed test substance is collected on absorbent paper. Next, the second developing solution is developed on the immunochromatographic test paper (FIG. 1 (c)). Here, when the test substance is contained in the first developing solution as shown in FIG. 1, the complex of the metal fine particle (a) and the molecule (b) is bound to the molecule (B) on the chromatographic carrier. It binds to the test substance (FIG. 1 (d)). When the test substance is contained in the first developing solution as shown in FIG. 1, the metal fine particles (a) remain in the determination portion, so that the test substance can be detected based on the color tone of the metal fine particles (a).

  FIG. 2 shows a schematic diagram of an immunochromatographic test using another embodiment of the immunochromatography kit of the present invention. The chromatographic carrier shown in FIG. 2 is the same as that shown in FIG. The developing solution shown in FIG. 2 is a developing solution containing a test substance, metal fine particles (a), molecules (b), and a buffer solution. Substances are bonded to form a complex (FIG. 2A). According to the immunochromatographic test shown in FIG. 2, a developing solution is developed on an immunochromatographic test paper (FIGS. 2B and 2C). Here, when the test substance is contained in the developing solution as shown in FIG. 2, the test substance binds to the molecule (b) immobilized on the chromatographic carrier, as shown in FIG. 2 (d). In addition, the complex remains in the determination part. Here, since the composite contains the metal fine particles (a), the test substance can be detected by the color tone of the metal fine particles (a).

  FIG. 3 shows a schematic diagram of an immunochromatographic test using another embodiment of the immunochromatography kit of the present invention. The chromatographic carrier shown in FIG. 3 (a) is the same as that shown in FIG. In addition, the fine metal particles (a) and the molecules (b) of the chromatographic carrier of FIG. 3 (a) are located upstream from the determination part (in FIG. 3 below the determination part) as viewed from the direction in which the developing solution is developed. A solution containing this complex was dropped and dried to produce a complex-supported immunochromatographic carrier (FIGS. 3B and 3C). In the chromatographic carrier of FIG. 3C, when the developing solution passes through the complex-supporting portion, the complex is also developed together with the developing solution. The developing solution shown in FIG. 3 is a developing solution containing a test substance and a buffer solution (actually, the above-described biological sample or the like may be used). According to the immunochromatographic test shown in FIG. 3, a developing solution is developed on an immunochromatographic test paper (FIGS. 3D and 3E). Here, when the test substance is contained in the developing solution as shown in FIG. 3, the test substance binds to the molecule (b) immobilized on the chromatographic carrier, while FIGS. 3 (e) and (f) As shown in the above, the complex remains in the determination part. Here, since the composite contains the metal fine particles (a), the test substance can be detected by the color tone of the metal fine particles (a).

FIG. 4 shows a schematic diagram of an immunochromatographic test using another embodiment of the immunochromatography kit of the present invention. The chromatographic carrier shown in FIG. 4 is a solution that can carry a complex of metal fine particles (a) and an antibody (b) at the end located upstream from the direction in which the developing solution develops and contains a test substance. An absorption portion of glass fiber for absorbing water is provided.
In the chromatographic carrier of FIG. 4 (a), a solution containing a complex of metal fine particles (a) and molecules (b) is dropped onto the glass fiber absorption part and dried to produce a complex-supported immunochromatographic carrier. .
The developing solution shown in FIG. 4 is a developing solution (in fact, the above-described biological sample or the like) containing a test substance and a buffer solution. According to the immunochromatographic test shown in FIG. 4, a developing solution is developed on an immunochromatographic test paper (FIGS. 4B, 4C, 4D and 4E). Here, when the test substance is contained in the developing solution as shown in FIG. 4, the test substance binds to the molecule (b) immobilized on the chromatographic carrier, but FIGS. 4 (e) and (f) As shown in the above, the complex remains in the determination part. Here, since the composite contains the metal fine particles (a), the test substance can be detected by the color tone of the metal fine particles (a).

  FIG. 5 shows a schematic diagram of an immunochromatographic test using another embodiment of the immunochromatography kit of the present invention. The chromatographic carrier shown in FIG. 5 is installed on a plastic, and an absorbent paper is provided on the upper end thereof (this is an immunochromatographic test paper). In this chromatographic carrier, a plurality of different kinds of molecules (b) 1-3 are linearly fixed at different positions to form a determination portion. The developing solution shown in FIG. 5 includes a first developing solution (FIGS. 5B and 5C) containing a plurality of different test substances 1-3 and a buffer solution, and a plurality of different metal fine particles (a) 1. -3, a second developing solution (FIG. 5 (e)) containing a plurality of different kinds of molecules (b) 1-3 (binding to the corresponding numbers of test substances 1-3) and a buffer, In the developing solution, a plurality of different kinds of fine metal particles (a) 1-3 and a plurality of different kinds of molecules (b) 1-3 are combined to form a complex. According to the immunochromatographic test shown in FIG. 5, first, the first developing solution is developed on the immunochromatographic test paper (FIGS. 5B and 5C). Here, when a plurality of different kinds of test substances 1-3 are contained in the first developing solution as shown in FIG. 5, the test substances are a plurality of different kinds of molecules (b) immobilized on a chromatographic carrier. Bind to the corresponding molecule (b) (FIG. 5C). Next, in order to remove the unbound test substance remaining on the chromatographic carrier, the chromatographic carrier is washed by developing a buffer solution (FIG. 5 (d)). The removed test substance is collected on absorbent paper. Next, the second developing solution is developed on the immunochromatographic test paper (FIG. 5 (e)). Here, when a plurality of different types of test substances are included in the first developing solution as shown in FIG. 5, a plurality of different types of metal fine particles (a) 1-3 and a plurality of different types of molecules (b) 1-3 are included. The complex binds to the test substance bound to each molecule (b) on the chromatographic carrier (FIG. 5 (e)). When a plurality of different kinds of test substances 1-3 are included in the first developing solution as shown in FIG. 5, since a plurality of different kinds of metal fine particles (a) 1-3 remain in a plurality of determination portions, A plurality of test substances can be detected at once by the color tone of a).

FIG. 6 shows a schematic diagram of an immunochromatographic test using another embodiment of the immunochromatography kit of the present invention. The chromatographic carrier shown in FIG. 6 is placed on a plastic, and an absorbent paper is provided on the upper end thereof (this is an immunochromatographic test paper). In this chromatographic carrier, a plurality of different kinds of molecules (b) 1-3 are linearly fixed at different positions to form a determination portion. The developing solution shown in FIG. 6 includes a first developing solution (FIGS. 6B and 6C) including a plurality of different test substances 1-3 and a buffer solution, and a plurality of different metal fine particles (a) 1. -3, a second developing solution (FIG. 6 (e)) containing a plurality of different kinds of molecules (b) 1-3 (binding to the corresponding numbers of test substances 1-3) and a buffer solution, In the developing solution, a plurality of different kinds of metal fine particles (a) 1-3 and a plurality of different kinds of molecules (b) 1-3 are combined to form a complex, but the metal fine particles (a) -1 Forms a complex with molecule (b) -1 or molecule (b) -3, and metal fine particle (a) -2 forms a complex with molecule (b) -1 or molecule (b) -2. The metal fine particles (a) -3 form a complex together with the molecule (b) -2 or the molecule (b) -3.
According to the immunochromatographic test shown in FIG. 6, first, the first developing solution is developed on the immunochromatographic test paper (FIGS. 6B and 6C). Here, when a plurality of different kinds of test substances 1-3 are contained in the first developing solution as shown in FIG. 6, the test substances are a plurality of different kinds of molecules (b) immobilized on a chromatographic carrier. It binds to the corresponding molecule (b) among 1-3 (FIG. 6 (c)). Next, in order to remove the unbound test substance remaining on the chromatographic carrier, the chromatographic carrier is washed by developing a buffer solution (FIG. 6 (d)). The removed test substance is collected on absorbent paper. Next, the second developing solution is developed on the immunochromatographic test paper (FIG. 6 (e)). Here, when a plurality of different kinds of test substances 1-3 are included in the first developing solution as shown in FIG. 6, a plurality of different kinds of metal fine particles (a) 1-3 and a plurality of different kinds of molecules (b) 1 -3 binds to the test substance bound to each molecule (b) on the chromatographic carrier (FIG. 6 (e)). When a plurality of different kinds of test substances 1-3 are included in the first developing solution as shown in FIG. 6, a plurality of different kinds of metal fine particles (a) remain in one determination portion, so that the metal fine particles (a) 1- 3 color mixture detection lines can be confirmed.
Next, an example of a method for preparing the second developing solution used in FIG. 6 will be described below. When the metal fine particles (a) -1 are yellow, the metal fine particles (a) -2 are magenta, and the metal fine particles (a) -3 are cyan, the metal fine particles (a) -1 and molecules (b) -1, the metal fine particles (a) -2 and the molecule (b) -1 are respectively complexed separately, and an equal amount of these complexes is mixed to prepare a second developing solution in red tone. Similarly, metal fine particles (a) -2 and molecules (b) -2, metal fine particles (a) -3 and molecules (b) -2 are respectively complexed separately, and then these two types of complexes are equivalent. By mixing each one, a second developing solution having a blue tone can be prepared. Also, the metal fine particles (a) -1 and the molecule (b) -3, the metal fine particles (a) -3 and the molecule (b) -3 are respectively complexed separately, and then these two types of complexes are each equivalent. By mixing, a second developing solution having a green tone can be prepared. A black second developing solution can be prepared by mixing the same amount of these three types of second developing solutions, and when this is developed on the immunochromatographic test paper of FIG. 6, the molecule (b) 1 immobilized on the immunochromatographic paper. -3 shows a mixed color detection line of the metal fine particles (a). FIG. 7 shows an example of a second developing solution that can be used in the immunochromatography kit shown in FIG. Note that the types of the metal fine particles (a) and the molecules (b) are not limited to the above-described three types, and can be increased without any upper limit, so that the color of the detection portion can be freely controlled.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

(Preparation of seed particles of plate-like silver nanoparticles)
To 20 mL of 2.5 mM sodium citrate aqueous solution, 1 mL of 0.5 g / L molecular weight 70,000 polystyrenesulfonic acid aqueous solution and 1.2 mL of 10 mM sodium borohydride aqueous solution were added, and then at 20 mL / min. While stirring, 50 mL of 0.5 mM aqueous silver nitrate solution was added. The obtained solution was allowed to stand in an incubator (30 ° C.) for 60 minutes to prepare an aqueous dispersion of seed particles of plate-like silver nanoparticles. The optical characteristics of the prepared aqueous dispersion (stock solution) are shown in FIG. The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. The wavelength exhibiting the maximum absorption was 396 nm (extinction degree 3.3) which is the LSPR of spherical silver nanoparticles. In addition, the extinction degree of this invention is the value of the light absorbency when measuring a dispersion liquid with a spectrophotometer. An SEM photograph is shown in FIG. A scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used for analysis of the SEM photograph. The particle diameter was mainly plate-like particles having a size of 3 nm or more and less than 10 nm.

(Preparation of plate-like silver nanoparticles A)
To 200 ml of distilled water, 4.5 mL of a 10 mM ascorbic acid aqueous solution was added, and 12 ml of a seed particle dispersion (hereinafter referred to as seed particle aqueous dispersion) of the plate-like silver nanoparticles was added. To the resulting solution, 120 mL of 0.5 mM aqueous silver nitrate solution was added with stirring at 30 mL / min. Stirring was stopped 4 minutes after the addition of the aqueous silver nitrate solution, 20 ml of 25 mM aqueous sodium citrate solution was added, and the resulting solution was allowed to stand for 100 hours in an incubator (30 ° C.) under atmospheric conditions. An aqueous dispersion of silver nanoparticles A was prepared. FIG. 9 shows the optical characteristics of an aqueous dispersion obtained by diluting the prepared dispersion with distilled water to 4 volumes. The wavelength exhibiting the maximum absorption was 454 nm (quenching 1.0). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. When the plate-like silver nanoparticles A in the aqueous dispersion were observed by SEM, the plate-like silver nanoparticles A had an average particle diameter of 18 nm, an average thickness of 8 nm, and an aspect ratio of 2.2. A scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used for analysis of the SEM photograph.

(Preparation of plate-like silver nanoparticles B)
An aqueous dispersion of plate-like silver nanoparticles B was prepared in the same manner as the preparation of plate-like silver nanoparticles A, except that the amount of seed particle aqueous dispersion added was changed from 12 ml to 4 ml. FIG. 9 shows the optical characteristics of an aqueous dispersion obtained by diluting the prepared dispersion with distilled water to 4 volumes. The wavelength exhibiting the maximum absorption was 526 nm (quenching level 1.1). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. When the plate-like silver nanoparticles B in the aqueous dispersion were observed by SEM, the plate-like silver nanoparticles B had an average particle size of 31 nm, an average thickness of 8 nm, and an aspect ratio of 3.8. A scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used for analysis of the SEM photograph.

(Preparation of plate-like silver nanoparticles C)
An aqueous dispersion of the plate-like silver nanoparticles C was prepared in the same manner as the preparation of the plate-like silver nanoparticles A, except that the amount of the seed particle aqueous dispersion added was changed from 12 ml to 2 ml. FIG. 9 shows the optical characteristics of an aqueous dispersion obtained by diluting the prepared dispersion with distilled water to 4 volumes. The wavelength exhibiting the maximum absorption was 626 nm (quenching level 1.1). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. When the plate-like silver nanoparticles C in the aqueous dispersion were observed by SEM, the plate-like silver nanoparticles C had an average particle diameter of 50 nm, an average thickness of 10 nm, and an aspect ratio of 5.0. A scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used for analysis of the SEM photograph.

(Preparation of plate-like silver nanoparticles D)
An aqueous dispersion of plate-like silver nanoparticles D was prepared in the same manner as the preparation of plate-like silver nanoparticles A, except that the amount of seed particle aqueous dispersion added was changed from 12 ml to 1 ml. FIG. 9 shows the optical characteristics of an aqueous dispersion obtained by diluting the prepared dispersion with distilled water to 4 volumes. The wavelength exhibiting the maximum absorption was 704 nm (extinction degree 1.0). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. When the plate-like silver nanoparticles D in the aqueous dispersion were observed by SEM, the plate-like silver nanoparticles D had an average particle diameter of 74 nm, an average thickness of 8 nm, and an aspect ratio of 9.2. A scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used for analysis of the SEM photograph.

(Preparation of metal fine particles A)
To 120 ml of the aqueous dispersion of plate-like silver nanoparticles A, 8 ml of a 5% by mass polyvinylpyrrolidone (PVP) aqueous solution was added, 1.2 ml of diethylamine was added, and 1.6 ml of 0.5 M ascorbic acid aqueous solution was added. Then, 9.6 ml of 0.16 mM chloroauric acid aqueous solution was added at 0.5 mL / min with stirring. The obtained solution was allowed to stand in an incubator (30 ° C.) for 24 hours to prepare an aqueous dispersion (yellow tone) of metal fine particles A in which the surface of the plate-like silver nanoparticles A was coated with gold. This aqueous dispersion of metal fine particles A was used as a stock solution. FIG. 10 shows the optical characteristics of the aqueous dispersion obtained by diluting the stock solution with distilled water to 4 volumes, and FIG. 33 shows the chromaticity coordinates of the stock solution in the CIE1931xy chromaticity diagram. The wavelength exhibiting the maximum absorption was 464 nm (extinction degree 0.8), and the chromaticity coordinates were x = 0.57070 and y = 0.4774. The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. The chromaticity coordinates are measured using the same spectrophotometer, with an optical path length of 1 cm and a spectrophotometer operation software “Color measurement software P / N 206-65207 (manufactured by Shimadzu Corporation)” illumination: D65, field of view : Performed under conditions set at 2 °. An SEM photograph is shown in FIG. A scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used for analysis of the SEM photograph.

(Preparation of metal fine particles B)
The surface of the plate-like silver nanoparticle B is the same as the preparation of the metal fine particles A except that 120 ml of the aqueous dispersion of the plate-like silver nanoparticle B is used instead of 120 ml of the aqueous dispersion of the plate-like silver nanoparticle A. An aqueous dispersion (magenta tone) of metal fine particles B coated with gold was prepared. An aqueous dispersion of the metal fine particles B was used as a stock solution. FIG. 10 shows the optical characteristics of the aqueous dispersion obtained by diluting the stock solution with distilled water to 4 volumes, and FIG. 33 shows the chromaticity coordinates of the stock solution in the CIE1931xy chromaticity diagram. The wavelength exhibiting the maximum absorption was 534 nm (quenching degree 0.9), and the chromaticity coordinates were x = 0.4276 and y = 0.1751. The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. The chromaticity coordinates are measured using the same spectrophotometer, with an optical path length of 1 cm and a spectrophotometer operation software “Color measurement software P / N 206-65207 (manufactured by Shimadzu Corporation)” illumination: D65, field of view : Performed under conditions set at 2 °. An SEM photograph is shown in FIG. A scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used for analysis of the SEM photograph.

(Preparation of metal fine particles C)
The surface of the plate-like silver nanoparticles C was prepared in the same manner as the preparation of the metal fine particles A, except that 120 ml of the aqueous dispersion of plate-like silver nanoparticles C was used instead of 120 ml of the aqueous dispersion of plate-like silver nanoparticles A. An aqueous dispersion (cyan tone) of metal fine particles C coated with gold was prepared. An aqueous dispersion of the metal fine particles C was used as a stock solution. FIG. 10 shows the optical characteristics of the aqueous dispersion obtained by diluting the stock solution with distilled water to 4 volumes, and FIG. 33 shows the chromaticity coordinates of the stock solution in the CIE1931xy chromaticity diagram. The wavelength exhibiting the maximum absorption was 634 nm (extinction degree 0.9), and the chromaticity coordinates were x = 0.1467 and y = 0.290. The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. The chromaticity coordinates are measured using the same spectrophotometer, with an optical path length of 1 cm and a spectrophotometer operation software “Color measurement software P / N 206-65207 (manufactured by Shimadzu Corporation)” illumination: D65, field of view : Performed under conditions set at 2 °. An SEM photograph is shown in FIG. A scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used for analysis of the SEM photograph.

(Preparation of color mixture with multiple types of metal fine particles)
When an aqueous dispersion of metal fine particles A and an aqueous dispersion of metal fine particles B, which are stock solutions, are mixed at an equal mass, a red-tone mixed liquid A (chromaticity coordinates x: 0.6057, y: 0.3317) is obtained. It was. When an aqueous dispersion of metal fine particles B and an aqueous dispersion of metal fine particles C, which are stock solutions, are mixed at an equal mass, a blue-tone mixed liquid B (chromaticity coordinates x: 0.1731, y: 0.0675) is obtained. It was. When an aqueous dispersion of metal fine particles A and an aqueous dispersion of metal fine particles C, which are stock solutions, are mixed at an equal mass, a green-tone mixed liquid C (chromaticity coordinates x: 0.2549, y: 0.5712) is obtained. It was. FIG. 34 shows the chromaticity coordinates of the liquid mixtures A, B, and C in the CIE 1931xy chromaticity diagram. The chromaticity coordinates are measured using the same spectrophotometer, with an optical path length of 1 cm and a spectrophotometer operation software “Color measurement software P / N 206-65207 (manufactured by Shimadzu Corporation)” illumination: D65, field of view : Performed under conditions set at 2 °.

(Preparation of metal fine particles D)
The surface of the plate-like silver nanoparticles D was prepared in the same manner as the preparation of the metal fine particles A except that 120 ml of the aqueous dispersion of plate-like silver nanoparticles D was used instead of 120 ml of the aqueous dispersion of plate-like silver nanoparticles A. An aqueous dispersion of metal fine particles D coated with gold was prepared. FIG. 10 shows the optical characteristics of an aqueous dispersion obtained by diluting the prepared dispersion with distilled water to 4 volumes. The wavelength exhibiting the maximum absorption was 714 nm (extinction degree 0.8). The optical properties were measured using an ultraviolet-visible near-infrared spectrophotometer MPC3100UV-3100PC manufactured by Shimadzu Corporation under the conditions of optical path length: 1 cm and measurement wavelength: 190-1300 nm. An SEM photograph is shown in FIG. A scanning electron microscope SU-70 manufactured by Hitachi, Ltd. was used for analysis of the SEM photograph.

(Stability test of metal fine particle B in buffer)
[Stability of plate-like silver nanoparticles in buffer]
1 mL of the previously prepared dispersion of plate-like silver nanoparticles B was added to 3 mL of distilled water and 10 mM PBS (+) buffer solution (0.133 g of calcium chloride dihydrate (manufactured by Kanto Chemical Co., Inc.), magnesium chloride hexahydrate. 0.1 g of the product (manufactured by Kanto Chemical Co., Ltd.) is dissolved in 800 mL of distilled water, 50 mL of a commercially available 200 mM PBS (−) solution (manufactured by Kanto Chemical Co., Ltd.) is added, and the total amount is adjusted to 1 L. A 4-fold dilution was used.
The extinction degree (Extinction) of the prepared solution was measured with an ultraviolet visible near infrared spectrophotometer (device name: ultraviolet visible near infrared spectrophotometer MPC3100UV-3100PC, manufacturer: Shimadzu Corporation). The quenching reduction rate of the plate-like silver nanoparticles B at a wavelength of 542 nm was about 90%, which was greatly reduced. The extinction degree measurement results are shown in FIG.

[Stability of gold-coated plate-like silver nanoparticles in buffer]
1 mL of the aqueous dispersion of metal fine particles B prepared above was added to 3 mL of distilled water and 10 mM PBS (+) buffer (calcium chloride dihydrate (manufactured by Kanto Chemical Co., Ltd.) 0.133 g, magnesium chloride hexahydrate ( 0.1 g of Kanto Chemical Co., Ltd.) was dissolved in 800 mL of distilled water, 50 mL of a commercially available 200 mM PBS (−) solution (manufactured by Kanto Chemical Co., Ltd.) was added, and the total amount was adjusted to 1 L. The dilution was doubled.
The extinction degree of the prepared solution was measured with an ultraviolet-visible near-infrared spectrophotometer (device name: UV-visible near-infrared spectrophotometer MPC3100UV-3100PC, manufacturer: Shimadzu Corporation). The reduction rate of the extinction degree of the metal fine particle B at a wavelength of 556 nm was about 1.4%, and was almost unchanged and stable. The extinction degree measurement results are shown in FIG.

  From the above, it was found that the metal fine particles B are excellent in stability in a biochemical buffer.

[Immunochromatographic test Example 1]
Immunochromatographic test of concanavalin A using metal fine particles A to D (Preparation of developing solutions A to D for concanavalin A immunochromatography test)
5 mM PBS (+) buffer (200 mM PBS solution (product name: PBS solution 20-fold concentrated solution, manufacturer: Kanto Chemical Co., Inc.) diluted to 40-fold to prepare 1 L of 5 mM PBS (-) buffer, 50 μg / concentration in 0.1 g of 1 g / mL aqueous solution of calcium (manufactured by Kanto Chemical Co., Ltd.) and 0.05 mL of 1 g / mL aqueous solution of magnesium chloride hexahydrate (manufactured by Kanto Chemical Co., Ltd.) 0.2 mL of a solution of mL of anti-concanavalin A antibody (product name: Anti Concanavalin A, manufacturer: EY Laboratories, Inc.) and 1.8 mL of the previously prepared dispersion of metal fine particles A were mixed, and the resulting mixture was obtained. Was shaken for 30 minutes at room temperature. Subsequently, centrifugation (75000 rpm, 4 ° C., 1 hour) was performed to precipitate the antibody-metal fine particle complex, and the supernatant was removed. Thereafter, the antibody-metal fine particle complex is redispersed in 500 μL of pure water and adjusted to an extinction level of 0.55 using an ultraviolet-visible spectrophotometer Agilent 8453 (manufactured by Agilent Technologies). Then, a developing solution A was prepared. Similarly, developing solutions B to D were prepared using dispersions of metal fine particles B to D.

(Immunochromatographic test 1 for concanavalin A)
An immunochromatographic test as shown in FIG. 1 was performed. An immunochromatographic test paper on which an anti-concanavalin A antibody was immobilized linearly was used. The immunochromatographic test paper used was purchased from a contract manufacturing company for immunochromatographic test paper (Biodevice Technology Co., Ltd.). When immobilizing the anti-concanavalin A antibody in a straight line, the anti-concanavalin A antibody solution was diluted with 5 mM PBS (−) buffer solution (prepared by diluting the above-mentioned commercially available 200 mM PBS solution 200 times with distilled water) to a concentration of 1 g / What was adjusted to mL was used. The first developing solution is a solution of concanavalin A (product name: Canavaria ensiformis (Jack Bean) [Con A], Jack bean (−), manufactured by J-Oil Mills Co., Ltd.) in 5 mM PBS (+) buffer. There were prepared solutions having concanavalin A concentrations of 6 μM, 0.60 μM, 0.06 μM, 6 nM, 0.6 nM, 0.06 nM and 0 M (pH 7.4). The developing solution for washing is a 5 mM PBS (+) buffer (pH 7.4). The above developing solutions A to D were used as the second developing solution (pH 7.0). Specifically, 15 μL of the first developing solution of each concentration was developed on the immunochromatographic test paper. Subsequently, 30 μL of 5 mM PBS (+) buffer, which is a developing solution for washing, was developed. Finally, 60 μL of various second developing solutions were developed. In all immunochromatographic tests using the developing solutions A to D, the detection of concanavalin A was visually confirmed in the developing solution A over the concentrations of 6 μM, 0.60 μM, 0.06 μM, 6 nM, 0.6 nM, and 0.06 nM. In the developing solutions B, C, and D, it was visually confirmed over the concentrations of 6 μM, 0.60 μM, 0.06 μM, 6 nM, and 0.6 nM. The results are shown in FIG.

(Immunochromatographic test 2 for concanavalin A)
An immunochromatographic test as shown in FIG. 2 was performed. The same immunochromatographic test paper as in immunochromatographic test 1 was used. The developing solution includes 60 μL of one type of developing solution among the developing solutions A to D and concentrations of 6 μM, 0.60 μM, 0.06 μM, 6 nM, 0.6 nM,. A solution mixed with 15 μL of a solution of 06 nM or 0 M Conacavalin A (pH 7.0) was used. Specifically, various developing solutions were developed on immunochromatographic test paper. In all the developing solutions, the detection of concanavalin A was visually confirmed over the concentrations of 6 μM, 0.60 μM, 0.06 μM, 6 nM, and 0.6 nM.

(Luminance analysis of immunochromatographic test 1 for concanavalin A)
Scan the immunochromatographic test paper after the test in immunochromatographic test 1 for concanavalin A (device name: Canon Scan LiDE500F, Canon Inc.), and determine the part other than the judgment part (immobilization part of anti-concanavalin A antibody) By measuring the minimum luminance with image analysis software (Image-J), the detection sensitivity can be quantified. Image-J is an open source and public image processing software developed by Wayne Rasband at the National Institutes of Health (http://imagej.nih.gov/ij/). The detection sensitivity can be the difference between the median values obtained by measuring each part five times. As a result of the luminance difference analysis, the detection of concanavalin A can be confirmed over the concentrations of 6 μM, 0.60 μM, 0.06 μM, 6 nM, 0.6 nM, and 0.06 nM in the metal fine particles A and B. The detection of concanavalin A was confirmed over concentrations of 6 μM, 0.60 μM, 0.06 μM, 6 nM, and 0.6 nM. 14 to 17 show the results of luminance analysis, and FIG. 19 shows a comparison with a spherical gold colloid described later.

[Immunochromatographic test Example 2]
Immunochromatographic test of hepatitis B virus antigen using metal fine particle B (Preparation of developing solution for hepatitis B virus antigen immunokumat test)
Centrifugation (25000 rpm, 4 ° C., 10 minutes) of the dispersion liquid of metal fine particles B prepared earlier was performed to precipitate metal fine particles, and 1.85 mL of the supernatant liquid was removed. Thereafter, the metal microparticles were redispersed with 1.85 mL of 5 mM PBS (−) buffer. This operation was repeated twice to adjust the pH of the dispersion to 7.4.
Anti-hepatitis B virus antigen antibody (product name: Goat anti HBsAg, manufacturer: Arista Biologicals, Inc.) at a concentration of 50 μg / mL in 5 mM PBS (−) buffer (prepared by diluting a commercially available 200 mM PBS solution 40-fold). ) And 1.8 mL of a dispersion of metal fine particles B whose pH was previously adjusted to 7.4 by centrifugation were mixed, and the resulting mixture was shaken at room temperature for 30 minutes. Subsequently, centrifugation (25000 rpm, 4 ° C., 10 minutes) was performed to precipitate the antibody-metal fine particle complex, and 1.85 mL of the supernatant was removed. Thereafter, the antibody-metal fine particle complex is redispersed with 5 mM PBS (−) buffer, and an extinction degree (Extinction) of 0.35 is obtained using an ultraviolet-visible spectrophotometer Agilent 8453 (manufactured by Agilent Technologies). Thus, a developing solution E was prepared.

(Immunochromatographic test for hepatitis B virus antigen)
An immunochromatographic test as shown in FIG. 1 was performed. An immunochromatographic test paper on which an anti-hepatitis B virus antigen antibody was immobilized linearly was used. The immunochromatographic test paper used was purchased from a contract manufacturing company for immunochromatographic test paper (Biodevice Technology Co., Ltd.). When the anti-hepatitis B virus antigen antibody is immobilized in a straight line, an anti-hepatitis B virus antigen antibody solution is prepared by diluting the above-mentioned commercially available 200 mM PBS solution 40 times with distilled water. ) Was used to adjust the concentration to 1 g / mL. The first developing solution is a solution of hepatitis B virus antigen (product name: HBsAg Protein (Subtype adr), manufacturer: Fitzgerald Industries International Inc.) in 1 mM PBS (−) buffer, and hepatitis B virus antigen Solution having a concentration of 6 μM, 0.60 μM, 0.06 μM, 6 nM, and 0 M (Blank) was prepared. The developing solution for washing is a 5 mM PBS (−) buffer. The above-mentioned developing solution E was used as the second developing solution. Specifically, 15 μL of the first developing solution of each concentration was developed on the immunochromatographic test paper. Subsequently, 30 μL of 5 mM PBS (−) buffer, which is a developing solution for washing, was developed. Finally, 60 μL of the second developing solution was developed. In this immunochromatographic test, detection of hepatitis B virus antigen was confirmed by visual observation over concentrations of 6 μM, 0.60 μM, and 0.06 μM. The results are shown in FIG.

(Luminance analysis of immunochromatographic test for hepatitis B virus antigen)
The immunochromatographic test paper after the test in the immunochromatographic test of hepatitis B virus antigen was scanned (device name: Cano Scan LiDE500F, manufacturer: Canon Inc.), and the determination part (anti-hepatitis B virus antigen antibody immobilization part) and The detection sensitivity can be quantified by measuring the minimum luminance of the portion other than the determination portion with image analysis software (Image-J). The detection sensitivity can be the difference between the median values obtained by measuring each part five times. As a result of luminance difference analysis, detection of hepatitis B virus antigen was confirmed over concentrations of 6 μM, 0.60 μM, and 0.06 μM. The result of luminance analysis is shown in FIG. 21, and the comparison with the spherical gold colloid is shown in FIG.

[Immunochromatographic test Example 3]
Multicolor immunochromatographic test (preparation of developing solution for multicolor immunochromatographic test)
Centrifugation (25000 rpm, 4 ° C., 10 minutes) of 2.0 mL of the previously prepared dispersion of metal fine particles A to C was performed to precipitate the metal fine particles, and 1.85 mL of the supernatant was removed. Thereafter, the metal microparticles were redispersed with 1.85 mL of 5 mM PBS (−) buffer. This operation was repeated twice to adjust the pH of the dispersion to 7.4.
Anti-hepatitis B virus antigen antibody (product name: Goat anti HBsAg, manufacturer: Arista Biologicals, Inc.) at a concentration of 50 μg / mL in 5 mM PBS (−) buffer (prepared by diluting a commercially available 200 mM PBS solution 40-fold). And 0.2 mL of a dispersion of metal fine particles A to C whose pH was previously adjusted to 7.4 by centrifugation, and the resulting mixture was shaken at room temperature for 30 minutes. . Subsequently, centrifugation (25000 rpm, 4 ° C., 10 minutes) was performed to precipitate the antibody-metal fine particle complex, and 1.85 mL of the supernatant was removed to prepare a 13.3 times concentrated solution of the antibody-metal fine particle complex. . Using a UV-visible spectrophotometer Agilent 8453 (manufactured by Agilent Technologies), the concentrated solution is diluted 14-fold with 5 mM PBS (−) buffer so as to have an extinction degree of 2.0. FH were prepared. Moreover, the above-mentioned concentrated liquid was diluted 7 times, 2 times concentration concentrated developing liquid FH was prepared, these were prepared according to FIG. 24, and developing liquid IL was prepared. The spectral characteristic measurement results of the developing solutions F to L are shown in FIGS. As shown in FIGS. 25 and 26, in the case of a developing solution using a spherical gold colloid, only a single color design is possible. However, in the case of using the metal fine particles A, B, and C as in the present invention, a multicolor design is possible. It was.

(Multicolor immunochromatographic test)
An immunochromatographic test as shown in FIG. 1 was performed. An immunochromatographic test paper on which an anti-hepatitis B virus antigen antibody was immobilized linearly was used. The immunochromatographic test paper used was purchased from a contract manufacturing company for immunochromatographic test paper (Biodevice Technology Co., Ltd.). When the anti-hepatitis B virus antigen antibody is immobilized in a straight line, an anti-hepatitis B virus antigen antibody solution is prepared by diluting the above-mentioned commercially available 200 mM PBS solution 40 times with distilled water. ) Was used to adjust the concentration to 1 g / mL. The first developing solution is a solution of hepatitis B virus antigen (product name: HBsAg Protein (Subtype adr), manufacturer: Fitzgerald Industries International Inc.) in 1 mM PBS (−) buffer, and hepatitis B virus antigen. Solution having a concentration of 6 μM, 0.60 μM, 0.06 μM, 6 nM, and 0 M (Blank) was prepared. The developing solution for washing is a 5 mM PBS (−) buffer. As the second developing solution, the developing solutions F to L described above were used. Specifically, 15 μL of the first developing solution of each concentration was developed on the immunochromatographic test paper. Subsequently, 30 μL of 5 mM PBS (−) buffer, which is a developing solution for washing, was developed. Finally, 60 μL of the second developing solution was developed. In this immunochromatographic test, detection of hepatitis B virus antigen was confirmed by visual observation over concentrations of 6 μM, 0.60 μM, and 0.06 μM. The color of the detection line varies depending on the developing solution. When developing solution F is used, yellow tone, when developing solution G is used, magenta tone, when developing solution H is used, cyan tone, when developing solution I is used, red tone, developing solution. When J was used, the color was blue, when the developing solution K was used, the color was green, and when using the developing solution L, the color was black.
Therefore, when performing an immunochromatographic test as shown in FIG. 5 or FIG. 6 (when a plurality of different kinds of molecules (b) are linearly immobilized at different positions on a chromatographic carrier and a determination portion is formed. ), It can be seen that each immobilization portion can function as a detection line exhibiting a different color by utilizing the difference in color tone of the metal fine particles (a) (multiple detection lines). This also shows that a plurality of test substances can be detected at a time.

(Color measurement of multicolor immunochromatographic test results)
The immunochromatographic paper obtained by the multicolor immunochromatographic test was scanned (device name: Canon Scan LiDE500F, manufacturer: Canon Inc.), and the obtained image was captured with the bitmap image editing software Adobe Photoshop, and the CIELAB D50 of the detection line was captured. It was measured. The result of color measurement is shown in FIG.

[Immunochromatographic test Example 4]
Two-sample two-color detection immunochromatographic test for hepatitis B virus antigen and human chorionic gonadotropin using metal microparticles B and C (complex dispersion A for hepatitis B virus antigen and human chorionic gonadotropin immunokumato test) Preparation)
Centrifugation (25000 rpm, 4 ° C., 10 minutes) of the dispersion liquid of metal fine particles B prepared earlier was performed to precipitate metal fine particles, and 1.85 mL of the supernatant liquid was removed. Thereafter, the metal microparticles were redispersed with 1.85 mL of 5 mM PBS (−) buffer. This operation was repeated twice to adjust the pH of the dispersion to 7.4. Anti-hepatitis B virus antigen antibody (product name: Goat anti HBsAg, manufacturer: Arista Biologicals, Inc.) at a concentration of 50 μg / mL in 5 mM PBS (−) buffer (prepared by diluting a commercially available 200 mM PBS solution 40-fold). ) And 1.8 mL of a dispersion of metal fine particles B whose pH was previously adjusted to 7.4 by centrifugation were mixed, and the resulting mixture was shaken at room temperature for 60 minutes. Thereafter, 100 μL of a 3.26 wt% bovine serum albumin (BSA) -5 mM PBS (−) solution was added, and the resulting mixture was shaken at room temperature for 60 minutes. Subsequently, centrifugation (25000 rpm, 4 ° C., 10 minutes) was performed to precipitate the antibody-metal fine particle complex, and 1.85 mL of the supernatant was removed. Thereafter, the antibody-metal fine particle complex is re-dispersed with 5 mM PBS (−) buffer, and the extinction degree is 1.0 using an ultraviolet-visible spectrophotometer Agilent 8453 (manufactured by Agilent Technologies). Thus, a composite dispersion A was prepared.

(Preparation of complex dispersion B for hepatitis B virus antigen and human chorionic gonadotropin immunokumato test)
Centrifugation (25000 rpm, 4 ° C., 10 minutes) of 2.0 mL of the previously prepared dispersion of metal fine particles C was performed to precipitate the metal fine particles, and 1.85 mL of the supernatant was removed. Thereafter, the metal microparticles were redispersed with 1.85 mL of 5 mM PBS (−) buffer. This operation was repeated twice to adjust the pH of the dispersion to 7.4. Anti-human chorionic gonadotropin antibody (product name: MONOCLONAL ANTI-HUMAN CHORONIC GONADOTROPIN, manufacturer: Medix) in a concentration of 50 μg / mL in 5 mM PBS (−) buffer (prepared by diluting a commercially available 200 mM PBS solution 40-fold) Biochemica solution (0.2 mL) and the metal fine particle C dispersion (1.8 mL) previously adjusted to pH 7.4 by centrifugation were mixed, and the resulting mixture was shaken at room temperature for 30 minutes. Then, it was allowed to stand at 4 ° C. for 24 hours. Subsequently, centrifugation (25000 rpm, 4 ° C., 10 minutes) was performed to precipitate the antibody-metal fine particle complex, and 1.85 mL of the supernatant was removed. Thereafter, the antibody-metal fine particle complex is re-dispersed with 5 mM PBS (−) buffer, and the extinction degree is 1.0 using an ultraviolet-visible spectrophotometer Agilent 8453 (manufactured by Agilent Technologies). Thus, a composite dispersion B was prepared.

(Preparation of developing solution M for hepatitis B virus antigen and human chorionic gonadotropin immunokumato test)
Centrifugation (25000 rpm, 4 ° C., 10 minutes) of 2.0 mL of the previously prepared dispersion of metal fine particles A was performed to precipitate the metal fine particles, and 1.85 mL of the supernatant was removed. Thereafter, the metal microparticles were redispersed with 1.85 mL of 5 mM PBS (−) buffer. This operation was repeated twice to adjust the pH of the dispersion to 7.4. Thereafter, the dispersion of the metal fine particles A adjusted to pH 7.4 is adjusted using an ultraviolet-visible spectrophotometer Agilent 8453 (manufactured by Agilent Technologies) so that the extinction degree is 1.0. Preparation dispersion A was prepared. The pH adjustment dispersion A, the composite dispersion A, and the composite dispersion B were mixed at an equal volume ratio to prepare a developing liquid M (color tone: black tone).

(Immunochromatographic test for hepatitis B virus antigen and human chorionic gonadotropin)
An immunochromatographic test as shown in FIG. 5 was performed. An immunochromatographic test paper in which an anti-hepatitis B virus antigen antibody and an anti-human chorionic gonadotropin antibody are each immobilized in a straight line (the anti-human chorionic gonadotropin antibody is downstream of the developing solution in the direction of development) And an anti-hepatitis B virus antigen antibody is fixed upstream from the direction in which the developing solution develops). The immunochromatographic test paper used was purchased from a contract manufacturing company for immunochromatographic test paper (Biodevice Technology Co., Ltd.). When the anti-hepatitis B virus antigen antibody and the anti-human chorionic gonadotropin antibody are immobilized linearly, each antibody solution is diluted with 5 mM PBS (−) buffer (the above-mentioned commercially available 200 mM PBS solution is 40 times with distilled water. What was adjusted to a concentration of 1 g / mL in (prepared by dilution) was used. The first developing solution is a hepatitis B virus antigen (product name: HBsAg Protein (Subtype adr), manufacturer: Fitzgerald Industries International Inc.) and human chorionic gonadotropin (product name: 5B) in PBS (-) buffer. hCG Human (-), manufacturer: Meridian Life Science, Inc.), and both antigen concentrations were 0.3 μM, 0.03 μM, 0.003 M, and 0 M (Blank). The developing solution for washing is a 5 mM PBS (−) buffer. The developing solution M was used as the second developing solution. Specifically, 30 μL of the first developing solution at each concentration was developed on an immunochromatographic test paper. Subsequently, 30 μL of 5 mM PBS (−) buffer, which is a developing solution for washing, was developed. Finally, 120 μL of the second developing solution was developed. In this immunochromatographic test, the detection of hepatitis B virus antigen was confirmed visually over the concentrations of 0.3 μM, 0.030 μM and 0.003 μM, and the concentrations of human chorionic gonadotropin 0.3 μM and 0.03 μM. Was confirmed. The results are shown in FIG. The color of the detection line was cyan in the immobilized part of the anti-human chorionic gonadotropin antibody and magenta in the immobilized part of the anti-hepatitis B virus antigen antibody.

(Color measurement of immunochromatographic test results of two-sample two-color detection of hepatitis B virus antigen and human chorionic gonadotropin)
The immunochromatographic paper obtained by the two-sample two-color detection immunochromatographic test was scanned (device name: Canon Scan LiDE500F, manufacturer: Canon Inc.), and the obtained image was captured by Adobe Photoshop, a bitmap image editing software. CIELAB D50 was measured. The result of color measurement is shown in FIG.

(Luminance analysis of two-sample two-color immunochromatography test for hepatitis B virus antigen and human chorionic gonadotropin)
The immunochromatographic test paper after the test in the two-sample two-color detection immunochromatography test was scanned (device name: Canon Scan LiDE500F, manufacturer: Canon Inc.), and the determination part (anti-hepatitis B virus antigen antibody and anti-human chorionic gonad stimulation) The detection sensitivity can be quantified by measuring the minimum luminance of portions other than the determination portion of the hormone antibody) and image analysis software (Image-J). The detection sensitivity can be the difference between the median values obtained by measuring each part five times. As a result of the luminance difference analysis, the detection of hepatitis B virus antigen was confirmed over the concentrations of 0.3 μM, 0.03 μM and 0.003 μM, and the concentrations of human chorionic gonadotropin 0.3 μM and 0.03 μM were confirmed. Confirmed. The result of the luminance analysis is shown in FIG.

[Immunochromatographic test Example 5]
High-resolution multicolor immunochromatography test (Preparation of developing solution for high-color multicolor immunochromatography test)
Centrifugation (25000 rpm, 4 ° C., 10 minutes) of 2.0 mL of the previously prepared dispersion of metal fine particles A to C was performed to precipitate the metal fine particles, and 1.85 mL of the supernatant was removed. Thereafter, the metal microparticles were redispersed with 1.85 mL of 5 mM PBS (−) buffer. This operation was repeated twice to adjust the pH of the dispersion to 7.4.
Anti-hepatitis B virus antigen antibody (product name: Goat anti HBsAg, manufacturer: Arista Biologicals, Inc.) at a concentration of 50 μg / mL in 5 mM PBS (−) buffer (prepared by diluting a commercially available 200 mM PBS solution 40-fold). And 0.2 mL of a dispersion of metal fine particles A to C whose pH was previously adjusted to 7.4 by centrifugation, and the resulting mixture was shaken at room temperature for 30 minutes. . Thereafter, 100 μL of a 3.26 wt% bovine serum albumin (BSA) -5 mM PBS (−) solution was added to the mixture, and the resulting mixture was shaken at room temperature for 60 minutes. Subsequently, centrifugation (25000 rpm, 4 ° C., 10 minutes) is performed to precipitate the antibody-metal fine particle complex, 1.75 mL of the supernatant is removed, and then 0.16 wt% BSA-5 mM PBS (− ) 0.75 mL of buffer solution was added to prepare a 2-fold concentrated solution of antibody-metal fine particle complex. The concentrated solution was diluted 3.0 times with 0.16 wt% BSA-5 mM PBS (−) buffer to prepare developing solutions N to P. Moreover, the above-mentioned concentrate was prepared according to FIG. 38 and the developing liquids QT were prepared.

(Highly detailed multicolor immunochromatographic test)
An immunochromatographic test as shown in FIG. 1 was performed. An immunochromatographic test paper on which an anti-hepatitis B virus antigen antibody was immobilized linearly was used. The immunochromatographic test paper used was purchased from a contract manufacturing company for immunochromatographic test paper (Biodevice Technology Co., Ltd.). When the anti-hepatitis B virus antigen antibody is immobilized in a straight line, an anti-hepatitis B virus antigen antibody solution is prepared by diluting the above-mentioned commercially available 200 mM PBS solution 40 times with distilled water. ) Was used to adjust the concentration to 1 g / mL. The first developing solution is a solution of hepatitis B virus antigen (product name: HBsAg Protein (Subtype adr), manufacturer: Fitzgerald Industries International Inc.) in 1 mM PBS (−) buffer, and hepatitis B virus antigen A solution having a concentration of 0.60 μM and 0M (Blank) was prepared. The developing solution for washing is a 5 mM PBS (−) buffer. The above-described developing solutions N to T were used as the second developing solution. Specifically, 15 μL of the first developing solution of each concentration was developed on the immunochromatographic test paper. Subsequently, 30 μL of 5 mM PBS (−) buffer, which is a developing solution for washing, was developed. Finally, 60 μL of the second developing solution was developed. In this immunochromatographic test, the detection of hepatitis B virus antigen was confirmed visually at a concentration of 0.6 μM, not at a concentration of 0 μM (Blank), and it was confirmed that there was no non-specific detection. The color of the detection line differs depending on the developing solution. When developing solution N is used, yellow tone, when developing solution O is used, magenta tone, when developing solution P is used, cyan tone, when developing solution Q is used, red tone, developing solution When R was used, the color was blue, when using the developing solution S, the color was green, and when using the developing solution T, the color was black.

(Color measurement of high-resolution multicolor immunochromatographic test results)
The immunochromatographic paper obtained by the high-definition and multicolor immunochromatographic test was scanned (device name: Canon Scan LiDE220, manufacturer: Canon Inc.), and the obtained image was captured by Adobe Photoshop, a bitmap image editing software, and CIELAB of the detection line D50 was measured. The result of color measurement is shown in FIG.

[Immunochromatographic test Comparative Example 1]
Immunochromatographic test of concanavalin A using spherical gold colloid (preparation of developing solution for immunokumato test of concanavalin A using spherical gold colloid)
A commercially available spherical gold colloid dispersion (product name: Au colloid solution-SC, particle size: 40 nm, manufacturer: Tanaka Kikinzoku Co., Ltd.) 10 mL was added with 5 mL of a 5% by mass polyvinylpyrrolidone (PVP) aqueous solution. It left still and produced gold colloid adjustment liquid.
Centrifugation (25000 rpm, 4 ° C., 10 minutes) of 2.0 mL of the above gold colloid adjustment solution was performed to precipitate gold fine particles, and 1.85 mL of the supernatant was removed. Thereafter, the gold microparticles were diluted to 40 mM with 5 mM PBS (+) buffer (200 mM PBS solution (product name: PBS solution 20-fold concentrated solution, manufacturer: Kanto Chemical Co., Ltd.)), and 5 mM PBS (-) buffer solution was added. Prepare 1 L and add 0.1 mL of 1 g / mL aqueous solution of calcium chloride (manufactured by Kanto Chemical Co., Ltd.) and 0.05 mL of 1 g / mL aqueous solution of magnesium chloride hexahydrate (manufactured by Kanto Chemical Co., Ltd.) Redispersed with 1.85 mL. This operation was repeated twice to adjust the pH of the dispersion to 7.4.
Dispersion of gold colloid prepared previously in 0.2 mL of a solution of anti-concanavalin A antibody (product name: Anti Concanavalin A, manufacturer: EY Laboratories, Inc.) at a concentration of 50 μg / mL in 5 mM PBS (+) buffer The liquid 1.8mL was mixed and the obtained mixture was shaken for 30 minutes at room temperature. Subsequently, centrifugation (75000 rpm, 4 ° C., 1 hour) was performed to precipitate the antibody-gold colloid complex, and the supernatant was removed. Thereafter, the antibody-gold colloid complex is re-dispersed in 500 μL of pure water and adjusted to an extinction level of 0.35 using an ultraviolet-visible spectrophotometer Agilent 8453 (manufactured by Agilent Technologies). Then, an antibody-gold colloid developing solution I was prepared.

(Immunochromatographic test of concanavalin A using gold colloid)
An immunochromatographic test as shown in FIG. 1 was performed. An immunochromatographic test paper on which an anti-concanavalin A antibody was immobilized linearly was used. The immunochromatographic test paper used was purchased from a contract manufacturing company for immunochromatographic test paper (Biodevice Technology Co., Ltd.). When the anti-concanavalin A antibody was fixed linearly, an anti-concanavalin A antibody solution adjusted to a concentration of 1 g / mL with 5 mM PBS (+) buffer was used. The first developing solution is a solution of concanavalin A (product name: Canavaria ensiformis (Jack Bean) [Con A], Jack bean (−), manufactured by J-Oil Mills Co., Ltd.) in 5 mM PBS (+) buffer. Yes, solutions with concanavalin A concentrations of 6 μM, 0.60 μM, 0.06 μM, 6 nM, 0.6 nM, 0.06 nM and 0 M were prepared. The developing solution for washing is a 5 mM PBS (+) buffer. The above-described antibody-gold colloid developing solution I was used as the second developing solution. Specifically, 15 μL of the first developing solution of each concentration was developed on the immunochromatographic test paper. Subsequently, 30 μL of 5 mM PBS (+) buffer, which is a developing solution for washing, was developed. Finally, 60 μL of antibody-gold colloid developing solution I as the second developing solution was developed. As a result of the immunochromatographic test, the detection of concanavalin A was visually confirmed over the concentrations of 6 μM, 0.60 μM, 0.06 μM and 6 nM. The results are shown in FIG.

(Luminance analysis of immunochromatographic test of concanavalin A using spherical gold colloid)
The immunochromatographic test paper after the test in the immunochromatographic test of concanavalin A using a spherical gold colloid was scanned (device name: Canon Scan LiDE500F, Canon Inc.) and judged as a judgment part (anti-concanavalin A antibody immobilization part). The detection sensitivity can be quantified by measuring the minimum luminance of the portion other than the portion with image analysis software (Image-J). The detection sensitivity can be the difference between the median values obtained by measuring each part five times. As a result of the luminance difference analysis, the detection of concanavalin A was confirmed over the concentrations of 6 μM, 0.60 μM, 0.06 μM, and 6 nM in the spherical gold colloid. The result of the luminance analysis is shown in FIG. 18, and the comparison with the metal fine particles A to D is shown in FIG.

[Immunochromatographic test comparative example 2]
Immunochromatographic test of hepatitis B virus antigen using spherical gold colloid (Preparation of developing solution for hepatitis B virus antigen immunokumat test using spherical gold colloid)
A commercially available spherical gold colloid dispersion (product name: Au colloid solution-SC, particle size: 40 nm, manufacturer: Tanaka Kikinzoku Co., Ltd.) 10 mL was added with 5 mL of a 5% by mass polyvinylpyrrolidone (PVP) aqueous solution. It left still and produced gold colloid adjustment liquid.
Centrifugation (25000 rpm, 4 ° C., 10 minutes) of 2.0 mL of the above gold colloid adjustment solution was performed to precipitate gold fine particles, and 1.85 mL of the supernatant was removed. Thereafter, the gold microparticles were redispersed with 1.85 mL of 5 mM PBS (−) buffer. This operation was repeated twice to adjust the pH of the dispersion to 7.4.
Anti-hepatitis B virus antigen antibody (product name: Goat anti HBsAg, manufacturer: Arista Biologicals, Inc.) at a concentration of 50 μg / mL in 5 mM PBS (−) buffer (prepared by diluting a commercially available 200 mM PBS solution 200-fold). And 0.2 mL of a gold colloid adjustment solution whose pH was previously adjusted to 7.4 by centrifugation, and the resulting mixture was shaken at room temperature for 30 minutes. Subsequently, centrifugation (25000 rpm, 4 ° C., 10 minutes) is performed to precipitate the antibody-gold colloid complex, 1.85 mL of the supernatant is removed, and a 13.3 times concentrated solution of the antibody-gold colloid complex is prepared. did. Then, the concentrated solution was diluted with 5 mM PBS (−) buffer, adjusted to have an extinction degree of 0.35 using an ultraviolet-visible spectrophotometer Agilent 8453 (manufactured by Agilent Technologies), Antibody-gold colloid developing solution II was prepared. Further, the concentrated solution was diluted 3 times to prepare an antibody-gold colloid developing solution III having a quenching degree of 2.0 and used for an immunochromatographic test used for color measurement of the hepatitis B virus antigen immunochromatographic test result described later.

(Immunochromatographic test of hepatitis B virus antigen using colloidal gold)
An immunochromatographic test as shown in FIG. 1 was performed. An immunochromatographic test paper on which an anti-hepatitis B virus antigen antibody was immobilized linearly was used. The immunochromatographic test paper used was purchased from a contract manufacturing company for immunochromatographic test paper (Biodevice Technology Co., Ltd.). When the anti-hepatitis B virus antigen antibody is immobilized in a straight line, the anti-hepatitis B virus antigen antibody solution is prepared by diluting the above-mentioned commercially available 200 mM PBS solution 200-fold with distilled water (200 mM PBS). ) Was used to adjust the concentration to 1 g / mL. The first developing solution is a solution of hepatitis B virus antigen (product name: HBsAg Protein (Subtype adr), manufacturer: Fitzgerald Industries International Inc.) in 1 mM PBS (−) buffer, and hepatitis B virus antigen Solution having a concentration of 6 μM, 0.60 μM, 0.06 μM, 6 nM, and 0 M (Blank) was prepared. The developing solution for washing is a 1 mM PBS (−) buffer. As the second developing solution, the previously prepared antibody-gold colloid developing solution II adjusted to a quenching degree of 0.35 was used. Specifically, 15 μL of the first developing solution of each concentration was developed on the immunochromatographic test paper. Subsequently, 30 μL of 1 mM PBS (−) buffer, which is a developing solution for washing, was developed. Finally, 60 μL of the second developing solution was developed. In this immunochromatography test, the detection of hepatitis B virus antigen was confirmed visually over the concentrations of 6 μM and 0.60 μM. The results are shown in FIG. The color of the detection line was reddish purple.

(Luminance analysis of immunochromatographic test for hepatitis B virus antigen using spherical gold colloid)
The immunochromatographic test paper after the test in the immunochromatographic test of the hepatitis B virus antigen using a spherical gold colloid was scanned (device name: Canon Scan LiDE500F, manufacturer: Canon Inc.), and the determination part (anti-hepatitis B virus antigen antibody) The detection sensitivity can be quantified by measuring the minimum luminance of the portion other than the determination portion and the determination portion with image analysis software (Image-J). The detection sensitivity can be the difference between the median values obtained by measuring each part five times. As a result of luminance difference analysis, detection of hepatitis B virus antigen was confirmed over concentrations of 6 μM and 0.60 μM. The result of the luminance analysis is shown in FIG. 22, and the comparison with the metal fine particle B is shown in FIG.

[Immunochromatographic test comparative example 3]
(Color measurement of hepatitis B virus antigen immunochromatography test results using colloidal gold)
Scanning immunochromatographic paper obtained by the hepatitis B virus antigen immunochromatographic test using the antibody-gold colloid developing solution III adjusted to the extinction degree of 2.0 prepared previously (device name: Cano Scan LiDE500F, manufacturer: Canon Inc.) The acquired image was taken in with Adobe Photoshop, a bitmap image editing software, and CIELAB D50 of the detection line was measured. FIG. 25 shows the spectral characteristics of the antibody-gold colloid developing solution III, and FIG. 27 shows the results of colorimetry.

Claims (4)

  Plate-shaped silver nanoparticles and metal fine particles (a) made of gold covering the surface of the plate-shaped silver nanoparticles, and having a specific binding property to a test substance and to the metal fine particles (a) An immunochromatography kit comprising a molecule (b) having binding properties. An immunochromatography kit comprising a chromatographic carrier exhibiting one or more detection lines after an immunochromatographic test,
The immunochromatography kit according to claim 1, wherein the color of the detection line is based on a color of one kind of metal fine particles (a) or a mixed color of a plurality of kinds of metal fine particles (a) exhibiting different colors. An immunochromatography kit comprising a chromatographic carrier that exhibits a plurality of detection lines after an immunochromatographic test,
The immunochromatography kit according to claim 1 or 2, wherein a plurality of types of molecules (b) having binding properties to different test substances are fixed to the chromatographic carrier at different positions.   The water-soluble polymer (c) having no binding property to the test substance and having binding property to the metal fine particles (a) is further provided. The immunochromatography kit according to item.