WO2016002743A1 - 標識物質、免疫学的測定法、免疫学的測定用試薬、アナライトの測定方法、アナライト測定用キット、及び、ラテラルフロー型クロマト用テストストリップ - Google Patents
標識物質、免疫学的測定法、免疫学的測定用試薬、アナライトの測定方法、アナライト測定用キット、及び、ラテラルフロー型クロマト用テストストリップ Download PDFInfo
- Publication number
- WO2016002743A1 WO2016002743A1 PCT/JP2015/068759 JP2015068759W WO2016002743A1 WO 2016002743 A1 WO2016002743 A1 WO 2016002743A1 JP 2015068759 W JP2015068759 W JP 2015068759W WO 2016002743 A1 WO2016002743 A1 WO 2016002743A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- resin
- particles
- analyte
- gold
- metal
- Prior art date
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Definitions
- the present invention is a labeling substance that can be used for immunological measurement and has excellent sensitivity, durability, and visibility, an immunological measurement method, a reagent for immunological measurement, an analyte measurement method, an analyte, and the like.
- the present invention relates to a light measurement kit and a test strip for lateral flow chromatography.
- immunoassay This is also called an immunological measurement method, and is a method for qualitatively and quantitatively analyzing a trace component by utilizing a specific reaction between an antigen and an antibody, which is one of immune reactions.
- the antigen-antibody reaction is widely used in the above field because of its high sensitivity and high selectivity of the reaction.
- EIA enzyme immunoassay
- RIA radioimmunoassay
- CLIA chemiluminescence immunoassay
- FIA fluorescence immunoassay
- LIA latex agglutination
- ICA immunochromatography
- HA hemagglutination method
- HI hemagglutination inhibition method
- an antigen or antibody is detected qualitatively or quantitatively from a change (a change in the concentration of the antigen, antibody or complex) when the antigen and the antibody react to form a complex.
- a change a change in the concentration of the antigen, antibody or complex
- the detection sensitivity is increased by binding a labeling substance to the antibody, antigen or complex. Therefore, it can be said that the labeling ability of the labeling substance is an important factor affecting the detection ability in the immunoassay.
- red blood cells in the case of HA
- latex particles in the case of LIA
- fluorescent dyes in the case of FIA
- radioactive elements in the case of RIA
- enzymes in the case of EIA
- Chemiluminescent materials in the case of CLIA
- colored fine particles when used as the labeling substance, detection can be confirmed by visual observation without using a special analyzer, so that simpler measurement is expected.
- colored fine particles include colloidal particles of metals and metal oxides, latex particles colored with a pigment, and the like (Patent Document 1, Patent Document 4 and the like).
- Patent Document 1 Patent Document 4 and the like.
- the color tone of the colloidal particles is determined depending on the particle diameter and preparation conditions, it is difficult to obtain a desired vivid dark color tone, that is, there is a problem that visibility is insufficient.
- the colored latex particles have a problem that the coloring effect by the coloring matter is low and the visual judgment is insufficient.
- the coloring amount of the dye is increased, the dye covers the surface of the latex, and the original surface state of the latex particles is damaged, so that it is difficult to bind the antigen or antibody. was there. Also, it is not always necessary to improve the performance by clogging the pores of a chromatographic medium such as a membrane filter or causing non-specific aggregation of latex particles and increasing the coloring amount of the dye to increase the coloring. There was also a problem of not sticking.
- a colored latex comprising gold nanoparticles bonded to the surface of polymer latex particles is disclosed (Patent Document 3).
- the gold nanoparticles themselves serve as a colorant to improve visual judgment and detection sensitivity.
- the gold nanoparticles themselves also bind to antigens or antibodies. Since it is excellent, it is said that a sufficient amount of antigen or antibody can be bound even if gold nanoparticles are bound to a sufficiently dark color.
- the colored latex is one in which gold nanoparticles are bonded to the surface of the latex by irradiating a dispersion of HAuCl, which is a precursor of styrene-acrylic acid copolymer latex and gold nanoparticles, with gamma rays.
- HAuCl a precursor of styrene-acrylic acid copolymer latex and gold nanoparticles
- Patent Document 4 discloses polymer latex particles coated with metallic gold, suggesting application to reagents that can be used in microscopy and immunoassay methods.
- the polymer latex particles coated with the above metal gold do not disclose the material and particle size of the polymer latex particles. Furthermore, there is no verification of the effect as a reagent that can be used in immunoassay methods. Therefore, the effect as a reagent in metal gold and polymer latex particles is unknown.
- latex particles to which gold nanoparticles are bound or coated are expected as reagents for immunological measurement, but the durability and visibility are not sufficient with conventional techniques. Moreover, even if the visibility is high, applicable applications and use environments are limited.
- An object of the present invention is a labeling substance that can be used for immunological measurement, has excellent sensitivity, durability, and visibility, and enables high-sensitivity determination without requiring addition of a special device or work process. Is to provide.
- the labeling substance of the present invention is a labeling substance comprising a resin-metal complex having a structure in which metal particles are immobilized on resin particles, and has the following constitution (A) or (B): : (A) The average particle size of the resin-metal composite exceeds 300 nm; Or (B) The average particle diameter of the metal particles is in the range of more than 20 nm and less than 70 nm; It is characterized by having.
- the average particle diameter of the metal particles may be in the range of 1 nm to 80 nm in the case of (A).
- the resin-metal composite may have an average particle diameter of more than 300 nm and 1000 nm or less in the case of (A).
- the metal particles may be gold particles.
- the average particle size of the resin-metal complex may be in the range of 100 nm to 1000 nm in the case of (B).
- the metal particles may be gold particles.
- the labeling substance of the present invention may be one obtained by dispersing the resin-metal complex in water.
- the labeling substance of the present invention may be used by adsorbing an antigen or antibody on the surface of the resin-metal complex.
- the immunological measurement method of the present invention is characterized by using any of the above-mentioned labeling substances.
- the reagent for immunological measurement of the present invention includes any one of the labeling substances described above.
- the analyte measurement method of the present invention is an analyte measurement method for detecting or quantifying an analyte contained in a sample.
- the method for measuring an analyte of the present invention uses a lateral flow type chromatographic test strip including a membrane and a determination unit in which a capture ligand that specifically binds to the analyte is fixed to the membrane.
- the analyte measuring method of the present invention comprises the following steps (I) to (III): Step (I): a step of bringing the analyte contained in a sample into contact with a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with any of the labeling substances described above, Step (II): A step of bringing the complex containing the analyte and the labeled antibody formed in Step (I) into contact with a capture ligand in the determination unit, Step (III): measuring a color intensity derived from localized surface plasmon resonance of the resin-metal complex in the labeling substance, The process including this is performed.
- the analyte measurement kit of the present invention is an analyte measurement kit for detecting or quantifying an analyte contained in a sample using a lateral flow type chromatographic test strip.
- the analyte measurement kit of the present invention includes a membrane, and a test strip for lateral flow type chromatography including a determination unit in which a capture ligand that specifically binds to the analyte is fixed to the membrane; An antibody that specifically binds to the analyte, and a detection reagent comprising a labeled antibody labeled with the labeling substance according to any one of claims 1 to 8, Is included.
- the test strip for lateral flow chromatography of the present invention is a test strip for lateral flow chromatography for detecting or quantifying an analyte contained in a sample.
- the test strip for lateral flow chromatography of the present invention is A membrane, A determination unit in which a capture ligand that specifically binds to the analyte is fixed to the membrane in a direction in which the sample is developed, A reaction part containing a labeled antibody labeled with the labeling substance according to any one of claims 1 to 8, wherein the antibody that specifically binds to the analyte is upstream of the determination part; Is included.
- the labeling substance of the present invention includes a resin-metal composite having a structure in which metal particles are immobilized on resin particles. For this reason, there is a large amount of metal particles that express localized surface plasmon absorption in resin particles. Therefore, the labeling substance of the present invention is excellent in durability and visibility, and as an excellent material capable of highly sensitive determination without requiring addition of a special device or work process, for example, EIA, RIA, CLIA, It can be preferably applied to immunological measurements such as FIA, LIA, PA, ICA, HA, and HI.
- FIG. 2 is a schematic diagram showing a cross-sectional structure of a resin-metal composite that constitutes a labeling substance according to an embodiment of the present invention. It is explanatory drawing which shows the outline
- mold chromatography which concerns on one embodiment of this invention.
- 2 is a scanning electron microscope (SEM) photograph of the resin-metal composite obtained in Example 1.
- FIG. 4 is a scanning electron microscope (SEM) photograph of the resin-gold composite obtained in Example 9.
- 4 is a scanning transmission electron microscope (STEM) photograph of a cross section of the resin-gold composite obtained in Example 9.
- 2 is a scanning electron microscope (SEM) photograph of the resin-gold composite obtained in Example 10.
- 2 is a scanning transmission electron microscope (STEM) photograph of a cross section of the resin-gold composite obtained in Example 10.
- the labeling substance according to the first embodiment of the present invention is a labeling substance provided with a resin-metal complex having a structure in which metal particles are immobilized on resin particles, and is an average of the resin-metal complex.
- the particle diameter exceeds 300 nm.
- FIG. 1 is a schematic cross-sectional view of a resin-metal complex constituting the labeling substance according to the present embodiment.
- the resin-metal composite 100 includes resin particles 10 and metal particles 20.
- the labeling substance of the present embodiment having the resin-metal complex 100 can be preferably used, for example, as an immunoassay reagent or a material thereof.
- the metal particles 20 are dispersed or fixed to the resin particles 10. Further, in the resin-metal composite 100, a part of the metal particles 20 is distributed two-dimensionally or three-dimensionally in the surface layer portion 60 of the resin particle 10, and a part of the metal particles 20 distributed three-dimensionally. Is partially exposed outside the resin particle 10, and the remaining part is encapsulated in the resin particle 10.
- the metal particles 20 include metal particles completely encapsulated in the resin particles 10 (hereinafter also referred to as “encapsulated metal particles 30”), portions embedded in the resin particles 10, and outside the resin particles 10. It is also referred to as a metal particle having an exposed portion (hereinafter, “partially exposed metal particle 40”). ) And metal particles adsorbed on the surface of the resin particle 10 (hereinafter also referred to as “surface adsorbed metal particles 50”).
- an antibody or an antigen is immobilized on the partially exposed metal particles 40 or the surface-adsorbed metal particles 50 and used. At this time, the antibody or antigen is immobilized on the partially exposed metal particles 40 and the surface-adsorbed metal particles 50, but not immobilized on the encapsulated metal particles 30.
- all of the metal particles 20 including the encapsulated metal particles 30 exhibit localized surface plasmon absorption, not only the partially exposed metal particles 40 and the surface adsorbed metal particles 50 but also the encapsulated metal particles 30 are labeled substances. Contributes to improved visibility.
- the partially exposed metal particles 40 and the encapsulated metal particles 30 have a large contact area with the resin particles 10 as compared with the surface-adsorbed metal particles 50, and also have a physical adsorption force such as an anchor effect due to the embedded state. Strong and difficult to desorb from the resin particles 10. Therefore, durability and stability as a labeling substance using the resin-metal composite 100 can be improved.
- the encapsulated metal particles 30 are all covered with the resin constituting the resin particles 10 on the entire surface.
- the partially exposed metal particles 40 are those in which 5% or more and less than 100% of the surface area is covered with the resin constituting the resin particles 10. From the viewpoint of durability, the lower limit is preferably 20% or more, more preferably 30% or more of the surface area.
- the surface-adsorbed metal particles 50 are covered with the resin constituting the resin particles 10 with more than 0% and less than 5% of the surface area thereof.
- the loading amount of the metal particles 20 (the total of the encapsulated metal particles 30, the partially exposed metal particles 40, and the surface adsorbed metal particles 50) on the resin-metal composite 100 is based on the weight of the resin-metal composite 100. It is preferably 5 wt% to 70 wt%. Within this range, the resin-metal composite 100 is excellent in visibility as a labeling substance, visual judgment and detection sensitivity. When the loading amount of the metal particles 20 is less than 5 wt%, the amount of antibody or antigen immobilized is decreased, and the detection sensitivity tends to decrease.
- the supported amount of the metal particles 20 is more preferably 15 wt% to 70 wt%.
- 10 wt% to 90 wt% of the metal particles 20 are preferably partially exposed metal particles 40 and surface adsorbed metal particles 50. Within this range, a sufficient amount of antibody or antigen immobilized on the metal particles 20 can be secured, and the sensitivity as a labeling substance is high. More preferably, 20 wt% to 80 wt% of the metal particles 20 are the partially exposed metal particles 40 and the surface adsorbed metal particles 50, and from the viewpoint of durability, the surface adsorbed metal particles 50 are more preferably 20 wt% or less. .
- 60 wt% to 100 wt%, preferably 75 to 100 wt%, more preferably 85 to 100 wt% of the metal particles 20 are present in the surface layer portion 60. More preferably, it exists in the range of 40% of the particle radius in the depth direction from the surface of the resin particle 10.
- 5 wt% to 90 wt% of the metal particles 20 present on the surface layer portion 60 are partially exposed metal particles 40 or surface adsorbed metal particles 50, which indicates that the amount of antibody or antigen immobilized on the metal particles 20 is fixed. Since sufficient sensitivity can be secured, the sensitivity as a labeling substance is high, which is preferable.
- 10 wt% to 95 wt% of the metal particles 20 existing in the surface layer portion 60 are the encapsulated metal particles 30.
- the “surface layer portion” refers to the resin particle 10 based on the outermost position of the resin-metal composite 100 (that is, the protruding end portion of the partially exposed metal particle 40 or the surface-adsorbed metal particle 50). Means a range of 50% of the particle radius in the depth direction.
- the “two-dimensional distribution” means that the metal particles 20 are distributed in the surface direction of the resin particles 10.
- the “three-dimensional distribution” means that the metal particles 20 are distributed not only in the surface direction of the resin particles 10 but also in the depth direction. It is preferable that the metal particles 20 are “three-dimensionally distributed” because the metal particles 20 are less likely to be detached from the resin particles 10 and the amount of the metal particles 20 supported is increased.
- the average particle size of the resin-metal composite 100 exceeds 300 nm.
- the average particle size of the resin-metal composite 100 is preferably more than 300 nm and 1000 nm or less, and more preferably 340 nm or more and less than 650 nm.
- the particle diameter of the resin-metal composite 100 means a value obtained by adding the length of the protruding portion of the partially exposed metal particle 40 or the surface-adsorbed metal particle 50 to the particle diameter of the resin particle 10. / Measured by scattering method, dynamic light scattering method, or centrifugal sedimentation method.
- the resin particles 10 are preferably polymer particles having a substituent in the structure capable of adsorbing metal ions.
- nitrogen-containing polymer particles are preferable.
- the nitrogen atom in the nitrogen-containing polymer is preferable because it is easy to chemisorb an anionic metal ion that is a precursor of metal particles such as gold and palladium, which has excellent visibility and can easily immobilize an antigen or antibody.
- metal ions adsorbed in the nitrogen-containing polymer are reduced to form metal nanoparticles, so that part of the generated metal particles 20 becomes encapsulated metal particles 30 or partially exposed metal particles 40. .
- carboxylic acid and the like can adsorb cationic metal ions, so it is easy to adsorb cationic metal ions that are precursors of metal particles such as silver, nickel, copper, etc.
- the metal particles 20 such as nickel and copper can be formed, and an alloy with the metal such as gold and palladium can be formed.
- resin particles other than the nitrogen-containing polymer having a substituent capable of adsorbing metal ions, such as polystyrene it is difficult to adsorb the metal ions inside the resin. As a result, most of the generated metal particles 20 become surface-adsorbed metal particles 50.
- the nitrogen-containing polymer is a resin having a nitrogen atom in the main chain or side chain, and examples thereof include polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, and polyaniline.
- polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, poly-4-vinylpyridine and the like are preferable.
- an acrylic resin, a phenol resin, an epoxy resin, etc. can be used widely.
- silver, nickel, copper, gold, palladium or the like can be applied to the metal particles 20.
- Gold and palladium are preferable because they are excellent in visibility and can easily immobilize antigens or antibodies. These are preferable because they express absorption derived from localized surface plasmon resonance. More preferably, it is gold with good storage stability.
- These metals can be used as a simple substance or a composite such as an alloy.
- a gold alloy means an alloy composed of gold and a metal species other than gold and containing 10% by weight or more of gold.
- the average particle diameter of the metal particles 20 measured by scanning electron microscope (SEM) observation is preferably 1 to 80 nm, for example.
- the average particle diameter of the metal particles 20 is less than 1 nm or exceeds 80 nm, the localized surface plasmon is less likely to be expressed, and thus the sensitivity tends to decrease.
- the metal particle 20 is a gold particle
- the average particle size of the metal particle 20 in the first embodiment is preferably 20 nm or more and less than 70 nm, and more preferably 22 nm or more and less than 50 nm.
- the labeling substance of the second embodiment of the present invention is a labeling substance comprising a resin-metal complex having a structure in which metal particles are immobilized on resin particles, and the average particle diameter of the metal particles is 20 nm. And within the range of less than 70 nm.
- the resin-metal complex constituting the labeling substance according to the present embodiment is different from the first embodiment except that the average particle diameter range of the metal particles and the average particle diameter range of the resin-metal complex are different. This is the same as the resin-metal composite 100 in FIG. Hereinafter, the difference from the first embodiment will be mainly described with reference to FIG.
- the average particle diameter of the metal particles 20 measured by scanning electron microscope (SEM) observation is, for example, more than 20 nm and less than 70 nm.
- SEM scanning electron microscope
- the average particle diameter of the metal particles 20 is more preferably 22 nm or more and less than 50 nm.
- the average particle diameter of the resin-metal composite 100 is preferably 100 to 1000 nm, for example.
- the average particle diameter of the resin-metal composite 100 is less than 100 nm, for example, when gold particles are used as the metal particles 20, the amount of gold particles tends to be reduced.
- it exceeds 1000 nm when it is used as a reagent, it tends to be clogged in the pores of a chromatographic medium such as a membrane filter, and the dispersibility tends to decrease.
- the average particle diameter of the resin-metal composite 100 is preferably 100 nm or more and less than 700 nm, and more preferably 340 nm or more and less than 650 nm.
- the particle diameter of the resin-metal composite 100 means a value obtained by adding the length of the protruding portion of the partially exposed metal particle 40 or the surface-adsorbed metal particle 50 to the particle diameter of the resin particle 10. / Measured by scattering method, dynamic light scattering method, or centrifugal sedimentation method.
- the configuration other than the above is the same as that of the resin-metal composite 100 used in the first embodiment. Omitted.
- the method for producing the resin-metal composite 100 used for the labeling substances of the first and second embodiments is not particularly limited.
- a solution containing metal ions is added to a dispersion of resin particles 10 produced by an emulsion polymerization method, and the metal ions are adsorbed on the resin particles 10 (hereinafter referred to as “metal ion adsorption resin particles”).
- metal ion adsorption resin particles the metal ion adsorption resin particles
- the metal ions are reduced to generate metal particles 20, and the resin-metal composite 100 is obtained.
- examples of the solution containing metal ions include chloroauric acid (HAuCl 4 ) aqueous solution.
- Hauric acid HuCl 4
- water-containing alcohol or alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, t-butanol, alcohol, hydrochloric acid, sulfuric acid, nitric acid, instead of water
- An acid such as the above may be used.
- water-soluble polymer compounds such as polyvinyl alcohol, surfactants, alcohols; ethers such as tetrahydrofuran, diethyl ether, diisopropyl ether; alkylene glycol, polyalkylene glycol, these Additives such as various water-miscible organic solvents such as polyols such as monoalkyl ether or dialkyl ether, glycerol, etc .; ketones such as acetone and methyl ethyl ketone may be added. Such an additive is effective for accelerating the reduction reaction rate of metal ions and controlling the size of the metal particles 20 to be produced.
- a well-known thing can be used for a reducing agent.
- sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, sodium phosphinate, hydroquinone, Rochelle salt, etc. can be mentioned.
- sodium borohydride, dimethylamine borane, and citric acid are preferable.
- a surfactant can be added to the reducing agent solution, and the pH of the solution can be adjusted.
- the pH can be adjusted with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.
- a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.
- the particle size of the metal particles to be formed can be controlled by adjusting the reduction rate of the metal ions depending on the temperature of the reducing agent solution.
- the metal ion adsorption resin particles may be added to the reducing agent solution, or the reducing agent may be added to the metal ion adsorption resin. Although it may be added to the particles, the former is preferable from the viewpoint of easy generation of the encapsulated metal particles 30 and the partially exposed metal particles 40.
- the dispersibility of the resin-metal composite 100 in water for example, citric acid, poly-L-lysine, polyvinyl pyrrolidone, polyvinyl pyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, DISPERBYK184 (Big Chemy Japan)
- a dispersant may be added.
- the dispersibility can be maintained by adjusting the pH with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.
- the resin-metal complex 100 having the above configuration is used as a labeling substance, for example, by adsorbing an antigen or antibody on the surface of the metal particle 20, for example, EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA. It is preferably applicable to immunological measurement methods such as HI. In particular, it can be preferably applied as a labeling substance excellent in visual judgment in a low concentration region (high sensitivity region).
- the form of the labeling substance is not particularly limited.
- the labeling substance can be used as a dispersion liquid in which the resin-metal complex 100 is dispersed in water or a pH-adjusted buffer.
- the method for adsorbing an antigen or antibody on the surface of the metal particle 20 is not particularly limited, and known physical adsorption and chemical adsorption methods can be used.
- physical adsorption such as immersing the resin-metal complex 100 in a buffer containing the antigen or antibody and incubating it, or introducing SH groups into the antigen or antibody and reacting with the resin-metal complex 100 to cause Au
- chemisorption such as formation of —SH bond. Of these, chemisorption is preferred because the bond between the metal particles 20 and the antigen or antibody is strong.
- test strip for lateral flow chromatography
- test strip a lateral flow type chromatographic test strip (test strip) according to an embodiment of the present invention will be described with reference to FIG.
- the test strip 200 can be preferably used in the analyte measuring method of one embodiment of the present invention.
- the test strip 200 includes a membrane 110.
- the membrane 110 is provided with a sample addition unit 120, a determination unit 130, and a liquid absorption unit 140 in order in the sample development direction.
- a membrane used as a membrane material in a general test strip can be applied.
- the membrane 110 is formed of an inert substance (a substance that does not react with the analyte 160, various ligands, etc.) made of a microporous material that exhibits a capillary phenomenon and develops the sample at the same time as the sample is added. It is.
- Specific examples of the membrane 110 include a fibrous or non-woven fibrous matrix composed of polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, cellulose derivatives, etc., membrane, filter paper, glass fiber filter paper, cloth, Cotton etc. are mentioned.
- membranes composed of cellulose derivatives and nylon, filter paper, glass fiber filter paper, etc. are preferably used, more preferably nitrocellulose membrane, mixed nitrocellulose ester (mixture of nitrocellulose and cellulose acetate) membrane, nylon membrane Filter paper is used.
- the test strip 200 preferably includes a support that supports the membrane 110 in order to make the operation easier.
- a support for example, plastic can be used.
- the test strip 200 may have a sample adding unit 120 for adding a sample including the analyte 160.
- the sample addition unit 120 is a part for receiving a sample including the analyte 160 in the test strip 200.
- the sample addition unit 120 may be formed on the membrane 110 upstream of the determination unit 130 in the direction in which the sample is developed, or, for example, cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon
- a sample addition pad made of a material such as cotton cloth may be provided on the membrane 110 to constitute the sample addition unit 120.
- a capture ligand 131 that specifically binds to the analyte 160 is fixed to the determination unit 130.
- the capture ligand 131 can be used without particular limitation as long as it forms a specific bond with the analyte 160.
- an antibody against the analyte 160 can be preferably used.
- the capture ligand 131 is immobilized so as not to move from the determination unit 130 even when a sample is provided to the test strip 200.
- the capture ligand 131 may be fixed directly or indirectly to the membrane 110 by physical or chemical bonding or adsorption.
- the determination unit 130 is not particularly limited as long as the complex 170 including the labeled antibody 150 and the analyte 160 is in contact with the capture ligand 131 that specifically binds to the analyte 160.
- the capture ligand 131 may be directly fixed to the membrane 110, or the capture ligand 131 may be fixed to a pad made of cellulose filter paper, glass fiber, nonwoven fabric, or the like fixed to the membrane 110. .
- the liquid-absorbing part 140 is formed of a pad of a water-absorbing material such as cellulose filter paper, non-woven fabric, cloth, cellulose acetate or the like.
- the moving speed of the sample after the development front (front line) of the added sample reaches the liquid absorption part 140 varies depending on the material, size, and the like of the liquid absorption part 140. Therefore, the optimum speed for detection / quantification of the analyte 160 can be set by selecting the material, size, etc. of the liquid absorption part 140.
- the liquid absorption part 140 is an arbitrary configuration and may be omitted.
- the test strip 200 may further include arbitrary parts such as a reaction part and a control part as necessary.
- the test strip 200 may be formed with a reaction part including the labeled antibody 150 on the membrane 110.
- the reaction unit can be provided upstream of the determination unit 130 in the direction in which the sample flows.
- the test strip 200 has a reaction part, if the sample containing the analyte 160 is supplied to the reaction part or the sample addition part 120, the analyte 160 contained in the sample and the labeled antibody 150 can be brought into contact with each other in the reaction part. it can.
- the complex 170 containing the analyte 160 and the labeled antibody 150 can be formed by simply supplying the sample to the reaction part or the sample addition part 120, so-called one-step type immunochromatography is possible. Become.
- the reaction part is not particularly limited as long as it includes the labeled antibody 150 that specifically binds to the analyte 160, but may be one in which the labeled antibody 150 is directly applied to the membrane 110.
- the reaction part may be formed by, for example, immobilizing the membrane 110 with a pad (conjugate pad) made of cellulose filter paper, glass fiber, nonwoven fabric, or the like impregnated with the labeled antibody 150.
- the test strip 200 may be formed with a control unit in which a capture ligand that specifically binds to the labeled antibody 150 is fixed to the membrane 110 in the direction in which the sample is developed.
- a control unit By measuring the color intensity at the control unit together with the determination unit 130, the sample provided for the test strip 200 is developed and reaches the reaction unit and the determination unit 130 to confirm that the test has been performed normally. be able to.
- the control unit is prepared in the same manner as the determination unit 130 described above except that another type of capture ligand that specifically binds to the labeled antibody 150 is used instead of the capture ligand 131. The configuration can be taken.
- the measuring method of the analyte 160 of the present embodiment is a measuring method of the analyte 160 that detects or quantifies the analyte 160 contained in the sample.
- the method for measuring the analyte 160 according to the embodiment uses the test strip 200 including the membrane 110 and the determination unit 130 in which the capture ligand 131 that specifically binds to the analyte 160 is fixed to the membrane 110.
- Step (I) is a step of bringing the analyte 160 contained in the sample into contact with the labeled antibody 150.
- the mode of contact is not particularly limited.
- the sample may be provided to the sample addition unit 120 or the reaction unit (not shown) of the test strip 200, and the analyte 160 may be brought into contact with the labeled antibody 150 in the reaction unit, or before the sample is applied to the test strip 200.
- the analyte 160 in the sample may be brought into contact with the labeled antibody 150.
- the composite 170 formed in step (I) expands and moves on the test strip 200 and reaches the determination unit 130.
- step (II) in the determination unit 130 of the test strip 200, the complex 170 formed in step (I) and including the analyte 160 and the labeled antibody 150 is brought into contact with the capture ligand 131.
- the capture ligand 131 specifically binds to the analyte 160 of the complex 170.
- the complex 170 is captured by the determination unit 130.
- the capture ligand 131 does not specifically bind to the labeled antibody 150, when the analyte 160 and the unbound labeled antibody 150 reach the determination unit 130, the analyte 160 and the unbound labeled antibody 150 are unbound. Passes through the determination unit 130.
- a control unit (not shown) to which another capture ligand that specifically binds to the labeled antibody 150 is fixed is formed on the test strip 200, the labeled antibody 150 that has passed through the determination unit 130 is developed. Then, bind to the other capture ligand at the control unit. As a result, the labeled antibody 150 that does not form the complex 170 with the analyte 160 is captured by the control unit.
- step (II) if necessary, before step (III), for example, the test strip 200 is washed with a buffer solution commonly used in biochemical tests such as water, physiological saline, and phosphate buffer solution.
- a cleaning step may be performed.
- the labeled antibody 150 (the labeled antibody 150 that is not bound to the analyte 160 and does not form the complex 170) that has not been captured by the determination unit 130 or the determination unit 130 and the control unit is removed. be able to.
- the background Color development intensity can be reduced, the signal / background ratio can be increased, and detection sensitivity and quantitativeness can be further improved.
- Step (III) is a step of measuring the color intensity derived from the localized surface plasmon resonance of the resin-metal composite 100. After the step (II) or the washing step as necessary, the color intensity derived from the localized surface plasmon resonance of the resin-metal composite 100 is measured on the test strip 200.
- step (II) When a control part is formed on the test strip 200, in step (II), the labeled antibody 150 is captured by another capture ligand in the control part to form a complex. Therefore, in step (III), in the test strip 200, color development by localized surface plasmon resonance can be generated not only in the determination unit 130 but also in the control unit. In this way, by measuring the color intensity in the control unit as well as the determination unit 130, it is possible to confirm whether the sample provided for the test strip 200 has developed normally and reached the reaction unit and the determination unit 130.
- the sample in the analyte measurement method of the present embodiment is not particularly limited as long as it includes a substance that can be an antigen such as a protein as the analyte 160.
- a biological sample containing the analyte 160 of interest ie, whole blood, serum, plasma, urine, saliva, sputum, nasal or throat swab, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, skin exudate , Extracts from tissues, cells and stool, etc.
- food extracts ie, whole blood, serum, plasma, urine, saliva, sputum, nasal or throat swab, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, skin exudate , Extracts from tissues, cells and stool, etc.
- the analyte 160 contained in the sample is pretreated prior to the step (I). May be.
- the pretreatment include chemical treatment using various chemicals such as acid, base, and surfactant, and physical treatment using heating, stirring, ultrasonic waves, and the like.
- the analyte 160 is a substance that is not normally exposed on the surface, such as an influenza virus NP antigen, it is preferable to perform treatment with a surfactant or the like.
- a nonionic surfactant can be used in consideration of specific binding reaction, for example, binding reactivity between the ligand 160 and the analyte 160 such as antigen-antibody reaction. .
- the sample may be appropriately diluted with a solvent (water, physiological saline, buffer, or the like) used in a normal immunological analysis method or a water-miscible organic solvent.
- a solvent water, physiological saline, buffer, or the like
- analyte 160 examples include tumor markers, signaling substances, proteins such as hormones (including polypeptides and oligopeptides), nucleic acids (single-stranded or double-stranded DNA, RNA, polynucleotides, oligos) Other molecules such as nucleotides, PNA (including peptide nucleic acids)) or nucleic acid-containing substances, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or sugar chains, lipids, and the like.
- proteins such as hormones (including polypeptides and oligopeptides), nucleic acids (single-stranded or double-stranded DNA, RNA, polynucleotides, oligos)
- Other molecules such as nucleotides, PNA (including peptide nucleic acids)) or nucleic acid-containing substances, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or
- CEA carcinoembryonic antigen
- HER2 protein HER2 protein
- PSA prostate specific antigen
- CA19-9 ⁇ -fetoprotein
- IPA Immunosuppressive acidic protein
- CA15-3 CA125
- estrogen receptor progesterone Scepter
- HCG human chorionic gonadotropin
- LH luteinizing hormone
- FSH follicle stimulating hormone
- syphilis antibody influenza virus human hemoglobin, chlamydia antigen
- examples include group A ⁇ -streptococcal antigens, HBs antibodies, HBs antigens, rotaviruses, adenoviruses, albumin, glycated albumin and the like.
- antigens solubilized by nonionic surfactants are preferable, and
- the labeled antibody 150 is used to contact the analyte 160 contained in the sample to form a complex 170 including the analyte 160 and the labeled antibody 150.
- the labeled antibody 150 is obtained by labeling an antibody that specifically binds to the analyte 160 with the resin-metal complex 100 having a structure in which a plurality of metal particles 20 are immobilized on the resin particles 10.
- labeling means that the resin-metal complex 100 is directly attached to the antibody to the extent that the resin-metal complex 100 is not detached from the labeled antibody 150 in steps (I) to (III). It means that it is fixed indirectly by chemical or physical bonding or adsorption.
- the labeled antibody 150 may be one in which the resin-metal complex 100 is directly bonded to the antibody, or the antibody and the resin-metal complex 100 are bonded via any linker molecule. Or those fixed to insoluble particles.
- the “antibody” is not particularly limited.
- a monoclonal antibody an antibody obtained by genetic recombination, an antibody fragment [for example, an H chain , L chain, Fab, F (ab ′) 2 etc.] can be used.
- IgG, IgM, IgA, IgE, or IgD may be used as the immunoglobulin.
- the animal species that produce the antibody may be humans or animals other than humans (eg, mice, rats, rabbits, goats, horses, etc.).
- Specific examples of the antibody include anti-PSA antibody, anti-AFP antibody, anti-CEA antibody, anti-adenovirus antibody, anti-influenza virus antibody, anti-HCV antibody, anti-IgG antibody, and anti-human IgE antibody.
- the production of the labeled antibody 150 is at least the following step A; Step A) including the step of obtaining the labeled antibody 150 by mixing and binding the resin-metal complex 100 with the antibody under a first pH condition, and preferably, further includes Step B; Step B) A step of treating the labeled antibody 150 under a second pH condition can be included.
- Step A the labeled antibody 150 is obtained by mixing the resin-metal complex 100 with an antibody under the first pH condition.
- the solid resin-metal complex 100 is preferably contacted with the antibody in a state dispersed in the liquid phase.
- the first pH condition varies depending on the metal species of the metal particle 20 in the resin-metal composite 100.
- the first pH condition is that the binding with the antibody is the resin-metal composite 100.
- the metal particles 20 of the resin-metal composite 100 are gold particles (including gold alloy particles; the same applies hereinafter)
- the first pH condition is that the binding with the antibody is the resin-metal composite 100.
- conditions within the range of pH 2 to 7 are preferable, and acidic conditions such as pH 2.5 to 5.5 are preferred. Within the range is more preferable.
- the metal particles 20 are gold particles, if the resin-metal complex 100 and the antibody are bonded under conditions where the pH is less than 2, the antibody may be altered and deactivated due to strong acidity.
- the metal complex 100 and the antibody are mixed, they aggregate and become difficult to disperse. However, if the antibody is not inactivated by strong acidity, the treatment can be performed even at a pH of less than 2.
- the first pH condition is that the binding with the antibody is the resin-metal composite.
- a condition within the range of pH 2 to 10 is preferable, and for example, a range of pH 5 to 9 is more preferable.
- the metal particles 20 are particles other than gold, if the conditions for bonding the resin-metal complex 100 and the antibody are less than pH 2, the antibody may be denatured and deactivated due to strong acidity.
- the resin-metal complex 100 and the antibody are mixed, they aggregate and become difficult to disperse. However, if the antibody is not inactivated by strong acidity, the treatment can be performed even at a pH of less than 2.
- Step A is preferably performed in a binding buffer adjusted to the first pH condition.
- a predetermined amount of the resin-metal complex 100 is mixed in the binding buffer adjusted to the above pH and mixed thoroughly.
- a boric acid solution adjusted to a predetermined concentration can be used as the binding buffer.
- the pH of the binding buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide.
- a labeled antibody-containing liquid can be obtained by adding a predetermined amount of the antibody to the obtained mixed liquid, sufficiently stirring and mixing.
- the labeled antibody-containing liquid thus obtained can be fractionated only with the labeled antibody 150 as a solid part by solid-liquid separation means such as centrifugation.
- Step B the labeled antibody 150 obtained in the process A is treated under the second pH condition to perform blocking that suppresses nonspecific adsorption to the labeled antibody 150.
- the labeled antibody 150 separated by the solid-liquid separation means is dispersed in the liquid phase under the second pH condition.
- This blocking condition varies depending on the metal species of the metal particle 20 in the resin-metal composite 100.
- the second pH condition is, for example, within the range of pH 2 to 9 from the viewpoint of maintaining the activity of the antibody and suppressing the aggregation of the labeled antibody 150.
- acidic conditions for example, in the range of pH 2 to 6, are more preferable. If the blocking condition is less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and if it exceeds pH 9, the labeled antibody 150 aggregates, making dispersion difficult.
- the second pH condition is, for example, from pH 2 to pH 2 from the viewpoint of maintaining antibody activity and suppressing aggregation of the labeled antibody 150.
- the range of 10 is preferable, and the range of pH 5 to 9 is more preferable from the viewpoint of suppressing nonspecific adsorption of the labeled antibody 150.
- the blocking condition is less than pH 2, the antibody may be denatured and deactivated due to strong acidity, and if it exceeds pH 10, the labeled antibody 150 aggregates, making dispersion difficult.
- Step B is preferably performed using a blocking buffer adjusted to the second pH condition.
- the blocking buffer adjusted to the above pH is added to a predetermined amount of labeled antibody 150, and the labeled antibody 150 is uniformly dispersed in the blocking buffer.
- the blocking buffer for example, a protein solution that does not bind to the detection target is preferably used.
- proteins that can be used in the blocking buffer include bovine serum albumin, ovalbumin, casein, and gelatin. More specifically, it is preferable to use a bovine serum albumin solution adjusted to a predetermined concentration.
- the pH of the blocking buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide.
- a dispersing means such as ultrasonic treatment. In this way, a dispersion in which the labeled antibody 150 is uniformly dispersed is obtained.
- a dispersion of labeled antibody 150 is obtained. From this dispersion, only the labeled antibody 150 can be fractionated as a solid part by solid-liquid separation means such as centrifugation. Moreover, a cleaning process, a preservation
- a washing buffer solution is added to the labeled antibody 150 sorted by the solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the washing buffer solution.
- a dispersion means such as ultrasonic treatment is preferably used.
- the washing buffer is not particularly limited, and for example, a Tris buffer solution, a glycinamide buffer solution, an arginine buffer solution, or the like having a predetermined concentration adjusted within a pH range of 8 to 9 may be used. it can.
- the pH of the washing buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide.
- the washing treatment of the labeled antibody 150 can be repeated a plurality of times as necessary.
- a storage buffer is added to the labeled antibody 150 collected by the solid-liquid separation means, and the labeled antibody 150 is uniformly dispersed in the storage buffer.
- a dispersion means such as ultrasonic treatment is preferably used.
- the storage buffer for example, a solution obtained by adding a predetermined concentration of an anti-aggregation agent and / or stabilizer to the washing buffer can be used.
- the aggregation inhibitor for example, saccharides typified by sucrose, maltose, lactose, trehalose, polyhydric alcohols typified by glycerin, polyvinyl alcohol, and the like can be used.
- the stabilizer is not particularly limited. For example, proteins such as bovine serum albumin, ovalbumin, casein, and gelatin can be used.
- save process of the labeled antibody 150 can be performed.
- a surfactant or a preservative such as sodium azide or paraoxybenzoate can be used as necessary.
- An analyte measurement kit uses, for example, a lateral flow type chromatographic test strip 200 and an analyte 160 contained in a sample based on the analyte measurement method of the present embodiment. It is a kit for detecting or quantifying.
- the kit of this embodiment is A lateral flow type chromatographic test strip 200 including a membrane 110 and a determination unit 130 in which a capture ligand that specifically binds to the analyte 160 is fixed to the membrane 110; A detection reagent comprising a labeled antibody 150 in which an antibody that specifically binds to the analyte 160 is labeled with a resin-metal complex 100 having a structure in which a plurality of metal particles 20 are immobilized on resin particles 10; Is included.
- the kit of the present embodiment may further include other components as necessary.
- the reaction section or the sample addition section 120 of the test strip 200 is performed.
- the step (II) and the step (III) may be sequentially performed by supplying a sample.
- a detection reagent is applied to the upstream side of the determination unit 130 of the test strip 200 and appropriately dried to form a reaction part, the formed reaction part or a position upstream of the reaction part (for example, The sample may be added to the sample addition unit 120) and the steps (I) to (III) may be performed sequentially.
- the absorbance of the resin-metal composite was measured by placing a resin-metal composite dispersion liquid (dispersion medium: water) prepared at 0.01 wt% into an optical white glass cell (optical path length 10 mm), and an instantaneous multi-photometry system (Otsuka In the case of gold, absorbance at 570 nm was measured using MCPD-3700) manufactured by Denki Co. In the case of gold, the absorbance at 570 nm is 0.9 (good), 0.5 to less than 0.9 is ⁇ (good), and less than 0.5 is x (impossible).
- Solid content concentration (wt%) [weight after drying (g) / weight before drying (g)] ⁇ 100
- Metal loading (wt%) [Weight after heat treatment at 500 ° C. (g) / Weight before heat treatment at 500 ° C. (g)] ⁇ 100
- the average particle size of the metal particles was measured by dropping a resin-metal composite dispersion liquid onto a metallic mesh with a carbon support film, using a field emission scanning electron microscope (STEM; manufactured by Hitachi High-Technologies Corporation, SU The area average diameter of the metal particles was measured from the image observed by ⁇ 9000).
- This gold ion adsorbing resin particle dispersion (45 g) was dropped into a 10 mM dimethylamine borane aqueous solution (450 ml) over 8 minutes, and then stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 0.6 ⁇ m.
- This resin-gold complex is precipitated by centrifugation (3000 rpm, 120 minutes), and after removing the supernatant, it is dispersed again in 37 g of pure water and purified with an ultrafiltration membrane to obtain 1 wt% resin-gold.
- a composite dispersion was obtained.
- the absorbance of the resin-gold complex in this resin-gold complex dispersion was 1.20 as a result of measurement according to the above method.
- the average particle size of the gold particles in the resin-gold composite was 22.0 nm, and the amount of gold supported was 49.4 wt%.
- a scanning electron microscope (SEM) photograph of the prepared resin-gold composite is shown in FIG.
- Example 2 In the same manner as in Example 1, except that 10 mM aqueous chloroauric acid solution (56 g) was added to the resin particle dispersion (3.05 g) obtained in Example 1, gold ion adsorption resin particle dispersion, average particle A resin-gold composite having a diameter of 0.6 ⁇ m and a 1 wt% resin-gold composite dispersion were obtained. The absorbance of the resin-gold complex in this resin-gold complex dispersion was 1.04. In addition, the average particle size of gold particles in the resin-gold composite was 7.61 nm, and the amount of gold supported was 36.8 wt%.
- This gold ion-adsorbed resin particle dispersion (1.5 g) was dropped into a 10 mM dimethylamine borane aqueous solution (65 ml) over 2 minutes, and then stirred at room temperature for 2 hours to give a resin having an average particle size of 0.22 ⁇ m. A gold composite was obtained.
- 600 ⁇ l of 10 wt% dispersant (BYK194) was added and stirred for 1 hour, and then precipitated by centrifugation (9000 rpm, 10 minutes), and the supernatant was removed. Thereafter, an appropriate amount of pure water was added and dispersed again, followed by purification with an ultrafiltration membrane to obtain a 1 wt% resin-gold composite dispersion.
- the absorbance of the resin-gold complex in this resin-gold complex dispersion was 1.12.
- the average particle diameter of the gold particles in the resin-gold composite was 22.6 nm, and the amount of gold supported was 37.0 wt%.
- the evaluation was performed using a monochrome screen for influenza A evaluation (manufactured by Adtech), and the coloring levels after 5 minutes, 10 minutes and 15 minutes were compared. In the performance evaluation, a 2-fold dilution series (1-fold to 1024-fold) of influenza A positive control (APC) was used as the antigen (the virus concentration before APC dilution was 5000 FFU / ml).
- APC influenza A positive control
- ⁇ Evaluation of immunochromatography> 100 ⁇ g of influenza antibody was mixed with 1 ml of the obtained gold colloid (OD 10) and stirred at room temperature for about 3 hours to bind the antibody to the gold colloid.
- the bovine serum albumin solution was added so that the final concentration was 1%, and the mixture was stirred at room temperature for 2 hours to block the colloidal gold surface.
- the gold colloid-labeled antibody was prepared by centrifuging at 12000 rpm and 4 ° C. for 5 minutes and then suspending in a buffer containing 0.2% bovine serum albumin. Using the prepared colloidal gold labeled antibody, the performance of the colloidal gold was evaluated by measuring by the following immunochromatography method.
- the evaluation was performed using a monochrome screen for influenza A evaluation (manufactured by Adtech), and the coloring levels after 5 minutes, 10 minutes and 15 minutes were compared.
- a 2-fold dilution series (1-fold to 1024-fold) of influenza A positive control (APC) was used as the antigen (the virus concentration before APC dilution was 5000 FFU / ml).
- APC influenza A positive control
- 3 ⁇ l of colloidal gold-labeled antibody was added to each well of a 96-well plate, and 100 ⁇ l each of a 2-fold dilution series (1-fold to 1024-fold) of APC and a negative control was mixed.
- 50 ⁇ l was added to a monochrome screen for influenza A evaluation, and the color development level after 5 minutes, 10 minutes and 15 minutes was evaluated. The results are shown below.
- the 2.5 wt% gold ion adsorbing resin particle dispersion (42.4 ml) is added to 1580 ml of pure water and stirred at 160 rpm at 20 ° C., 528 mM dimethylamine borane aqueous solution (10 ml) and 528 mM boric acid.
- a mixed solution of an aqueous solution (10 ml) was added dropwise over 4 minutes and then stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 250 nm.
- the resin-gold complex labeled antibody dispersion was prepared by centrifuging at 12000 pm for 5 minutes at 4 ° C. and then suspending in a buffer containing 0.2% bovine serum albumin. Using the prepared resin-gold complex labeled antibody dispersion, measurement was performed by immunochromatography to evaluate the performance of the resin-gold complex dispersion. The results are shown below.
- the 2.5 wt% gold ion adsorption resin particle dispersion (42.4 ml) is added to 1580 ml of pure water, and a 528 mM dimethylamine borane aqueous solution (10 ml) is added over 2 minutes while stirring at 160 rpm and 20 ° C. After the dropwise addition, the mixture was stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 510 nm. After the resin-gold complex is precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant is removed, and the operation of re-dispersing in pure water is repeated three times, followed by purification by dialysis and concentration adjustment.
- a 1 wt% resin-gold composite dispersion was obtained.
- the absorbance of the produced resin-gold composite was 1.01 as a result of measurement according to the above method.
- the formed gold particles had an average particle size of 46.3 nm and a gold loading amount of 54.2 wt%.
- the gold particles are: encapsulated metal particles that are completely encapsulated in the resin particles; Surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles.
- Example 5 Aliquat 336 (manufactured by Aldrich) (0.50 g) and polyethylene glycol methyl ethyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 2-vinylpyridine (2-VP, 48.00 g) and Divinylbenzene (DVB, 2.00 g) was added, and the mixture was stirred at 150 rpm, 30 ° C. for 50 minutes, and then at 60 ° C. for 30 minutes under a nitrogen stream.
- PEGMA polyethylene glycol methyl ethyl ether methacrylate
- the 2.5 wt% gold ion adsorption resin particle dispersion (42.4 ml) is added to 1580 ml of pure water, and 528 mM dimethylamine borane aqueous solution (10 ml) is added over 2 minutes while stirring at 160 rpm at 3 ° C. After the dropwise addition, the mixture was stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 625 nm. After the resin-gold complex is precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant is removed, and the operation of re-dispersing in pure water is repeated three times, followed by purification by dialysis and concentration adjustment.
- a 1 wt% resin-gold composite dispersion was obtained.
- the absorbance of the produced resin-gold composite was 0.98 as a result of measurement according to the above method.
- the formed gold particles had an average particle diameter of 25.0 nm and a gold loading amount of 55.3 wt%.
- the gold particles are: encapsulated metal particles that are completely encapsulated in the resin particles; Surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles.
- the 2.5 wt% gold ion adsorption resin particle dispersion (42.4 ml) is added to 1580 ml of pure water, and 528 mM dimethylamine borane aqueous solution (10 ml) is added over 2 minutes while stirring at 160 rpm at 3 ° C. After the dropwise addition, the mixture was stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 448 nm. After the resin-gold complex is precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant is removed, and the operation of re-dispersing in pure water is repeated three times, followed by purification by dialysis and concentration adjustment.
- a 1 wt% resin-gold composite dispersion was obtained.
- the absorbance of the produced resin-gold composite was measured according to the above method and was 0.99.
- the formed gold particles had an average particle diameter of 24.0 nm and a gold loading amount of 55.7 wt%.
- the gold particles are: encapsulated metal particles that are completely encapsulated in the resin particles; Surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles.
- Example 7 Aliquat 336 (manufactured by Aldrich) (1.00 g) and polyethylene glycol methyl ethyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 3-vinylpyridine (3-VP, 48.00 g) and Divinylbenzene (DVB, 2.00 g) was added, and the mixture was stirred at 150 rpm, 30 ° C. for 50 minutes, and then at 60 ° C. for 30 minutes under a nitrogen stream.
- PEGMA polyethylene glycol methyl ethyl ether methacrylate
- the 2.5 wt% gold ion adsorption resin particle dispersion (42.4 ml) is added to 1580 ml of pure water, and 528 mM dimethylamine borane aqueous solution (10 ml) is added over 2 minutes while stirring at 160 rpm at 3 ° C. After the dropwise addition, the mixture was stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 436 nm. After the resin-gold complex is precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant is removed, and the operation of re-dispersing in pure water is repeated three times, followed by purification by dialysis and concentration adjustment.
- a 1 wt% resin-gold composite dispersion was obtained.
- the absorbance of the produced resin-gold composite was 1.03 as a result of measurement according to the above method.
- the formed gold particles had an average particle size of 24.3 nm and a gold loading amount of 55.5 wt%.
- the gold particles are: encapsulated metal particles that are completely encapsulated in the resin particles; Surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles.
- Example 8 2- (Diisopropylamino) ethyl methacrylate (DPA, 10.3 g), poly (propylene glycol) diacrylate (0.2 g) and polyethylene glycol methyl ethyl ether methacrylate (PEGMA, 2.0 g) were dissolved in 85 g of pure water. Thereafter, the mixture was stirred at 150 rpm at 30 ° C. for 50 minutes and then at 70 ° C. for 30 minutes under a nitrogen stream. After stirring, ammonium peroxodisulfate (ASP, 0.10 g) dissolved in 2.00 g of pure water was added dropwise over 2 minutes, and the mixture was stirred at 150 rpm and 70 ° C.
- DPA Diisopropylamino) ethyl methacrylate
- PEGMA polyethylene glycol methyl ethyl ether methacrylate
- the 2.5 wt% gold ion adsorption resin particle dispersion (42.4 ml) is added to 1580 ml of pure water, and 528 mM dimethylamine borane aqueous solution (10 ml) is added over 2 minutes while stirring at 160 rpm at 3 ° C. After the dropwise addition, the mixture was stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 345 nm. After the resin-gold complex is precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant is removed, and the operation of re-dispersing in pure water is repeated three times, followed by purification by dialysis and concentration adjustment.
- a 1 wt% resin-gold composite dispersion was obtained.
- the absorbance of the produced resin-gold composite was 0.96 as a result of measurement according to the above method.
- the formed gold particles had an average particle diameter of 24.6 nm and a gold loading amount of 48.5 wt%.
- the gold particles are: encapsulated metal particles that are completely encapsulated in the resin particles; Surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles.
- the 2.5 wt% gold ion adsorption resin particle dispersion (42.4 ml) is added to 1580 ml of pure water, and a 528 mM dimethylamine borane aqueous solution (10 ml) is added over 2 minutes while stirring at 160 rpm and 20 ° C. After the dropwise addition, the mixture was stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 393 nm. After the resin-gold complex is precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant is removed, and the operation of re-dispersing in pure water is repeated three times, followed by purification by dialysis and concentration adjustment.
- a 1 wt% resin-gold composite dispersion was obtained.
- the absorbance of the produced resin-gold composite was 0.92 as a result of measurement according to the above method.
- the formed gold particles had an average particle diameter of 14.9 nm and a gold loading amount of 55.8 wt%.
- a scanning electron microscope (SEM) photograph of the surface of the obtained resin-gold composite is shown in FIG. 4, and a scanning transmission electron microscope (STEM) photograph of the cross section thereof is shown in FIG.
- the gold particles are: encapsulated metal particles that are completely encapsulated in the resin particles; Surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles.
- the 2.5 wt% gold ion adsorption resin particle dispersion (42.4 ml) is added to 1580 ml of pure water, and a 528 mM dimethylamine borane aqueous solution (10 ml) is added over 2 minutes while stirring at 160 rpm and 20 ° C. After the dropwise addition, the mixture was stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 399 nm. After the resin-gold complex is precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant is removed, and the operation of re-dispersing in pure water is repeated three times, followed by purification by dialysis and concentration adjustment.
- a 1 wt% resin-gold composite dispersion was obtained.
- the absorbance of the produced resin-gold composite was 0.96 as a result of measurement according to the above method.
- the formed gold particles had an average particle size of 25.0 nm and a gold loading amount of 53.2 wt%.
- a scanning electron microscope (SEM) photograph of the surface of the obtained resin-gold composite is shown in FIG. 6, and a scanning transmission electron microscope (STEM) photograph of the cross section thereof is shown in FIG.
- the gold particles are: encapsulated metal particles that are completely encapsulated in the resin particles; Surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles.
- Example 10 Comparing the cross-sectional images of FIG. 5 of Example 9 and FIG. 7 of Example 10, 60 to 100%, preferably 75 to 100% of the gold particles are 50% of the particle radius in the depth direction from the surface of the resin particles. In Example 10, the detection sensitivity of immunochromatography was superior.
- a resin-gold composite D-1 having a thickness of 38 ⁇ m was obtained.
- the D-1 is precipitated by centrifugation (3000 rpm, 120 minutes), the supernatant is removed, an appropriate amount of pure water is added and dispersed again, and purified by ultrafiltration membrane to obtain 1 wt% resin-gold.
- a composite dispersion E-1 was obtained.
- the absorbance of the resin-gold complex F-1 in E-1 was 1.0 as a result of measurement according to the above method. Further, the average particle size of gold particles in F-1 was 22.0 nm, and the amount of gold supported was 49.1 wt%.
- Binding buffer a 100 mM A boric acid solution was adjusted to pH ⁇ 3 with HCl.
- Binding buffer b 100 mM boric acid solution was adjusted to pH ⁇ 4 with HCl.
- Binding buffer c 100 mM boric acid solution was adjusted to pH ⁇ 5 with HCl.
- Binding buffer d 100 mM boric acid solution pH ⁇ 6.5
- Binding buffer e 100 mM boric acid solution was adjusted to pH ⁇ 7.5 with NaCl.
- Binding buffer f 100 mM boric acid solution was adjusted to pH ⁇ 8.5 with NaCl.
- Binding buffer g 50 mM 2-morpholinoethanesulfonic acid solution pH ⁇ 3.8
- Blocking buffer a 1% by weight bovine serum albumin solution was adjusted to pH ⁇ 5 with HCl.
- Blocking buffer b A 1% by weight bovine serum albumin solution was adjusted to pH ⁇ 7 with HCl.
- Blocking buffer c 1% by weight bovine serum albumin solution was adjusted to pH ⁇ 8.5 with HCl.
- Blocking buffer d A 1% by weight bovine serum albumin solution was adjusted to pH ⁇ 9.5 with HCl.
- Washing buffer A 5 mM Tris solution was adjusted to pH ⁇ 8.5 with HCl.
- Influenza A positive control Influenza A virus inactivated antigen (manufactured by Adtech Co., Ltd.) was prepared by diluting 100 times with a sample treatment solution (manufactured by Adtech Co., Ltd.). The antigen concentration of APC corresponds to 5000 FFU / ml.
- Negative control Sample treatment solution (manufactured by Adtec Corporation)
- AuNCP beads Resin-gold composite obtained in Preparation Example 1 (1% by weight; average particle size 380 nm)
- Test Example 2 In the binding step of Test Example 1, labeled antibody-containing solutions A-2, B-2, and C-2 were obtained in the same manner as in Test Example 1 except that the binding buffer b was used instead of the binding buffer a. .
- Test Example 3 Labeled antibody-containing solutions A-3, B-3, and C-3 were obtained in the same manner as in Test Example 1 except that the binding buffer c was used instead of the binding buffer a in the binding step of Test Example 1. .
- Test Example 4 In the binding step of Test Example 1, labeled antibody-containing solutions A-4, B-4, and C-4 were obtained in the same manner as in Test Example 1 except that the binding buffer d was used instead of the binding buffer a. .
- Test Example 5 Labeled antibody-containing solutions B-5 and C-5 were obtained in the same manner as in Test Example 1 except that the block buffer b was used instead of the block buffer a in the block step of Test Example 1.
- Test Example 7 In the binding step of Test Example 1, labeled antibody-containing solutions A-7, B-7, and C-7 were obtained in the same manner as in Test Example 1 except that the binding buffer g was used instead of the binding buffer a. .
- ⁇ Evaluation method> The evaluation was performed using a monochrome screen for influenza A evaluation (manufactured by Adtech), and the coloring levels after 5 minutes, 10 minutes and 15 minutes were compared. The color development level was determined using a color sample for gold colloid determination (manufactured by Adtech).
- the influenza A positive control (APC) was used as the antigen.
- the antigen used was a 2-fold dilution series (1-fold to 1024-fold dilution) of APC.
- SYMBOLS 10 Resin particle, 20 ... Metal particle, 30 ... Encapsulated metal particle, 40 ... Partially exposed metal particle, 50 ... Surface adsorbed metal particle, 60 ... Surface layer part, 100 ... Resin-metal composite, 110 ... Membrane, 120 ... Sample addition unit, 130 ... determination unit, 131 ... capture ligand, 140 ... liquid absorption part, 150 ... labeled antibody, 160 ... analyte, 170 ... complex, 200 ... test strip
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Nanotechnology (AREA)
- Inorganic Chemistry (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
Description
そのため、標識物質の標識能力は、イムノアッセイにおける検出能力を左右する重要な要素であるといえる。上記に例示したイムノアッセイにおいても、標識物質として、赤血球(HAの場合)、ラテックス粒子(LIAの場合)、蛍光色素(FIAの場合)、放射性元素(RIAの場合)、酵素(EIAの場合)、化学発光物質(CLIAの場合)等が用いられている。
しかし、上記コロイド状粒子は、粒子径及び調製条件によって色調が決定されてしまうため、所望の鮮明な濃い色調のものを得難い、つまり視認性が不十分であるという問題がある。
また、上記着色したラテックス粒子は、色素による着色の効果が低く、目視判定性が不十分であるという問題がある。なお、この問題を解消するために色素の着色量を増やそうとすると、色素がラテックスの表面を覆い、ラテックス粒子本来の表面状態が損なわれるため、抗原又は抗体を結合させるのが困難になるという問題があった。また、メンブランフィルター等のクロマトグラフ媒体の細孔内に詰まったり、ラテックス粒子が非特異凝集を起こしたりして、色素の着色量を増やすことにより濃く着色することが、必ずしも、性能の向上に結び付かない、という問題もあった。
(A)前記樹脂-金属複合体の平均粒子径が300nmを超える;
又は、
(B)前記金属粒子の平均粒子径が20nmを超え70nm未満の範囲内である;
を備えていることを特徴とする。
本発明のアナライトの測定方法は、メンブレン、及び当該メンブレンに前記アナライトと特異的に結合する捕捉リガンドが固定されてなる判定部を含むラテラルフロー型クロマト用テストストリップを用いる。
そして、本発明のアナライトの測定方法は、下記工程(I)~(III);
工程(I):試料に含まれる前記アナライトと、該アナライトに特異的に結合する抗体を、上記いずれかの標識物質で標識した標識抗体と、を接触させる工程、
工程(II):前記判定部にて、工程(I)において形成された、アナライトと標識抗体とを含む複合体を、捕捉リガンドに接触させる工程、
工程(III):前記標識物質における前記樹脂-金属複合体の局在型表面プラズモン共鳴に由来する発色強度を測定する工程、
を含む工程を行うことを特徴とする。
本発明のアナライト測定用キットは、メンブレン、及び当該メンブレンに、前記アナライトと特異的に結合する捕捉リガンドが固定されてなる判定部を含むラテラルフロー型クロマト用テストストリップと、
前記アナライトに特異的に結合する抗体を、請求項1~8のいずれか1項に記載の標識物質で標識した標識抗体を含む検出試薬と、
を含むものである。
本発明のラテラルフロー型クロマト用テストストリップは、
メンブレンと、
前記メンブレンに、前記試料が展開する方向において、前記アナライトと特異的に結合する捕捉リガンドが固定されてなる判定部と、
当該判定部よりも上流側に、前記アナライトに特異的に結合する抗体を請求項1~8のいずれか1項に記載の標識物質で標識した標識抗体が含まれる反応部と、
を含むものである。
本発明の第1の実施の形態に係る標識物質は、樹脂粒子に金属粒子が固定化された構造を有する樹脂-金属複合体を備えた標識物質であって、前記樹脂-金属複合体の平均粒子径が300nmを超えるものである。図1は、本実施の形態に係る標識物質を構成する樹脂-金属複合体の断面模式図である。樹脂-金属複合体100は、樹脂粒子10と、金属粒子20と、を備えている。樹脂-金属複合体100を有する本実施の形態の標識物質は、例えば免疫学的測定用試薬又はその材料として好ましく用いることができる。
一方、金属イオンを吸着することが可能な置換基を構造に有する含窒素ポリマー以外の樹脂粒子、例えばポリスチレン等の場合、前記金属イオンを樹脂内部に吸着しにくい。その結果、生成した金属粒子20の大部分は、表面吸着金属粒子50となる。上記のとおり、表面吸着金属粒子50は、樹脂粒子10との接触面積が小さいため、樹脂と金属の接着力が小さく、樹脂粒子10から金属粒子20が脱離する影響が大きい傾向にある。
上記含窒素ポリマーは、主鎖または側鎖に窒素原子を有する樹脂であり、例えば、ポリアミン、ポリアミド、ポリペプチド、ポリウレタン、ポリ尿素、ポリイミド、ポリイミダゾール、ポリオキサゾール、ポリピロール、ポリアニリン等がある。これらの中でも、好ましくは、ポリ-2-ビニルピリジン、ポリ-3-ビニルピリジン、ポリ-4-ビニルピリジン等のポリアミンである。また、側鎖に窒素原子を有する場合は、例えば、アクリル樹脂、フェノール樹脂、エポキシ樹脂等幅広く利用することが可能である。
本発明の第2の実施の形態の標識物質は、樹脂粒子に金属粒子が固定化された構造を有する樹脂-金属複合体を備えた標識物質であって、前記金属粒子の平均粒子径が20nmを超え70nm未満の範囲内である。本実施の形態に係る標識物質を構成する樹脂-金属複合体は、金属粒子の平均粒子径の範囲、及び樹脂-金属複合体の平均粒子径の範囲が異なる以外は、第1の実施の形態(図1)の樹脂-金属複合体100と同様である。以下、図1も参照しながら、第1の実施の形態との相違点を中心に説明する。
上記第1及び第2の実施の形態の標識物質に使用される樹脂-金属複合体100の製造方法は、特に限定されない。例えば、乳化重合法により製造した樹脂粒子10の分散液に、金属イオンを含有する溶液を加えて、金属イオンを樹脂粒子10に吸着させる(以下、「金属イオン吸着樹脂粒子」という。)。さらに、金属イオン吸着樹脂粒子を還元剤溶液中に加えることで、金属イオンを還元して金属粒子20を生成させ、樹脂-金属複合体100を得る。
また、金属イオンを含有する溶液の溶媒として、水の代わりに、メタノール、エタノール、n-プロパノール、イソプロパノール、n-ブタノール、sec-ブタノール、t-ブタノール等の含水アルコール又はアルコール、塩酸、硫酸、硝酸等の酸等を用いても良い。
また、前記溶液に、必要に応じて、例えば、ポリビニルアルコール等の水溶性高分子化合物、界面活性剤、アルコール類;テトラヒドロフラン、ジエチルエーテル、ジイソプロピルエーテル等のエーテル類;アルキレングリコール、ポリアルキレングリコール、これらのモノアルキルエーテル又はジアルキルエーテル、グリセリン等のポリオール類;アセトン、メチルエチルケトン等のケトン類等の各種水混和性有機溶媒等の添加剤を添加してもよい。このような添加剤は、金属イオンの還元反応速度を促進し、また生成される金属粒子20の大きさを制御するのに有効となる。
さらに還元剤溶液の温度により、金属イオンの還元速度を調整することで、形成する金属粒子の粒径をコントロールすることが出来る。
さらにホウ酸やリン酸等の緩衝剤、塩酸や硫酸などの酸、水酸化ナトリウムや水酸化カリウムなどのアルカリによりpHを調整し、分散性を保持することが出来る。
まず、図2を参照しながら、本発明の一実施の形態に係るラテラルフロー型クロマト用テストストリップ(テストストリップ)について説明する。このテストストリップ200は、後述するように、本発明の一実施の形態のアナライトの測定方法に好ましく使用できるものである。
テストストリップ200に使用されるメンブレン110としては、一般的なテストストリップにおいてメンブレン材料として使用されるものを適用可能である。メンブレン110は、例えば毛管現象を示し、試料を添加すると同時に、試料が展開するような微細多孔性物質からなる不活性物質(アナライト160、各種リガンドなどと反応しない物質)で形成されているものである。メンブレン110の具体例としては、ポリウレタン、ポリエステル、ポリエチレン、ポリ塩化ビニル、ポリフッ化ビニリデン、ナイロン、セルロース誘導体等で構成される繊維状又は不織繊維状マトリクス、膜、濾紙、ガラス繊維濾紙、布、綿等が挙げられる。これらの中でも、好ましくはセルロース誘導体やナイロンで構成される膜、濾紙、ガラス繊維濾紙等が用いられ、より好ましくはニトロセルロース膜、混合ニトロセルロースエステル(ニトロセルロースと酢酸セルロースの混合物)膜、ナイロン膜、濾紙が用いられる。
テストストリップ200は、アナライト160を含む試料を添加するための試料添加部120を有していてもよい。試料添加部120は、テストストリップ200に、アナライト160を含む試料を受け入れるための部位である。試料添加部120は、試料が展開する方向において、判定部130よりも上流側のメンブレン110に形成されていてもよいし、あるいは、例えばセルロース濾紙、ガラス繊維、ポリウレタン、ポリアセテート、酢酸セルロース、ナイロン、綿布などの材料で構成された試料添加パッドがメンブレン110に設けられて試料添加部120を構成していてもよい。
判定部130には、アナライト160と特異的に結合する捕捉リガンド131が固定されている。捕捉リガンド131は、アナライト160と特異的な結合を形成するものであれば特に制限なく使用でき、例えばアナライト160に対する抗体などを好ましく用いることができる。捕捉リガンド131は、テストストリップ200に試料を提供した場合においても、判定部130から移動することないように不動化している。捕捉リガンド131は、物理的又は化学的な結合や吸着等によって、メンブレン110に直接的又は間接的に固定されていればよい。
吸液部140は、例えば、セルロ-ス濾紙、不織布、布、セルロースアセテート等の吸水性材料のパッドにより形成される。添加された試料の展開前線(フロントライン)が吸液部140に届いてからの試料の移動速度は、吸液部140の材質、大きさなどにより異なるものとなる。従って、吸液部140の材質、大きさなどの選定により、アナライト160の検出・定量に最適な速度を設定することができる。なお、吸液部140は任意の構成であり、省略してもよい。
図示は省略するが、テストストリップ200には、メンブレン110に、標識抗体150を含む反応部が形成されていてもよい。反応部は、試料が流れる方向において、判定部130よりも上流側に設けることができる。なお、図2における試料添加部120を反応部として利用してもよい。テストストリップ200が反応部を有する場合、アナライト160を含む試料を、反応部又は試料添加部120に供すると、反応部において、試料に含まれるアナライト160と標識抗体150とを接触させることができる。この場合、試料を、単に反応部又は試料添加部120に供することで、アナライト160と標識抗体150とを含む複合体170を形成させることができるので、いわゆる1ステップ型のイムノクロマトグラフが可能になる。
図示は省略するが、テストストリップ200は、メンブレン110に、試料が展開する方向において、標識抗体150と特異的に結合する捕捉リガンドが固定されてなるコントロール部が形成されていてもよい。判定部130とともに、コントロール部でも発色強度が測定されることにより、テストストリップ200に供した試料が展開して、反応部及び判定部130に到達し、検査が正常に行われたことを確認することができる。なお、コントロール部は、捕捉リガンド131の代わりに、標識抗体150と特異的に結合する別の種類の捕捉リガンドを用いることを除いては、上述の判定部130と同様にして作製され、同様の構成を採ることができる。
次に、テストストリップ200を用いて行われる本発明の一実施の形態のアナライト160の測定方法について説明する。
工程(I):試料に含まれる前記アナライト160と、該アナライト160に特異的に結合する抗体を、樹脂粒子10に複数の金属粒子20が固定化された構造を有する樹脂-金属複合体100で標識した標識抗体150と、を接触させる工程、
工程(II):判定部130にて、工程(I)において形成された、アナライト160と標識抗体150とを含む複合体を、捕捉リガンド131に接触させる工程、
工程(III):樹脂-金属複合体100の局在型表面プラズモン共鳴に由来する発色強度を測定する工程、
を含むことができる。
工程(I)は、試料に含まれるアナライト160を、標識抗体150に接触させる工程である。アナライト160と標識抗体150とを含む複合体170を形成する限り、接触の態様は特に限定されるものではない。例えば、テストストリップ200の試料添加部120又は反応部(図示省略)に試料を供し、当該反応部においてアナライト160を標識抗体150に接触させてもよいし、テストストリップ200に試料を供する前に、試料中のアナライト160を標識抗体150に接触させてもよい。
工程(II)は、テストストリップ200の判定部130において、工程(I)において形成された、アナライト160と標識抗体150とを含む複合体170を、捕捉リガンド131に接触させる。複合体170を、捕捉リガンド131に接触させると、捕捉リガンド131は、複合体170のアナライト160に特異的に結合する。その結果、複合体170が判定部130において捕捉される。
工程(III)は、樹脂-金属複合体100の局在型表面プラズモン共鳴に由来する発色強度を測定する工程である。上記工程(II)又は必要に応じて洗浄工程を実施した後、テストストリップ200において、樹脂-金属複合体100の局在型表面プラズモン共鳴に由来する発色強度を測定する。
本実施の形態のアナライトの測定方法における試料は、アナライト160として、蛋白質などの抗原となり得る物質を含むものである限り特に限定されるものではない。例えば、目的のアナライト160を含む生体試料(すなわち、全血、血清、血漿、尿、唾液、喀痰、鼻腔又は咽頭拭い液、髄液、羊水、乳頭分泌液、涙、汗、皮膚からの浸出液、組織や細胞及び便からの抽出液等)や食品の抽出液等が挙げられる。必要に応じて、標識抗体150及び捕捉リガンド131とアナライト160との特異的な結合反応が生じやすくするために、上記工程(I)に先立って、試料に含まれるアナライト160を前処理してもよい。ここで、前処理としては、酸、塩基、界面活性剤等の各種化学薬品等を用いた化学的処理や、加熱・撹拌・超音波等を用いた物理的処理が挙げられる。特に、アナライト160がインフルエンザウィルスNP抗原等の、通常は表面に露出していない物質である場合、界面活性剤等による処理を行うことが好ましい。この目的に使用される界面活性剤として、特異的な結合反応、例えば、抗原抗体反応等のリガンドとアナライト160との結合反応性を考慮して、非イオン性界面活性剤を用いることができる。
標識抗体150は、工程(I)において、試料に含まれるアナライト160に接触させて、アナライト160と標識抗体150とを含む複合体170を形成するために使用される。標識抗体150は、アナライト160に特異的に結合する抗体を、樹脂粒子10に複数の金属粒子20が固定化された構造を有する樹脂-金属複合体100で標識化してなるものである。ここで、「標識化」とは、工程(I)~(III)において、標識抗体150から樹脂-金属複合体100が脱離しない程度に、抗体に樹脂-金属複合体100が直接的に又は間接的に、化学的又は物理的な結合や吸着等で固定されていることを意味する。例えば、標識抗体150は、抗体に樹脂-金属複合体100が直接結合してなるものであってもよいし、抗体と樹脂-金属複合体100とが、任意のリンカー分子を介して結合してなるものや、それぞれが不溶性粒子に固定されてなるものであってもよい。
次に、標識抗体150の好ましい作製方法を挙げて説明する。標識抗体150の製造は、少なくとも、次の工程A;
工程A)樹脂-金属複合体100を第1のpH条件で抗体と混合して結合させることによって、標識抗体150を得る工程
を含み、好ましくは、さらに工程B;
工程B)標識抗体150を第2のpH条件で処理する工程
を含むことができる。
工程Aでは、樹脂-金属複合体100を第1のpH条件で抗体と混合して標識抗体150を得る。工程Aは、固体状の樹脂-金属複合体100を液相中に分散させた状態で抗体と接触させることが好ましい。第1のpH条件は、樹脂-金属複合体100における金属粒子20の金属種によって異なる。
工程Bでは、工程Aで得られた標識抗体150を第2のpH条件で処理することによって、標識抗体150への非特異的な吸着を抑制するブロッキングを行う。この場合、固液分離手段によって分取しておいた標識抗体150を、第2のpH条件で液相中に分散させる。このブロッキングの条件は、樹脂-金属複合体100における金属粒子20の金属種によって異なる。
洗浄処理は、固液分離手段によって分取した標識抗体150に洗浄用緩衝液を添加し、洗浄用緩衝液中で標識抗体150を均一に分散させる。分散には、例えば超音波処理などの分散手段を用いることが好ましい。洗浄用緩衝液としては、特に限定されるものではないが、例えばpH8~9の範囲内に調整した所定濃度の、トリス(Tris)緩衝液、グリシンアミド緩衝液、アルギニン緩衝液などを用いることができる。洗浄用緩衝液のpHの調整は、例えば塩酸、水酸化ナトリウムなどを用いて行うことができる。標識抗体150の洗浄処理は、必要に応じて複数回を繰り返し行うことができる。
保存処理は、固液分離手段によって分取した標識抗体150に保存用緩衝液を添加し、保存用緩衝液中で標識抗体150を均一に分散させる。分散には、例えば超音波処理などの分散手段を用いることが好ましい。保存用緩衝液としては、例えば、洗浄用緩衝液に、所定濃度の凝集防止剤及び/又は安定剤を添加した溶液などを用いることができる。凝集防止剤としては、例えば、スクロース、マルトース、ラクトース、トレハロースに代表される糖類や、グリセリン、ポリビニルアルコールに代表される多価アルコールなどを用いることができる。安定剤としては、特に限定されるものではないが、例えば牛血清アルブミン、卵白アルブミン、カゼイン、ゼラチンなどの蛋白質を用いることができる。このようにして標識抗体150の保存処理を行うことができる。
本発明の一実施の形態に係るアナライト測定用キットは、例えばラテラルフロー型クロマト用テストストリップ200を用いて、本実施の形態のアナライトの測定方法に基づき、試料中に含まれるアナライト160の検出又は定量するためのキットである。
メンブレン110と
メンブレン110に、前記アナライト160と特異的に結合する捕捉リガンドが固定されてなる判定部130を含むラテラルフロー型クロマト用テストストリップ200と、
アナライト160に特異的に結合する抗体を樹脂粒子10に複数の金属粒子20が固定化された構造を有する樹脂-金属複合体100で標識した標識抗体150を含む検出試薬と、
を含んでいる。本実施の形態のキットは、必要に応じて、さらにその他の構成要素を含むものであってもよい。
樹脂-金属複合体の吸光度は、光学用白板ガラス製セル(光路長10mm)に0.01wt%に調製した樹脂-金属複合体分散液(分散媒:水)を入れ、瞬間マルチ測光システム(大塚電子社製、MCPD-3700)を用いて、金の場合570nmの吸光度を測定した。金の場合570nmでの吸光度が0.9以上を○(良好)、0.5~0.9未満を△(可)、0.5未満を×(不可)とした。
磁製るつぼに濃度調整前の分散液1gを入れ、70℃、3時間熱処理を行った。熱処理前後の重量を測定し、下記式により固形分濃度を算出した。
金属担持量(wt%)=
[500℃加熱処理後の重量(g)/500℃加熱処理前の重量(g)]×100
ディスク遠心式粒度分布測定装置(CPS Disc Centrifuge DC24000 UHR、CPS instruments, Inc.社製)を用いて測定した。測定は、樹脂-金属複合体を水に分散させた状態で行った。
各実施例等で作製した樹脂-金属複合体標識抗体分散液を用いて、下記に示すイムノクロマト法での測定を行って樹脂-金属複合体分散液の性能を評価した。
(評価方法)
評価は、インフルエンザA型評価用モノクロスクリーン(アドテック社製)を用い、5分後、10分後、15分後の発色レベルを比較した。性能評価において、抗原はインフルエンザA型陽性コントロール(APC)の2倍希釈列(1倍~1024倍)を用いた(APC希釈前のウィルスの濃度は5000FFU/ml)。
(評価手順)
96ウェルプレートの各ウェルに、樹脂-金属複合体標識抗体分散液を3μlずつ入れ、APCの2倍希釈列(1倍~1024倍)及び陰性コントロールを、それぞれ100μlを混和した。次に、インフルエンザA型評価用モノクロスクリーンに50μl添加し、5分後、10分後、15分後の発色レベルを評価した。発色レベルは金コロイド判定用色見本(アドテック社製)を用いて判定した。
金属粒子の平均粒子径の測定は、樹脂-金属複合体分散液をカーボン支持膜付き金属性メッシュへ滴下して作製した基板を、電界放出形走査電子顕微鏡(STEM;日立ハイテクノロジーズ社製、SU-9000)により観測した画像から、金属粒子の面積平均径を測定した。
<樹脂粒子の合成>
Aliquat 336[アルドリッチ社製](1.00g)及びポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、2.00g)を80gの純水に溶解した後、2-ビニルピリジン(2-VP、9.90g)及びジビニルベンゼン(DVB、0.100g)を加え、窒素気流下において250rpm、60℃で30分間撹拌した。撹拌後、9.00gの純水に溶解した2,2-アゾビス(2-メチルプロピオンアミジン)二塩酸塩(AIBA、0.100g)を5分かけて滴下し、250rpm、60℃で6時間撹拌することで、平均粒子径0.45μmの樹脂粒子を得た。この樹脂粒子を遠心分離(9000rpm、10分)により沈殿させ、上澄みを除去した後、純水に再度分散させ、10wt%の樹脂粒子分散液を得た。
前記樹脂粒子分散液(3.05g)に30mM塩化金酸水溶液(80g)を加え、室温で24時間放置した。その後、遠心分離(3000rpm、10分)により樹脂粒子を沈殿させ、上澄みを除去することで余分な塩化金酸を除去した後、55gの純水に再度分散させ、金イオン吸着樹脂粒子分散液を調製した。この金イオン吸着樹脂粒子分散液45g)を10mMのジメチルアミンボラン水溶液(450ml)に8分かけて滴下した後、室温で2時間撹拌することで、平均粒子径0.6μmの樹脂-金複合体を得た。この樹脂-金複合体を遠心分離(3000rpm、120分)により沈殿させ、上澄みを除去した後、37gの純水に再度分散させ、限外濾過膜により精製することで、1wt%の樹脂-金複合体分散液を得た。この樹脂-金複合体分散液中の樹脂-金複合体の吸光度は上記方法に従って測定した結果、1.20であった。また、樹脂-金複合体における金粒子の平均粒子径は22.0nm、金の担持量は49.4wt%であった。作製した樹脂-金複合体の走査型電子顕微鏡(SEM)写真を図3に示した。
実施例1で得た樹脂粒子分散液(3.05g)に10mMの塩化金酸水溶液(56g)を加えた他は、実施例1と同様の方法で、金イオン吸着樹脂粒子分散液、平均粒子径0.6μmの樹脂-金複合体及び1wt%の樹脂-金複合体分散液を得た。この樹脂-金複合体分散液中の樹脂-金複合体の吸光度は1.04であった。また、樹脂-金複合体における金粒子の平均粒子径は7.61nm、金の担持量は36.8wt%であった。
<樹脂粒子の合成>
2-VP(9.90g)及びDVB(0.100g)を450gの純水に加え、窒素気流下において250rpm、60℃で30分間撹拌した。30分撹拌した後、9.00gの純水に溶解したAIBA(0.100g)を5分かけて滴下し、250rpmで6時間撹拌することで平均粒子径0.10μmの樹脂粒子を得た。この樹脂粒子を遠心分離(9000rpm、20分)により沈殿させ、上澄みを除去した後、純水に再度分散させ、10wt%の樹脂粒子分散液を得た。
<樹脂-金属複合体の合成>
樹脂粒子分散液(5.0g)に30mMの塩化金酸水溶液(198g)を加え、室温で24時間放置した。その後、遠心分離(3000rpm、10分)により樹脂粒子を沈殿させ、上澄みを除去することで余分な塩化金酸を除去した後、1.5gの純水に再度分散させ、金イオン吸着樹脂粒子分散液を調製した。この金イオン吸着樹脂粒子分散液(1.5g)を10mMのジメチルアミンボラン水溶液(65ml)に2分かけて滴下した後、室温で2時間撹拌することで、平均粒子径0.22μmの樹脂-金複合体を得た。この樹脂-金複合体に10wt%の分散剤(BYK194)600μlを加え1時間撹拌した後、遠心分離(9000rpm、10分)により沈殿させ、上澄みを除去した。その後、適量の純水を加え再度分散させ、限外濾過膜により精製し、1wt%の樹脂-金複合体分散液を得た。この樹脂-金複合体分散液中の樹脂-金複合体の吸光度は1.12であった。また、樹脂-金複合体における金粒子の平均粒子径は22.6nm、金の担持量は37.0wt%であった。
<イムノクロマトの評価>
着色ラテックス(メルクミリポア社製、着色Estapor機能性粒子、K1030、平均粒子径;392nm、570nmでの吸光度は0.83、400nmでの吸光度は1.11)1ml(0.1wt%)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して着色ラテックスに抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して着色ラテックスをブロックした。12000rpm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して着色ラテックス標識抗体を作製した。
作製した着色ラテックス標識抗体を用いて、下記に示すイムノクロマト法での測定を行って着色ラテックスの性能を評価した。
(評価方法)
評価は、インフルエンザA型評価用モノクロスクリーン(アドテック社製)を用い、5分後、10分後、15分後の発色レベルを比較した。性能評価において、抗原はインフルエンザA型陽性コントロール(APC)の2倍希釈列(1倍~1024倍)を用いた(APC希釈前のウィルスの濃度は5000FFU/ml)。
(評価手順)
96ウェルプレートの各ウェルに、着色ラテックス標識抗体を3μlずつ入れ、APCの2倍希釈列(1倍~1024倍)及び陰性コントロールを、それぞれ100μlを混和した。次に、インフルエンザA型評価用モノクロスクリーンに50μl添加し、5分後、10分後、15分後の発色レベルを評価した。その結果を以下に示した。
〈金コロイドの合成〉
500ml三つ口丸底フラスコに1mM 塩化金酸水溶液を250ml入れ、加熱還流装置を用い、激しく攪拌しながら沸騰させ、沸騰後38.8mMクエン酸ナトリウム水溶液を25ml添加し、溶液が淡黄色から濃紅色に変化することを確認した。攪拌しながら10分間加熱を続けた後、室温で30分程度攪拌放冷をおこなった。孔径2μmのメンブランフィルターを用いて溶液をろ過し、三角フラスコに移し冷暗所で保存した。作製した粒子の平均粒径は12.3nmであった。
得られた金コロイド1ml(OD=10)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して金コロイドに抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して金コロイド表面をブロックした。12000rpm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して金コロイド標識抗体を作製した。
作製した金コロイド標識抗体を用いて、下記に示すイムノクロマト法での測定を行って金コロイドの性能を評価した。
(評価方法)
評価は、インフルエンザA型評価用モノクロスクリーン(アドテック社製)を用い、5分後、10分後、15分後の発色レベルを比較した。性能評価において、抗原はインフルエンザA型陽性コントロール(APC)の2倍希釈列(1倍~1024倍)を用いた(APC希釈前のウィルスの濃度は5000FFU/ml)。
(評価手順)
96ウェルプレートの各ウェルに、金コロイド標識抗体を3μlずつ入れ、APCの2倍希釈列(1倍~1024倍)及び陰性コントロールを、それぞれ100μlを混和した。次に、インフルエンザA型評価用モノクロスクリーンに50μl添加し、5分後、10分後、15分後の発色レベルを評価した。その結果を以下に示した。
<樹脂粒子の合成>
Aliquat 336[アルドリッチ社製](5.00g)及びポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、10.00g)を389.5gの純水に溶解した後、2-ビニルピリジン(2-VP、48.00g)及びジビニルベンゼン(DVB、2.00g)を加え、窒素気流下において150rpm、30℃で50分、次いで60℃で30分間撹拌した。撹拌後、50.00gの純水に溶解した2,2-アゾビス(2-メチルプロピオンアミジン)二塩酸塩(AIBA、0.500g)を2分かけて滴下し、150rpm、60℃で3.5時間撹拌することで、平均粒子径200nmの樹脂粒子を得た。遠心分離(9000rpm、60分)により沈殿させ、上澄みを除去した後、純水に再度分散させる操作を3回行った後、透析処理により不純物を除去した。その後、濃度調整を行い10wt%の樹脂粒子分散液を得た。
得られた樹脂―金複合体分散液1ml(0.1wt%)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して樹脂-金複合体に抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して樹脂-金複合体表面をブロックした。12000pm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して樹脂-金複合体標識抗体分散液を作製した。
作製した樹脂-金複合体標識抗体分散液を用いて、イムノクロマト法での測定を行って当該樹脂-金複合体分散液の性能を評価した。その結果を以下に示した。
Aliquat 336[アルドリッチ社製](1.00g)及びポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、10.00g)を300gの純水に溶解した後、2-ビニルピリジン(2-VP、48.00g)及びジビニルベンゼン(DVB、2.00g)を加え、窒素気流下において150rpm、30℃で50分、次いで60℃で30分間撹拌した。撹拌後、18.00gの純水に溶解した2,2-アゾビス(2-メチルプロピオンアミジン)二塩酸塩(AIBA、0.500g)を0.5分かけて滴下し、150rpm、60℃で3.5時間撹拌することで、平均粒子径500nmの樹脂粒子を得た。遠心分離(9000rpm、40分)により沈殿させ、上澄みを除去した後、純水に再度分散させる操作を3回行った後、透析処理により不純物を除去した。その後、濃度調整を行い10wt%の樹脂粒子分散液を得た。
得られた樹脂―金複合体分散液1ml(0.1wt%)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して樹脂-金複合体に抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して樹脂-金複合体表面をブロックした。12000rpm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して樹脂-金複合体標識抗体分散液を作製した。
作製した樹脂-金複合標識抗体分散液を用いて、イムノクロマト法での測定を行って当該樹脂-金複合体分散液の性能を評価した。その結果を以下に示した。
Aliquat 336[アルドリッチ社製](0.50g)及びポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、10.00g)を300gの純水に溶解した後、2-ビニルピリジン(2-VP、48.00g)及びジビニルベンゼン(DVB、2.00g)を加え、窒素気流下において150rpm、30℃で50分、次いで60℃で30分間撹拌した。撹拌後、18.00gの純水に溶解した2,2-アゾビス(2-メチルプロピオンアミジン)二塩酸塩(AIBA、0.500g)を0.5分かけて滴下し、150rpm、60℃で3.5時間撹拌することで、平均粒子径613nmの樹脂粒子を得た。遠心分離(9000rpm、40分)により沈殿させ、上澄みを除去した後、純水に再度分散させる操作を3回行った後、透析処理により不純物を除去した。その後、濃度調整を行い10wt%の樹脂粒子分散液を得た。
得られた樹脂―金複合体分散液1ml(0.1wt%)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して樹脂-金複合体に抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して樹脂-金複合体表面をブロックした。12000rpm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して樹脂-金複合体標識抗体分散液を作製した。
Aliquat 336[アルドリッチ社製](1.00g)及びポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、10.00g)を300gの純水に溶解した後、4-ビニルピリジン(4-VP、48.00g)及びジビニルベンゼン(DVB、2.00g)を加え、窒素気流下において150rpm、30℃で50分、次いで60℃で30分間撹拌した。撹拌後、18.00gの純水に溶解した2,2-アゾビス(2-メチルプロピオンアミジン)二塩酸塩(AIBA、0.500g)を2分かけて滴下し、150rpm、60℃で3.5時間撹拌することで、平均粒子径438nmの樹脂粒子を得た。遠心分離(9000rpm、45分)により沈殿させ、上澄みを除去した後、純水に再度分散させる操作を3回行った後、透析処理により不純物を除去した。その後、濃度調整を行い10wt%の樹脂粒子分散液を得た。
得られた樹脂―金複合体分散液1ml(0.1wt%)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して樹脂-金複合体に抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して樹脂-金複合体表面をブロックした。12000rpm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して樹脂-金複合体標識抗体分散液を作製した。
Aliquat 336[アルドリッチ社製](1.00g)及びポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、10.00g)を300gの純水に溶解した後、3-ビニルピリジン(3-VP、48.00g)及びジビニルベンゼン(DVB、2.00g)を加え、窒素気流下において150rpm、30℃で50分、次いで60℃で30分間撹拌した。撹拌後、18.00gの純水に溶解した2,2-アゾビス(2-メチルプロピオンアミジン)二塩酸塩(AIBA、0.500g)を2分かけて滴下し、150rpm、60℃で3.5時間撹拌することで、平均粒子径429nmの樹脂粒子を得た。遠心分離(9000rpm、45分)により沈殿させ、上澄みを除去した後、純水に再度分散させる操作を3回行った後、透析処理により不純物を除去した。その後、濃度調整を行い10wt%の樹脂粒子分散液を得た。
得られた樹脂―金複合体分散液1ml(0.1wt%)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して樹脂-金複合体に抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して樹脂-金複合体表面をブロックした。12000rpm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して樹脂-金複合体標識抗体分散液を作製した。
2-(ジイソプロピルアミノ)エチルメタクリレート(DPA、10.3g)、ポリ(プロピレングリコール)ジアクリレート(0.2g)とポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、2.0g)を85gの純水に溶解した後、窒素気流下において150rpm、30℃で50分、次いで70℃で30分間撹拌した。撹拌後、2.00gの純水に溶解したペルオキソ二硫酸アンモニウム(ASP、0.10g)を2分かけて滴下し、150rpm、70℃で3.5時間撹拌することで、平均粒子径338nmの樹脂粒子を得た。遠心分離(9000rpm、45分)により沈殿させ、上澄みを除去した後、純水に再度分散させる操作を3回行った後、透析処理により不純物を除去した。その後、濃度調整を行い10wt%の樹脂粒子分散液を得た。
得られた樹脂―金複合体分散液1ml(0.1wt%)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して樹脂-金複合体に抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して樹脂-金複合体表面をブロックした。12000rpm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して樹脂-金複合体標識抗体分散液を作製した。
<樹脂粒子の合成>
Aliquat 336[アルドリッチ社製](3.00g)及びポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、10.00g)を300gの純水に溶解した後、2-ビニルピリジン(2-VP、49.50g)及びジビニルベンゼン(DVB、0.50g)を加え、窒素気流下において150rpm、30℃で50分、次いで60℃で30分間撹拌した。撹拌後、18.00gの純水に溶解した2,2-アゾビス(2-メチルプロピオンアミジン)二塩酸塩(AIBA、0.250g)を2分かけて滴下し、150rpm、60℃で3.5時間撹拌することで、平均粒子径370nmの樹脂粒子を得た。遠心分離(9000rpm、60分)により沈殿させ、上澄みを除去した後、純水に再度分散させる操作を3回行った後、透析処理により不純物を除去した。その後、濃度調整を行い10wt%の樹脂粒子分散液を得た。
得られた樹脂―金複合体分散液1ml(0.1wt%)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して樹脂-金複合体に抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して樹脂-金複合体表面をブロックした。12000rpm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して樹脂-金複合体標識抗体分散液を作製した。
<樹脂粒子の合成>
Aliquat 336[アルドリッチ社製](2.00g)及びポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、10.00g)を300gの純水に溶解した後、2-ビニルピリジン(2-VP、48.00g)及びジビニルベンゼン(DVB、2.00g)を加え、窒素気流下において150rpm、30℃で50分、次いで60℃で30分間撹拌した。撹拌後、18.00gの純水に溶解した2,2-アゾビス(2-メチルプロピオンアミジン)二塩酸塩(AIBA、0.500g)を2分かけて滴下し、150rpm、60℃で3.5時間撹拌することで、平均粒子径380nmの樹脂粒子を得た。遠心分離(9000rpm、60分)により沈殿させ、上澄みを除去した後、純水に再度分散させる操作を3回行った後、透析処理により不純物を除去した。その後、濃度調整を行い10wt%の樹脂粒子分散液を得た。
得られた樹脂―金複合体分散液1ml(0.1wt%)にインフルエンザ抗体を100μg混合し、室温で約3時間攪拌して樹脂-金複合体に抗体を結合させた。終濃度が1%となるように牛血清アルブミン溶液を添加し、室温にて2時間攪拌して樹脂-金複合体表面をブロックした。12000rpm、4℃で5分間遠心分離を行って回収し、0.2%牛血清アルブミンを含む緩衝液に懸濁して樹脂-金複合体標識抗体分散液を作製した。
<樹脂粒子の合成>
Aliquat 336[アルドリッチ社製](1.00g)及びポリエチレングリコールメチルエチルエーテルメタクリレート(PEGMA、2.00g)を80gの純水に溶解した後、2-ビニルピリジン(2-VP、9.90g)及びジビニルベンゼン(DVB、0.100g)を加え、窒素気流下において250rpm、60℃で30分間撹拌した。撹拌後、9.00gの純水に溶解した2,2-アゾビス(2-メチルプロピオンアミジン)二塩酸塩(AIBA、0.100g)を5分かけて滴下し、250rpm、60℃で6時間撹拌することで、平均粒子径0.36μmの樹脂粒子A-1を得た。前記A-1を遠心分離(9000rpm、10分)により沈殿させ、上澄みを除去した後、純水に再度分散させ、2.1wt%の樹脂粒子分散液B-1を得た。
前記B-1(19.09g)に30mM塩化金酸水溶液(106.6g)を加え、室温で24時間放置した。その後、遠心分離(3000rpm、10分)により樹脂粒子を沈殿させ、上澄みを除去することで余分な塩化金酸を除去した後、40gの純水に再度分散させ、金イオン吸着樹脂粒子分散液C-1を調製した。前記C-1(20g)を3.3mMのジメチルアミンボラン水溶液(600ml)に4分かけて滴下した後、8℃で1時間撹拌し、さらに室温で5時間撹拌することで、平均粒子径0.38μmの樹脂-金複合体D-1を得た。前記D-1を遠心分離(3000rpm、120分)により沈殿させ、上澄みを除去した後、適量の純水を加えて再度分散させ、限外濾過膜により精製することで、1wt%の樹脂-金複合体分散液E-1を得た。E-1中の樹脂-金複合体F-1の吸光度は上記方法に従って測定した結果、1.0であった。また、F-1における金粒子の平均粒子径は22.0nm、金の担持量は49.1wt%であった。
試験例、参考試験例では以下の試薬等を使用した。
抗インフルエンザA型モノクローナル抗体(7.15mg/mL/PBS):アドテック株式会社製
結合用緩衝液a:100mM ホウ酸溶液をHClでpH≒3に調整した。
結合用緩衝液b:100mM ホウ酸溶液をHClでpH≒4に調整した。
結合用緩衝液c:100mM ホウ酸溶液をHClでpH≒5に調整した。
結合用緩衝液d:100mM ホウ酸溶液 pH≒6.5
結合用緩衝液e:100mM ホウ酸溶液をNaClでpH≒7.5に調整した。
結合用緩衝液f:100mM ホウ酸溶液をNaClでpH≒8.5に調整した。
結合用緩衝液g:50mM 2-モルフォリノエタンスルホン酸溶液 pH≒3.8
ブロック用緩衝液a:1重量%牛血清アルブミン溶液をHClでpH≒5に調整した。
ブロック用緩衝液b:1重量%牛血清アルブミン溶液をHClでpH≒7に調整した。
ブロック用緩衝液c:1重量%牛血清アルブミン溶液をHClでpH≒8.5に調整した。
ブロック用緩衝液d:1重量%牛血清アルブミン溶液をHClでpH≒9.5に調整した。
洗浄用緩衝液:5mMトリス溶液をHClでpH≒8.5に調整した。
保存用緩衝液:洗浄用緩衝液に、スクロースを10重量%濃度になるように添加した。
インフルエンザA型陽性コントロール(APC):インフルエンザA型ウィルス不活化抗原(アドテック株式会社製)を、検体処理液(アドテック株式会社製)を用いて100倍希釈して調製した。APCの抗原濃度は、5000FFU/mlに相当する。
陰性コントロール:検体処理液(アドテック株式会社製)
AuNCPビーズ:作製例1で得た樹脂-金複合体(1重量%;平均粒子径380nm)
マイクロチューブ[アイビス(登録商標;アズワン社製)2mL]に、樹脂-金属複合体としてAuNCPビーズ0.1mLを投入し、結合用緩衝液a0.9mLを添加した。転倒混和によって十分に混合した後、抗インフルエンザA型モノクローナル抗体100μgを添加し、室温で3時間かけて転倒撹拌を行い、樹脂-金属複合体で標識した抗インフルエンザA型モノクローナル抗体を含む標識抗体含有液A-1を得た。
次に、標識抗体含有液A-1を氷冷後、12000rpmで5分間かけて遠心分離を行い、上澄みを除去した後、固形分残渣にブロック用緩衝液a1mLを添加し、10~20秒間かけて超音波分散処理を行い、さらに、室温で2時間かけて転倒撹拌を行い、標識抗体含有液B-1を得た。
次に、標識抗体含有液B-1を氷冷後、12000rpmで5分間かけて遠心分離を行い、上澄みを除去した後、固形分残渣に洗浄用緩衝液1mLを添加し、10~20秒間かけて超音波分散処理を行った。この操作を3回繰り返し、洗浄処理とした。
次に、氷冷後、12000rpmで5分間かけて遠心分離を行い、上澄みを除去した後、固形分残渣に保存用緩衝液1mLを添加し、10~20秒間かけて超音波分散処理を行うことによって、標識抗体含有液C-1を得た。
試験例1の結合工程で結合用緩衝液aの代わりに結合用緩衝液bを用いる以外は試験例1と同様にして、標識抗体含有液A-2,B-2、C-2を得た。
試験例1の結合工程で結合用緩衝液aの代わりに結合用緩衝液cを用いる以外は試験例1と同様にして、標識抗体含有液A-3,B-3,C-3を得た。
試験例1の結合工程で結合用緩衝液aの代わりに結合用緩衝液dを用いる以外は試験例1と同様にして、標識抗体含有液A-4,B-4、C-4を得た。
試験例1の結合工程で結合用緩衝液aの代わりに結合用緩衝液eを用いた場合、樹脂-金属複合体が凝集してしまうため、標識抗体含有液を得ることが困難であった。
試験例1の結合工程で結合用緩衝液aの代わりに結合用緩衝液fを用いた場合、樹脂-金属複合体が凝集してしまうため、標識抗体含有液を得ることが困難であった。
試験例1のブロック工程でブロック用緩衝液aの代わりにブロック用緩衝液bを用いる以外は試験例1と同様にして、標識抗体含有液B-5、C-5を得た。
試験例1のブロック工程でブロック用緩衝液aの代わりにブロック用緩衝液cを用いる以外は試験例1と同様にして、標識抗体含有液B-6、C-6を得た。
試験例1のブロック工程でブロック用緩衝液aの代わりにブロック用緩衝液dを用いたところ、結合工程後の標識抗体は良好な分散性を示したが、ブロック工程後に標識抗体が凝集してしまい、標識抗体含有液を得ることが困難であった。
試験例1の結合工程で結合用緩衝液aの代わりに結合用緩衝液gを用いる以外は試験例1と同様にして、標識抗体含有液A-7,B-7、C-7を得た。
評価は、インフルエンザA型評価用モノクロスクリーン(アドテック社製)を用い、5分後、10分後、15分後の発色レベルを比較した。発色レベルは金コロイド判定用色見本(アドテック社製)を用いて判定した。スクリーニング評価において、抗原はインフルエンザA型陽性コントロール(APC)を用いた。性能評価において、抗原はAPCの2倍希釈列(1倍~1024倍希釈)を用いた。
96ウェルプレートの7ウェルに、試験例1~7で得られた標識抗体含有液C-1~7を3μLずつ入れ、それぞれにAPC100μLを混和した。次に、インフルエンザA型評価用モノクロスクリーンに50μLずつ添加し、5分後、10分後、15分後の発色レベルを評価した。その結果を表11に示した。なお、表11における数値が大きい程、発色レベルが高い(発色が強い)ことを意味する。
96ウェルプレートの12ウェルに、試験例1で得られた標識抗体含有液C-1を3μLずつ入れ、APCの2倍希釈列(1倍~1024倍希釈、それぞれAPC×1~APC×1024と表す)及び陰性コントロールを、それぞれ100μLを混和した。次に、インフルエンザA型評価用モノクロスクリーンに50μL添加し、5分後、10分後、15分後の発色レベルを評価した。その結果を表12に示した。なお、表12における数値が大きい程、発色レベルが高い(発色が強い)ことを意味する。
Claims (13)
- 樹脂粒子に金属粒子が固定化された構造を有する樹脂-金属複合体を備えた標識物質であって、
以下の(A)、(B)のいずれかの構成:
(A)前記樹脂-金属複合体の平均粒子径が300nmを超える;
又は、
(B)前記金属粒子の平均粒子径が20nmを超え70nm未満の範囲内である;
を備えていることを特徴とする標識物質。 - 前記(A)のとき、前記金属粒子の平均粒子径が1nm以上80nm以下の範囲内であることを特徴とする、請求項1に記載の標識物質。
- 前記(A)のとき、前記樹脂-金属複合体の平均粒子径が300nmを超え1000nm以下の範囲内であることを特徴とする、請求項1または2に記載の標識物質。
- 前記金属粒子が、金粒子である請求項3に記載の標識物質。
- 前記(B)のとき、前記樹脂-金属複合体の平均粒子径が100nm以上1000nm以下の範囲内であることを特徴とする、請求項1に記載の標識物質。
- 前記金属粒子が、金粒子である請求項5に記載の標識物質。
- 前記樹脂-金属複合体を水に分散したことを特徴とする、請求項1~6のいずれか1項に記載の標識物質。
- 前記樹脂-金属複合体の表面に、抗原または抗体を吸着させて使用するものである、請求項1~7のいずれか1項に記載の標識物質。
- 請求項1~8のいずれか1項に記載の標識物質を用いることを特徴とする、免疫学的測定法。
- 請求項1~8のいずれか1項に記載の標識物質を備えた免疫学的測定用試薬。
- 試料中に含まれるアナライトを検出又は定量するアナライトの測定方法であって、
メンブレン、及び当該メンブレンに前記アナライトと特異的に結合する捕捉リガンドが固定されてなる判定部を含むラテラルフロー型クロマト用テストストリップを用い、下記工程(I)~(III);
工程(I):試料に含まれる前記アナライトと、該アナライトに特異的に結合する抗体を、請求項1~8のいずれか1項に記載の標識物質で標識した標識抗体と、を接触させる工程、
工程(II):前記判定部にて、工程(I)において形成された、アナライトと標識抗体とを含む複合体を、捕捉リガンドに接触させる工程、
工程(III):前記標識物質における前記樹脂-金属複合体の局在型表面プラズモン共鳴に由来する発色強度を測定する工程、
を含む工程を行うことを特徴とするアナライトの測定方法。 - ラテラルフロー型クロマト用テストストリップを用いて、試料中に含まれるアナライトを検出又は定量するためのアナライト測定用キットであって、
メンブレン、及び当該メンブレンに、前記アナライトと特異的に結合する捕捉リガンドが固定されてなる判定部を含むラテラルフロー型クロマト用テストストリップと、
前記アナライトに特異的に結合する抗体を、請求項1~8のいずれか1項に記載の標識物質で標識した標識抗体を含む検出試薬と、
を含むアナライトを検出又は定量するためのアナライト測定用キット。 - 試料中に含まれるアナライトを検出又は定量するためのラテラルフロー型クロマト用テストストリップであって、
メンブレンと、
前記メンブレンに、前記試料が展開する方向において、前記アナライトと特異的に結合する捕捉リガンドが固定されてなる判定部と、
当該判定部よりも上流側に、前記アナライトに特異的に結合する抗体を請求項1~8のいずれか1項に記載の標識物質で標識した標識抗体が含まれる反応部と、
を含むラテラルフロー型クロマト用テストストリップ。
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP15814054.1A EP3165923B1 (en) | 2014-07-01 | 2015-06-30 | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral- flow chromatographic test strip |
US15/323,102 US10690665B2 (en) | 2014-07-01 | 2015-06-30 | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
CN201580035738.4A CN106662585B (zh) | 2014-07-01 | 2015-06-30 | 标记物质及其在免疫学、分析物及测试条的应用 |
EP19213455.9A EP3644059A1 (en) | 2014-07-01 | 2015-06-30 | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
JP2016531373A JP6353534B2 (ja) | 2014-07-01 | 2015-06-30 | 標識物質、免疫学的測定法、免疫学的測定用試薬、アナライトの測定方法、アナライト測定用キット、及び、ラテラルフロー型クロマト用テストストリップ |
US16/840,449 US11733241B2 (en) | 2014-07-01 | 2020-04-05 | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014136356 | 2014-07-01 | ||
JP2014136357 | 2014-07-01 | ||
JP2014-136356 | 2014-07-01 | ||
JP2014-136357 | 2014-07-01 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/323,102 A-371-Of-International US10690665B2 (en) | 2014-07-01 | 2015-06-30 | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
US16/840,449 Division US11733241B2 (en) | 2014-07-01 | 2020-04-05 | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016002743A1 true WO2016002743A1 (ja) | 2016-01-07 |
Family
ID=55019275
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2015/068759 WO2016002743A1 (ja) | 2014-07-01 | 2015-06-30 | 標識物質、免疫学的測定法、免疫学的測定用試薬、アナライトの測定方法、アナライト測定用キット、及び、ラテラルフロー型クロマト用テストストリップ |
Country Status (6)
Country | Link |
---|---|
US (2) | US10690665B2 (ja) |
EP (2) | EP3644059A1 (ja) |
JP (3) | JP6353534B2 (ja) |
CN (1) | CN106662585B (ja) |
TW (2) | TWI733048B (ja) |
WO (1) | WO2016002743A1 (ja) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017181368A (ja) * | 2016-03-31 | 2017-10-05 | 新日鉄住金化学株式会社 | 免疫測定方法、免疫測定用キット及びラテラルフロー型クロマトテストストリップ |
JP2017223604A (ja) * | 2016-06-17 | 2017-12-21 | 新日鉄住金化学株式会社 | 標識抗体、その製造方法及び免疫学的測定法 |
WO2018123952A1 (ja) * | 2016-12-28 | 2018-07-05 | 新日鉄住金化学株式会社 | 金属-樹脂複合体及びその利用 |
JP2018169373A (ja) * | 2017-03-30 | 2018-11-01 | 新日鉄住金化学株式会社 | 免疫測定方法、免疫測定用キット及びラテラルフロー型クロマトテストストリップ |
JP2019066323A (ja) * | 2017-09-29 | 2019-04-25 | 日鉄ケミカル&マテリアル株式会社 | 標識抗体、その製造方法及び免疫学的測定法 |
WO2024085240A1 (ja) * | 2022-10-21 | 2024-04-25 | ユニ・チャーム株式会社 | おりもの用テストストリップを有する吸収性物品、及び、吸収性物品で用いるためのおりもの用テストストリップ |
WO2024085239A1 (ja) * | 2022-10-21 | 2024-04-25 | ユニ・チャーム株式会社 | おりもの用テストストリップ |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3165560B1 (en) * | 2014-07-01 | 2022-04-27 | NIPPON STEEL Chemical & Material Co., Ltd. | Resin-metal composite, marker, immunoassay method, immunoassay reagent, method for measuring analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
US10690665B2 (en) | 2014-07-01 | 2020-06-23 | Nippon Steel Chemical & Materials Co., Ltd. | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
WO2017010391A1 (ja) * | 2015-07-11 | 2017-01-19 | 新日鉄住金化学株式会社 | 樹脂-白金複合体及びその利用 |
US20230104609A1 (en) * | 2021-10-04 | 2023-04-06 | Tintoria Piana, Inc | Diagnostic system and methods of using and manufacturing the same |
CN114410751B (zh) * | 2021-12-30 | 2024-01-12 | 安徽科技学院 | 标记物核酸探针及其制备方法、试纸条、聚吡咯纳米粒子的用途 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006115716A (ja) * | 2004-10-19 | 2006-05-11 | Matsushita Electric Ind Co Ltd | 抗c反応性タンパク質モノクローナル抗体産生細胞ライン、その作製方法、およびそれにより産生されたモノクローナル抗体 |
JP2007225576A (ja) * | 2006-02-27 | 2007-09-06 | Canon Inc | 検出試薬、検出素子、および検出方法 |
JP2009210411A (ja) * | 2008-03-04 | 2009-09-17 | Toyama Univ | 皮膚カテプシンの分析方法、皮膚の光ストレスの判定方法およびそのためのキット |
JP2012242277A (ja) * | 2011-05-20 | 2012-12-10 | Rohm Co Ltd | マイクロチップ、ならびに、それを用いた測定システムおよび測定方法 |
JP2013522653A (ja) * | 2010-03-24 | 2013-06-13 | ノースイースタン ユニヴァーシティ | 早期癌病巣のための多重モード診断技術 |
Family Cites Families (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57189122A (en) | 1981-05-18 | 1982-11-20 | Olympus Optical Co Ltd | Film winding device for camera |
CA2026409C (en) | 1989-09-29 | 2004-11-09 | Ernest G. Schutt | Method of producing a reagent containing a narrow distribution of colloidal particles of a selected size and the use thereof |
JP2955405B2 (ja) | 1990-08-31 | 1999-10-04 | ジェイエスアール株式会社 | イムノクロマトグラフ法 |
US5710005A (en) * | 1996-10-29 | 1998-01-20 | Biocode, Inc. | Analyte detection with a gradient lateral flow device |
WO1999064864A1 (en) | 1998-06-12 | 1999-12-16 | New Horizons Diagnostics Inc. | Optimizing sensitivity in colloidal colorimetric flow through and lateral flow tests |
NZ511705A (en) * | 2001-05-14 | 2004-03-26 | Horticulture & Food Res Inst | Methods and rapid immunoassay device for detecting progesterone and other steroids |
JP4426819B2 (ja) | 2002-10-21 | 2010-03-03 | 積水化学工業株式会社 | 磁性体内包粒子の製造方法 |
KR101115903B1 (ko) | 2003-04-16 | 2012-02-13 | 세키스이가가쿠 고교가부시키가이샤 | 자성체 내포 입자 및 그의 제조 방법, 면역 측정용 입자,및 면역 측정 방법 |
EP1664772A4 (en) | 2003-08-04 | 2007-01-03 | Univ Emory | POROUS MATERIALS ENCLOSED WITH NANO-SPECIES |
JP5035622B2 (ja) | 2008-01-11 | 2012-09-26 | 積水化学工業株式会社 | 着色ラテックス |
JP2009192270A (ja) | 2008-02-12 | 2009-08-27 | Bl:Kk | 高感度免疫測定法 |
JP5132664B2 (ja) | 2009-12-07 | 2013-01-30 | 富士フイルム株式会社 | イムノクロマトグラフ方法 |
CN102782024B (zh) * | 2010-03-01 | 2015-04-22 | 新日铁住金化学株式会社 | 金属微粒复合体及其制造方法 |
CN102253202B (zh) | 2010-05-17 | 2014-05-21 | 北京库尔科技有限公司 | 苏云金芽孢杆菌检测试剂盒 |
WO2011148870A1 (ja) * | 2010-05-28 | 2011-12-01 | 国立大学法人東京工業大学 | 金属微粒子複合体及びその製造方法 |
KR20140043323A (ko) * | 2011-02-09 | 2014-04-09 | 신닛테츠 수미킨 가가쿠 가부시키가이샤 | 금속 미립자 분산 복합체 및 그 제조 방법, 그리고 국재형 표면 플라즈몬 공명 발생 기판 |
JP2014136357A (ja) | 2013-01-16 | 2014-07-28 | Fujitsu Ltd | 電子機器の筐体及び該筐体の製造方法 |
JP5802689B2 (ja) | 2013-01-16 | 2015-10-28 | 日精樹脂工業株式会社 | 二液用射出機 |
EP3165560B1 (en) * | 2014-07-01 | 2022-04-27 | NIPPON STEEL Chemical & Material Co., Ltd. | Resin-metal composite, marker, immunoassay method, immunoassay reagent, method for measuring analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
US10690665B2 (en) * | 2014-07-01 | 2020-06-23 | Nippon Steel Chemical & Materials Co., Ltd. | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
WO2017010391A1 (ja) * | 2015-07-11 | 2017-01-19 | 新日鉄住金化学株式会社 | 樹脂-白金複合体及びその利用 |
-
2015
- 2015-06-30 US US15/323,102 patent/US10690665B2/en active Active
- 2015-06-30 CN CN201580035738.4A patent/CN106662585B/zh active Active
- 2015-06-30 WO PCT/JP2015/068759 patent/WO2016002743A1/ja active Application Filing
- 2015-06-30 EP EP19213455.9A patent/EP3644059A1/en active Pending
- 2015-06-30 EP EP15814054.1A patent/EP3165923B1/en active Active
- 2015-06-30 JP JP2016531373A patent/JP6353534B2/ja active Active
- 2015-07-01 TW TW107128916A patent/TWI733048B/zh active
- 2015-07-01 TW TW104121256A patent/TWI636257B/zh active
-
2018
- 2018-06-08 JP JP2018110063A patent/JP6847077B2/ja active Active
-
2020
- 2020-04-05 US US16/840,449 patent/US11733241B2/en active Active
- 2020-08-07 JP JP2020134848A patent/JP2020177035A/ja active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006115716A (ja) * | 2004-10-19 | 2006-05-11 | Matsushita Electric Ind Co Ltd | 抗c反応性タンパク質モノクローナル抗体産生細胞ライン、その作製方法、およびそれにより産生されたモノクローナル抗体 |
JP2007225576A (ja) * | 2006-02-27 | 2007-09-06 | Canon Inc | 検出試薬、検出素子、および検出方法 |
JP2009210411A (ja) * | 2008-03-04 | 2009-09-17 | Toyama Univ | 皮膚カテプシンの分析方法、皮膚の光ストレスの判定方法およびそのためのキット |
JP2013522653A (ja) * | 2010-03-24 | 2013-06-13 | ノースイースタン ユニヴァーシティ | 早期癌病巣のための多重モード診断技術 |
JP2012242277A (ja) * | 2011-05-20 | 2012-12-10 | Rohm Co Ltd | マイクロチップ、ならびに、それを用いた測定システムおよび測定方法 |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017181368A (ja) * | 2016-03-31 | 2017-10-05 | 新日鉄住金化学株式会社 | 免疫測定方法、免疫測定用キット及びラテラルフロー型クロマトテストストリップ |
JP2017223604A (ja) * | 2016-06-17 | 2017-12-21 | 新日鉄住金化学株式会社 | 標識抗体、その製造方法及び免疫学的測定法 |
JP7265357B2 (ja) | 2016-12-28 | 2023-04-26 | 日鉄ケミカル&マテリアル株式会社 | 金属-樹脂複合体及びその利用 |
CN110140053A (zh) * | 2016-12-28 | 2019-08-16 | 日铁化学材料株式会社 | 金属-树脂复合体及其应用 |
KR20190101992A (ko) * | 2016-12-28 | 2019-09-02 | 닛테츠 케미컬 앤드 머티리얼 가부시키가이샤 | 금속-수지 복합체 및 그 이용 |
JPWO2018123952A1 (ja) * | 2016-12-28 | 2019-10-31 | 日鉄ケミカル&マテリアル株式会社 | 金属−樹脂複合体及びその利用 |
EP3564675A4 (en) * | 2016-12-28 | 2020-08-26 | NIPPON STEEL Chemical & Material Co., Ltd. | METAL-RESIN COMPLEX AND ITS USE |
WO2018123952A1 (ja) * | 2016-12-28 | 2018-07-05 | 新日鉄住金化学株式会社 | 金属-樹脂複合体及びその利用 |
KR102640613B1 (ko) | 2016-12-28 | 2024-02-26 | 닛테츠 케미컬 앤드 머티리얼 가부시키가이샤 | 금속-수지 복합체 및 그 이용 |
JP2018169373A (ja) * | 2017-03-30 | 2018-11-01 | 新日鉄住金化学株式会社 | 免疫測定方法、免疫測定用キット及びラテラルフロー型クロマトテストストリップ |
JP2019066323A (ja) * | 2017-09-29 | 2019-04-25 | 日鉄ケミカル&マテリアル株式会社 | 標識抗体、その製造方法及び免疫学的測定法 |
WO2024085240A1 (ja) * | 2022-10-21 | 2024-04-25 | ユニ・チャーム株式会社 | おりもの用テストストリップを有する吸収性物品、及び、吸収性物品で用いるためのおりもの用テストストリップ |
WO2024084726A1 (ja) * | 2022-10-21 | 2024-04-25 | ユニ・チャーム株式会社 | おりもの用テストストリップを有する吸収性物品、及び、吸収性物品で用いるためのおりもの用テストストリップ |
WO2024085239A1 (ja) * | 2022-10-21 | 2024-04-25 | ユニ・チャーム株式会社 | おりもの用テストストリップ |
Also Published As
Publication number | Publication date |
---|---|
CN106662585A (zh) | 2017-05-10 |
TW201625948A (zh) | 2016-07-16 |
JP2020177035A (ja) | 2020-10-29 |
US11733241B2 (en) | 2023-08-22 |
EP3165923A1 (en) | 2017-05-10 |
US10690665B2 (en) | 2020-06-23 |
US20200240987A1 (en) | 2020-07-30 |
TWI636257B (zh) | 2018-09-21 |
CN106662585B (zh) | 2021-08-03 |
US20170168049A1 (en) | 2017-06-15 |
TW201840979A (zh) | 2018-11-16 |
JP2018136350A (ja) | 2018-08-30 |
EP3644059A1 (en) | 2020-04-29 |
EP3165923A4 (en) | 2017-11-29 |
JP6353534B2 (ja) | 2018-07-04 |
JPWO2016002743A1 (ja) | 2017-04-27 |
TWI733048B (zh) | 2021-07-11 |
JP6847077B2 (ja) | 2021-03-24 |
EP3165923B1 (en) | 2020-02-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6353534B2 (ja) | 標識物質、免疫学的測定法、免疫学的測定用試薬、アナライトの測定方法、アナライト測定用キット、及び、ラテラルフロー型クロマト用テストストリップ | |
JP6381642B2 (ja) | 樹脂−金属複合体、標識物質、免疫学的測定法、免疫学的測定用試薬、アナライトの測定方法、アナライト測定用キット、及び、ラテラルフロー型クロマト用テストストリップ | |
JP6800275B2 (ja) | 樹脂−白金複合体及びその利用 | |
WO2018123952A1 (ja) | 金属-樹脂複合体及びその利用 | |
JP2017181368A (ja) | 免疫測定方法、免疫測定用キット及びラテラルフロー型クロマトテストストリップ | |
JP7218052B2 (ja) | 検体処理液及びそれを用いたイムノクロマトキット | |
JP2017120242A (ja) | 樹脂−金属複合体、標識物質、免疫学的測定法、免疫学的測定用試薬、アナライトの測定方法、アナライト測定用キット、及び、ラテラルフロー型クロマト用テストストリップ | |
JP7265315B2 (ja) | 免疫学的測定用金属ナノ粒子-セルロース複合体、標識物質、免疫学的測定法、免疫学的測定用試薬、アナライトの測定方法、アナライト測定用キット、及び、ラテラルフロー型クロマト用テストストリップ |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15814054 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2016531373 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15323102 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2015814054 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2015814054 Country of ref document: EP |