Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
• • 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
• • 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
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

• the present invention relates to a method for detecting a substance to be measured using a specific surfactant.
• the present invention further relates to a kit for detecting a substance to be measured, which includes a specific surfactant.
• immunochromatography is widely used because it is easy to operate and can be measured in a short time.
• Competitive reactions or sandwich-type reactions are widely used as immune reactions in immunochromatography.
• sandwich-type reactions are the mainstream in immunochromatography, and in a typical example, the following operations are performed to detect the measured substance consisting of an antigen in a sample.
• a chromatographic carrier having a reaction site is prepared by immobilizing microparticles sensitized with an antibody against the antigen, which is the measured substance, as solid-phase microparticles on the chromatographic carrier, or by directly immobilizing the antibody itself on the chromatographic carrier.
• sensitized labeled microparticles are prepared by sensitizing labeled microparticles with an antibody against the measured substance.
• the sensitized labeled microparticles are chromatographically moved on the chromatographic carrier together with the sample.
• the immobilized antibody becomes an immobilized reagent at the reaction site formed on the chromatographic carrier, and the sensitized labeled microparticles specifically bind to it via the antigen, which is the measured substance.
• the presence or absence or amount of the substance to be measured in the sample can be measured by visually determining the presence or absence or the level of a signal generated by the capture of the sensitized labeled microparticles at the reaction site.
• Patent Document 1 describes a specimen suspension composition that contains a surfactant and has a pH of 3 to 8, and describes the use of the specimen suspension composition to detect an analyte by an immunoassay method.
• Patent Document 2 describes the use of an immunochromatographic reagent composition containing a nonionic surfactant such as polyoxyethylene 23 lauryl ether (also known as Brij 35), an N,N-bis(2-hydroxyethyl)glycine buffer, and casein as a sample treatment liquid or developing liquid when detecting a target substance in a sample by immunochromatography.
• a nonionic surfactant such as polyoxyethylene 23 lauryl ether (also known as Brij 35), an N,N-bis(2-hydroxyethyl)glycine buffer, and casein.
• Patent Document 2 does not focus on improving sensitivity, and does not describe silver-amplified immunochromatography.
• the problem to be solved by the present invention is to provide a method for detecting a substance to be measured and a kit for detecting the substance to be measured, in which signals in the background region are suppressed and highly sensitive detection is possible in silver amplification chromatography.
• the inventors conducted extensive research to solve the above problems and discovered that when silver amplified immunochromatography is performed using an extract to which one or more nonionic surfactants represented by the formula (1) below have been added, the background signal is low and pathogens can be detected with high sensitivity.
• the present invention was completed based on the above findings.
• a method for detecting a substance to be measured comprising the steps of: Formula (1): C n H 2n+1 (OCH 2 CH 2 ) m OH In the formula, C n H 2n+1 is a linear alkyl group, n is an integer of 10 to 20, and m is an integer of 18 to 25.
• ⁇ 2> The method according to ⁇ 1>, wherein the concentration of the nonionic surfactant in the extract is 0.001% by mass to 1.0% by mass.
• the substance to be measured is an antigen of one or more pathogens selected from SARS-CoV-2, influenza virus, RS virus, adenovirus, human metapneumovirus, mycoplasma, pneumococcus, group A streptococcus, norovirus, rotavirus, and Salmonella.
• the antigen is a nucleocapsid protein or a fragment thereof.
• ⁇ 5> The method according to any one of ⁇ 1> to ⁇ 4>, wherein at least one of the first binding substance and the second binding substance is an antibody.
• ⁇ 6> The method according to any one of ⁇ 1> to ⁇ 4>, wherein the signal of the captured labeling substance is amplified with silver using an amplification solution containing a silver-containing compound and a reducing agent for silver ions.
• the nonionic surfactant is one or more selected from the group consisting of polyoxyethylene 23 lauryl ether, polyoxyethylene 20 cetyl ether, and polyoxyethylene 20 stearyl ether.
• One or more nonionic surfactants represented by the following formula (1): a labeling substance including a metal modified with a first binding substance for the substance to be measured; an insoluble carrier containing a second binding substance for the analyte or a substance capable of binding to the first binding substance for the analyte; and an amplification solution for amplifying the signal of the labeling substance with silver.
• Formula (1) C n H 2n+1 (OCH 2 CH 2 ) m OH
• C n H 2n+1 is a linear alkyl group
• n is an integer of 10 to 20
• m is an integer of 18 to 25.
• the nonionic surfactant is provided as an extract containing the nonionic surfactant, and the concentration of the nonionic surfactant in the extract is 0.001% by mass to 1.0% by mass.
• the substance to be measured is an antigen of one or more pathogens selected from SARS-CoV-2, influenza virus, RS virus, adenovirus, human metapneumovirus, mycoplasma, pneumococcus, group A streptococcus, norovirus, rotavirus, and salmonella.
• ⁇ 11> The kit according to ⁇ 10>, wherein the antigen is a nucleocapsid protein or a fragment thereof.
• the amplification solution contains a silver-containing compound and a reducing agent for silver ions.
• ⁇ 14> The kit according to any one of ⁇ 8> to ⁇ 11>, wherein the nonionic surfactant is one or more selected from the group consisting of polyoxyethylene 23 lauryl ether, polyoxyethylene 20 cetyl ether, and polyoxyethylene 20 stearyl ether.
• the method for detecting a substance to be measured and the kit for detecting a substance to be measured according to the present invention suppress signals in the background region, enabling highly sensitive detection.
• FIG. 1 is a perspective view showing an example of an immunochromatography kit.
• FIG. 2 is an exploded schematic perspective view showing an example of an immunochromatography kit.
• FIG. 3 is a schematic side view showing the positional relationship between the test strip, the first pot, and the second pot.
• the numerical range indicated using “to” means a range that includes the numerical values before and after “to” as the minimum and maximum values, respectively.
• the method for detecting a substance to be measured comprises the steps of: developing a mixture of a specimen containing the substance to be measured and an extract on an insoluble carrier in a state where the mixture is in contact with a labeling substance containing a metal modified with a first binding substance for the substance to be measured; capturing a complex between the substance to be measured and the labeling substance at a detection site on an insoluble carrier containing a second binding substance for the substance to be measured or a substance having binding ability to the first binding substance for the substance to be measured; amplifying the signal of the captured labeled substance with silver; and detecting the amplified signal:
• the extract comprises one or more nonionic surfactants represented by the following formula (1):
• the kit for detecting a substance to be measured according to the present invention comprises one or more nonionic surfactants represented by the following formula (1): a labeling substance including a metal modified with a first binding substance for the substance to be measured; an insoluble carrier containing a second binding substance for the analyt
• the inventors performed immunochromatography without silver amplification using the extract, and the results were within the expected range.
• silver-amplified immunochromatography was performed using an extract to which the surfactant shown in formula (1) was added, the background signal was low and pathogens could be detected with high sensitivity, resulting in an unexpectedly advantageous effect.
• This effect is unexpectedly superior to the effect of using an extract to which Tween 80 was added as a surfactant.
• the chromatographic method is a method for easily, quickly and specifically determining and measuring a substance to be measured by the following method. That is, a binding substance immobilized membrane (insoluble carrier: porous carrier) capable of having a labeling substance capture region having at least one detection site having a binding substance (e.g., an antibody) capable of binding to the substance to be measured is used as a stationary phase. On this insoluble carrier, a liquid containing a labeling substance modified by a first binding substance for the substance to be measured is chromatographically moved as a moving layer, and the substance to be measured and the labeling substance are specifically bound to each other while reaching the labeling substance capture region having a detection site.
• a binding substance immobilized membrane insoluble carrier: porous carrier
• a binding substance e.g., an antibody
• a complex of the substance to be measured and the labeling substance is specifically bound to the immobilized second binding substance, and the labeling substance is concentrated in the second binding substance only when the substance to be measured is present in the test sample.
• two types of amplification reagents are used to amplify the signal of the labeled substance, specifically, a compound containing silver and a reducing agent capable of reducing silver ions, and the signal is amplified by an amplification reaction using the complex of the measured substance and the labeled substance bound to the immobilized reagent on the labeled substance capture area as the nucleus, thereby achieving high sensitivity.
• test sample that can be analyzed by the detection method and kit of the present invention is not particularly limited as long as it is a sample that may contain the substance to be measured.
• biological samples particularly body fluids (e.g., whole blood, serum, plasma, cerebrospinal fluid, tears, sweat, urine, pus, nasal discharge, nasopharyngeal swabs, pharyngeal swabs, nasal aspirates, saliva, or sputum) or excretions (e.g., feces) of animals (especially humans), organs, tissues, mucous membranes, skin, scraping specimens (swabs) that are thought to contain them, or animals and plants themselves or dried bodies thereof can be mentioned.
• body fluids e.g., whole blood, serum, plasma, cerebrospinal fluid, tears, sweat, urine, pus, nasal discharge, nasopharyngeal swabs, pharyngeal swabs, nasal aspirates, saliva, or sputum
• the substance to be measured in the present invention is not particularly limited, but may be, for example, an antigen of one or more pathogens selected from SARS-CoV-2, influenza virus, RS virus, adenovirus, human metapneumovirus, mycoplasma, pneumococcus, group A streptococcus, norovirus, rotavirus, and salmonella.
• pathogen antigens include, but are not limited to, nucleocapsid protein or a fragment thereof.
• test sample is spread on an insoluble carrier as a mixture with a suitable extraction solution, which contains a solvent used in a conventional immunological analysis (e.g., water, physiological saline, or a buffer solution) and one or more nonionic surfactants represented by formula (1).
• a solvent used in a conventional immunological analysis e.g., water, physiological saline, or a buffer solution
• nonionic surfactants represented by formula (1).
• the nonionic surfactant used in the present invention is one or more nonionic surfactants represented by the following formula (1).
• Formula (1) C n H 2n+1 (OCH 2 CH 2 ) m OH
• C n H 2n+1 is a linear alkyl group
• n is an integer of 10 to 20
• m is an integer of 18 to 25.
• the terminal carbon atom of C n H 2n+1 is bonded to (OCH 2 CH 2 ) m OH.
• n is preferably an integer of 12 to 18, and more preferably 12, 16 or 18.
• m is preferably an integer of 20 to 23, and more preferably 20 or 23.
• Particularly preferred examples of the one or more nonionic surfactants represented by formula (1) include one or more selected from the group consisting of polyoxyethylene 23 lauryl ether (trade name: Brij (registered trademark) 35), polyoxyethylene 20 cetyl ether (trade name: Brij (registered trademark) 58), and polyoxyethylene 20 stearyl ether (trade name: Brij (registered trademark) S20).
• Polyoxyethylene 23 lauryl ether C12H25 ( OCH2CH2 ) 23OH
• Polyoxyethylene 20 cetyl ether C16H33 ( OCH2CH2 ) 20OH
• Polyoxyethylene 20 stearyl ether C18H37 ( OCH2CH2 ) 20OH
• the concentration of the nonionic surfactant in the extract is preferably 0.0001% to 10% by mass, and more preferably 0.001% to 1.0% by mass.
• a chromatography strip can be incorporated and used.
• the chromatography strip that can be used is not particularly limited as long as it is a chromatography strip that can be used in a normal chromatography method.
• the chromatographic strip that can be used in the present invention has a labeled substance holding region and a labeled substance capture region from the upstream to downstream direction of the development direction of the test sample.
• the chromatographic strip further has a region having a color-developing reagent.
• the region having the color-developing reagent is located downstream of the labeled substance capture region, and a preferred embodiment is one in which a sample addition pad, a labeled substance holding pad having a labeled substance holding region (e.g., a gold colloid antibody holding pad), an antibody immobilized membrane that is an insoluble carrier (e.g., an antibody immobilized membrane that has a labeled substance capture region), and a water-absorbing pad are arranged in this order on an adhesive sheet.
• a sample addition pad e.g., a gold colloid antibody holding pad
• an antibody immobilized membrane that is an insoluble carrier e.g., an antibody immobilized membrane that has a labeled substance capture region
• a water-absorbing pad arranged in this order on an adhesive sheet.
• a marker substance-holding pad having a marker substance-holding region that can be used in the present invention can be prepared by preparing a suspension containing the marker substance, applying the suspension to a suitable water-absorbing pad (e.g., a glass fiber pad), and then drying it.
• a suitable water-absorbing pad e.g., a glass fiber pad
• the labeling substance used in the present invention is a labeling substance containing a metal.
• the types of metals that can be used in the present invention are preferably precious metals such as gold, silver, and platinum, iron, lead, copper, cadmium, bismuth, antimony, tin, and mercury, and compounds thereof can be used. More preferably, precious metals such as gold, silver, and platinum can be used.
• a metal colloid label or a metal sulfide label can be used as a preferred form of the labeling substance containing a metal that can be used in the present invention.
• the metal colloid label can preferably be platinum colloid, gold colloid, silver colloid, iron colloid, or aluminum hydroxide colloid
• the metal sulfide label can preferably be each sulfide of iron, silver, lead, copper, cadmium, bismuth, antimony, tin, or mercury. More preferably, platinum colloid, gold colloid, and silver colloid can be used in the present invention, and most preferably gold colloid can be used.
• gold colloid particles are used as the metal colloid label, a commercially available product may be used. Alternatively, gold colloid particles can be prepared by a conventional method, for example, a method in which chloroauric acid is reduced with sodium citrate (Nature Physical Science, 241 (1973) 20, etc.).
• the average particle size of the metal colloid is preferably about 1 nm to 500 nm, more preferably 3 to 100 nm, and particularly preferably 5 to 60 nm.
• the average particle size of the metal colloid can be measured using a commercially available particle size distribution analyzer. Methods for measuring particle size distribution include optical microscopy, confocal laser microscopy, electron microscopy, atomic force microscopy, static light scattering, laser diffraction, dynamic light scattering, centrifugal sedimentation, electric pulse measurement, chromatography, and ultrasonic attenuation, and devices corresponding to each principle are commercially available.
• the dynamic light scattering method is preferably used as a method for measuring the average particle size because of the particle size range and ease of measurement.
• Commercially available measuring devices that use dynamic light scattering include Nanotrac UPA (Nikkiso Co., Ltd.), Dynamic Light Scattering Particle Size Distribution Measuring Device LB-550 (Horiba Ltd.), and Concentrated Particle Size Analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.).
• the signal of the metal-based label can be amplified.
• the silver ions supplied from a compound containing silver, such as an inorganic silver salt or an organic silver salt, and a reducing agent capable of reducing the silver ions are brought into contact, and the silver ions are reduced by the reducing agent to generate silver particles.
• the silver particles are deposited on the metal-based label with the metal-based label as a nucleus, so that the metal-based label is amplified and the analysis of the substance to be measured can be performed with high sensitivity. That is, in the chromatographic method of the present invention, a reaction is carried out in which the silver particles generated by the reduction of the silver ions by the reducing agent are deposited on the label of the immune complex, and the amplified signal is analyzed.
• the labeling substance is modified with a first binding substance for the analyte.
• the first binding substance may be any compound that has affinity for the analyte, such as an antibody for the analyte (antigen), an antigen for the analyte (antibody), or an aptamer for the analyte (protein, low molecular weight compound, etc.).
• the kit of the present invention has a second binding substance for the analyte or a binding substance for the first binding substance in the labeling substance capture region.
• the second binding substance for the analyte may be any compound that has affinity for the analyte, such as an antibody for the analyte (antigen), an antigen for the analyte (antibody), or an aptamer for the analyte (protein, low molecular weight compound, etc.).
• the second binding substance may be different from the first binding substance, or may be the same.
• the binding substance for the first binding substance may be the analyte itself, or may be a compound having a site that is recognized by the first binding substance, such as a compound in which a derivative of the analyte is bound to a protein (e.g., BSA).
• a protein e.g., BSA
• At least one of the first binding substance and the second binding substance is an antibody. More preferably, the first binding substance is an antibody and the second binding substance is an antibody.
• the antibody against the substance to be measured is not particularly limited, but may be, for example, an antiserum prepared from the serum of an animal immunized with the substance to be measured, an immunoglobulin fraction purified from the antiserum, a monoclonal antibody obtained by cell fusion using spleen cells of an animal immunized with the substance to be measured, or a fragment thereof [e.g., F(ab')2, Fab, Fab', or Fv] or a single-chain antibody (such as scFv).
• an antiserum prepared from the serum of an animal immunized with the substance to be measured
• an immunoglobulin fraction purified from the antiserum e.g., a monoclonal antibody obtained by cell fusion using spleen cells of an animal immunized with the substance to be measured
• a fragment thereof e.g., F(ab')2, Fab, Fab', or Fv
• a single-chain antibody such as sc
• the method of modifying a labeling substance using a first binding substance can be carried out, for example, in the case of binding between a metal colloid and a binding substance, according to the conventionally known method described below (for example, The Journal of Histochemistry and Cytochemistry, 30, 7 (1982) 691-696).
• a metal colloid and a specific binding substance for example, an antibody
• the precipitate obtained by centrifugation is dispersed in a solution containing a dispersant such as polyethylene glycol, thereby obtaining the desired metal colloid-labeled specific binding substance.
• Insoluble carriers that can be used in the present invention are particularly preferably nitrocellulose carriers (such as nitrocellulose membranes), cellulose membranes, acetylcellulose membranes, polysulfone membranes, polyethersulfone membranes, nylon membranes, glass fibers, nonwoven fabrics, cloth, or threads.
• nitrocellulose carriers such as nitrocellulose membranes
• cellulose membranes such as nitrocellulose membranes
• acetylcellulose membranes such as nitrocellulose membranes
• polysulfone membranes such as polysulfone membranes, polyethersulfone membranes
• nylon membranes such as glass fibers, nonwoven fabrics, cloth, or threads.
• the insoluble carrier has a detection site in which a second binding substance for the substance to be measured is immobilized in the labeled substance capture region.
• the second binding substance for the substance to be measured may be directly immobilized to a part of the insoluble carrier by physical or chemical binding to form a detection site, or may be physically or chemically bound to fine particles such as latex particles, and the fine particles may be trapped and immobilized in a part of the insoluble carrier to form a detection site. It is preferable to use the insoluble carrier after immobilizing the second binding substance for the substance to be measured, and then subjecting it to a treatment to prevent nonspecific adsorption, such as treatment with an inactive protein.
• the insoluble carrier may also preferably have a plurality of binding sites, and may further have the above-mentioned control site as a part of the labeled substance capture region, if desired.
• a preferred embodiment is one in which a labeled substance holding pad having a labeled substance holding area, preferably a gold colloid holding pad, is incorporated into a chromatography kit for use.
• Preferred materials for the labeled substance holding pad include, for example, cellulose filter paper, glass fiber, and nonwoven fabric, and the labeled substance holding area can be formed by impregnating the pad with a certain amount of the labeled substance prepared as described above and drying it.
• the kit of the present invention is preferably further equipped with a sample addition pad.
• the sample addition pad is preferably not only capable of receiving a sample containing an added substance to be measured, but also capable of filtering insoluble particles and the like in the sample.
• materials for the sample addition pad include those having uniform properties, such as cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth.
• the material constituting the sample addition section may be subjected to a nonspecific adsorption prevention treatment before use.
• the sample addition pad may also serve as a labeling substance retention pad having a labeling substance retention region.
• the absorbent pad is a portion where the added sample is physically absorbed by chromatographic movement and where unreacted labeling substances and the like that are not insolubilized in the detection portion of the chromatographic carrier are absorbed and removed, and is made of absorbent materials such as cellulose filter paper, nonwoven fabric, cloth, and cellulose acetate.
• the speed of chromatography after the chromatographic tip of the added sample reaches the absorbent pad varies depending on the material and size of the absorbent pad, and a speed suitable for the measurement of the substance to be measured can be set by selecting the absorbent pad.
• the color-developing reagent may be supported on an insoluble carrier.
• the color-developing reagent for detecting a reducing agent capable of reducing silver ions for example, a compound that reacts with ions to develop color is preferably used.
• the first amplification reagent will be described later in this specification, but for example, when the first amplification reagent is a reagent containing divalent iron ions (Fe 2+ ), a compound that reacts with Fe 2+ ions to develop color can be used as the color-developing reagent.
• the compound that reacts with Fe 2+ ions to develop color a compound that can develop color by forming a complex with Fe 2+ ions can be used.
• compounds that react with Fe 2+ ions to develop color include compounds having a phenanthroline skeleton [e.g., 1,10-phenanthroline, 5-methylphenanthroline, 5-nitrophenanthroline, bathophenanthroline (4,7-diphenyl-1,10-phenanthroline), or bathophenanthroline disulfonic acid] or compounds having a bipyridine skeleton [e.g., 2,2'-bipyridine], and preferably compounds having a phenanthroline skeleton can be used.
• a phenanthroline skeleton e.g., 1,10-phenanthroline, 5-methylphenanthroline, 5-nitrophenanthroline, bathophenanthroline (4,7-diphenyl-1,10-phenanthroline), or bathophenanthroline disulfonic acid
• compounds having a bipyridine skeleton e.g., 2,2'-bipyridine
• a reagent that undergoes a structural change due to H + ions and changes color can be preferably used to detect the first amplification reagent.
• the aqueous solution containing the first amplification reagent is acidic (pH lower than 7, high H + ion concentration)
• a compound that reacts with H + ions to develop color which is a color-developing reagent known as a pH indicator for the acidic range (e.g., diazo-based color-developing reagents such as methyl orange, methyl red, Congo red, and methyl yellow, and sultone-based color-developing reagents such as thymol blue, bromocresol green, bromocresol purple, and bromothymol blue), appropriately selected according to the pH of the aqueous solution containing the amplification reagent.
• 1,10-phenanthroline, bathophenanthroline, or bromocresol green is more preferably used.
• the color-developing reagent is preferably one that does not substantially move in the insoluble carrier when either an aqueous solution containing the test sample or an aqueous solution containing a reducing agent capable of reducing silver ions is developed. Therefore, the LogP (partition coefficient in water and octanol) of the color-developing reagent is preferably 4.0 or more, and more preferably 5.0 or more. An actual measured value may be used for LogP, but a calculated value obtained from the chemical structure, etc., can also be used as a simple method of determination. The calculation method used in CambridgeSoft's ChemDrawPro version 12 is preferable as a method of calculating LogP. The responsiveness and LogP (according to ChemDrawPro version 12) of representative color-developing reagents are shown in Table 1 below.
• the region having the color-developing reagent is located downstream of the labeled substance capture region having the detection site of the insoluble carrier.
• Methods for retaining the color-developing reagent in the chromatography kit include a method of immersing an absorbent pad described below in the color-developing reagent solution and drying it under reduced pressure, and a method of applying the color-developing reagent in a line shape downstream of the labeled substance capture region of the insoluble carrier. If the color-developing reagent substantially migrates through the insoluble carrier when either the aqueous solution containing the test sample or the aqueous solution containing the first amplification reagent is developed, it is preferable to use the color-developing reagent contained in an absorbent pad.
• the color-developing reagent does not substantially move within the insoluble carrier when either an aqueous solution containing the test sample or an aqueous solution containing the first amplification reagent is developed, it is preferable to support the color-developing reagent on an insoluble carrier having a labeled substance capture region.
• an embodiment in which the color-developing reagent is supported on an insoluble carrier is more preferable.
• having a region where a mixture containing the substance to be measured is added and a labeled substance capture region in this order from upstream to downstream with respect to the direction of development of the mixture containing the substance to be measured is defined as the upstream direction and downstream direction with respect to the direction of development of the mixture containing the substance to be measured when the mixture containing the substance to be measured is developed using capillary action or the suction force when an absorbent pad is used.
• the direction of the labeled substance holding region is defined as the upstream direction and the direction of the labeled substance capture region is defined as the downstream direction.
• the first of the two types of amplification reagents used to amplify the signal of the labeled substance captured in the labeled substance capture region is expanded from the upstream direction of the labeled substance capture region to the downstream direction of the labeled substance capture region, and a physical or chemical change in the region having the color-developing reagent is detected to confirm that the labeled substance capture region is filled with the first amplification reagent.
• a physical or chemical change in the region having the color-developing reagent a change in color or fluorescence caused by the reaction between the first amplification reagent and the color-developing reagent can be detected.
• color development can be detected.
• Such physical or chemical changes may be detected visually or using a detection device.
• Detection method The detection method of the present invention will now be described with reference to a specific embodiment thereof, that is, the sandwich method.
• the analysis of the analyte can be performed, for example, by the following procedure, although it is not particularly limited.
• a first binding substance for the analyte and a second binding substance for the analyte are prepared in advance.
• a labeling substance is modified in advance with the first binding substance.
• the second binding substance is immobilized on a suitable chromatographic carrier (insoluble carrier) (e.g., nitrocellulose membrane, glass fiber membrane, nylon membrane, cellulose membrane, etc.) to form a labeling substance capture region, and is brought into contact with a mixture containing a test sample that may contain the analyte. If the analyte is present in the test sample, binding with the second binding substance (e.g., an antigen-antibody reaction with the second binding substance) occurs.
• a suitable chromatographic carrier insoluble carrier
• a suitable chromatographic carrier e.g., nitrocellulose membrane, glass fiber membrane, nylon membrane, cellulose membrane, etc.
• an excess amount of a labeling substance modified with the first binding substance is contacted, if the analyte is present in the test sample, a complex consisting of the immobilized second binding substance and the labeling substance modified with the first binding substance for the analyte is formed.
• the labeling substance that did not form an immune complex is removed, and then, for example, the labeling substance capture region of the insoluble carrier is observed as is to detect or quantify the labeling substance, thereby determining the presence or absence of the substance to be measured in the test sample, or measuring the amount.
• a reducing agent and a silver ion-containing compound are supplied to amplify and detect the signal from the labeling substance that has formed such a complex.
• the signal of the captured labeling substance is amplified using an amplification solution containing a silver-containing compound and a reducing agent for silver ions.
• the amplification reagent is a reagent that can generate a colored compound or luminescence by catalytic reaction with the action of a labeling substance or a substance to be measured, and can cause signal amplification, and can be used in the state of a solution containing the reagent, i.e., as an amplification solution.
• Examples include a silver ion solution that causes the precipitation of metallic silver by physical development on a metal label, and a solution of a phenylenediamine compound and a naphthol compound that becomes a dye by the action of a peroxidase label and hydrogen peroxide.
• so-called developing solutions such as those described in general books in the field of photographic chemistry (for example, "Revised Fundamentals of Photographic Engineering - Silver Halide Photography Edition” (edited by the Japan Society of Photography, Corona Publishing), “Chemistry of Photography” (Akira Sasai, Photographic Industry Publishing), and “Latest Prescription Handbook” (Shinichi Kikuchi et al., Amico Publishing)) can be used as the amplifying solution containing an amplifying reagent, and so-called physical developing solutions that contain silver ions in the solution and are reduced mainly by metal colloids that serve as the nuclei of development can be used as the amplifying solution without any particular limitations.
• the kit of the present invention includes a compound containing silver and a reducing agent capable of reducing silver ions.
• a specific example of the amplification solution is a combination of a first amplification solution containing a reducing agent capable of reducing silver ions and a second amplification solution containing a compound containing silver.
• the first amplification reagent is contained in the first amplification liquid and the second amplification reagent is contained in the second amplification liquid, and amplification is performed by sequentially adding the first amplification liquid and the second amplification liquid.
• the first amplification liquid is added to a pad for delivering a reducing agent solution, which is located upstream of the labeled substance retention pad and the sample addition pad.
• a silver ion-containing compound for example, an organic silver salt, an inorganic silver salt, or a silver complex
• a silver ion-containing compound having high solubility in a solvent such as water is preferable, and examples thereof include silver nitrate, silver acetate, silver lactate, silver butyrate, and silver thiosulfate. Silver nitrate is particularly preferable.
• a silver complex coordinated with a ligand having a water-soluble group such as a hydroxyl group or a sulfonic group is preferable, and examples thereof include silver hydroxythioether.
• the inorganic silver salt or silver complex is preferably contained in an amount of 0.001 mol/m 2 to 0.2 mol/m 2 , and more preferably 0.01 mol/m 2 to 0.05 mol/m 2 in terms of silver.
• the reducing agent capable of reducing silver ions can be any inorganic or organic material or a mixture thereof, so long as it can reduce silver ions to silver.
• inorganic reducing agents reducing metal salts and reducing metal complex salts whose valence can be changed by metal ions such as Fe2 + , V2 + , Ti3 + , etc. can be preferably mentioned.
• metal ions such as Fe2 + , V2 + , Ti3 + , etc.
• an inorganic reducing agent it is necessary to form a complex or reduce the oxidized ions, and remove or render them harmless.
• a complex of the oxide Fe3 + can be formed using citric acid or EDTA, and rendered harmless.
• it is preferable to use such an inorganic reducing agent and more preferably a metal salt of Fe2 + is preferable.
• Developing agents used in wet silver halide photographic materials e.g., methyl gallate, hydroquinone, substituted hydroquinones, 3-pyrazolidones, p-aminophenols, p-phenylenediamines, hindered phenols, amidoximes, azines, catechols, pyrogallols, ascorbic acid (or its derivatives), and leuco dyes
• methyl gallate, hydroquinone, substituted hydroquinones, 3-pyrazolidones, p-aminophenols, p-phenylenediamines, hindered phenols, amidoximes, azines, catechols, pyrogallols, ascorbic acid (or its derivatives), and leuco dyes can also be used.
• an ascorbic acid reducing agent is also preferred.
• useful ascorbic acid reducing agents include ascorbic acid and analogs, isomers and derivatives thereof, such as D- or L-ascorbic acid and its sugar derivatives (e.g., ⁇ -lactoascorbic acid, glucoascorbic acid, fucoascorbic acid, glucoheptoascorbic acid, maltoascorbic acid), sodium salt of ascorbic acid, potassium salt of ascorbic acid, isoascorbic acid (or L-erythroascorbic acid), salts thereof (e.g., alkali metal salts, ammonium salts or salts known in the art), enediol type ascorbic acid, enaminol type ascorbic acid, thioenol type ascorbic acid, etc., and in particular D, L or D,L-ascorbic acid (and its alkali metal salts) or isoascorbic acid (or its
• auxiliary agents in the amplification solution may include a buffer, a preservative, such as an antioxidant or an organic stabilizer, and a rate regulator.
• the buffer include a buffer using acetic acid, citric acid, sodium hydroxide or any of their salts, or tris(hydroxymethyl)aminomethane, and other buffers used in general chemical experiments.
• the amplification solution can be adjusted to an optimal pH.
• an antifogging agent an alkylamine can be used as an additive, and dodecylamine is particularly preferred.
• a surfactant can be used, and C 9 H 19 -C 6 H 4 -O-(CH 2 CH 2 O) 50 H is particularly preferred.
• a preferred method for spotting the amplification reagent onto the chromatography kit is to spot a reducing agent solution as a first amplification liquid onto a pad for delivering the reducing agent solution, and then spot a silver ion solution as a second amplification liquid from above onto an area including the labeled substance capture area, thereby allowing the silver ion solution to permeate the insoluble carrier in the thickness direction.
• a method for incorporating two types of amplification reagents in a chromatography kit is to place pots containing solutions containing each amplification reagent above the site where each amplification reagent is to be applied.
• the reducing agent solution (first amplification solution) above the pad for delivering the reducing agent solution, and place the pot containing the silver ion solution (second amplification solution) just above the silver ion solution filling hole.
• the liquid can flow by pressing each pot, and be applied to the specified site.
• the labeling substance modified with the first binding substance for the substance to be measured may be provided on the insoluble carrier in advance, or the labeling substance modified with the first binding substance for the substance to be measured may be provided separately from the insoluble carrier.
• the measurement can be performed by mixing the labeling substance modified with the first binding substance for the substance to be measured, which is provided separately from the insoluble carrier, with the test sample and then spreading it on the insoluble carrier.
• the kit of the present invention may include a housing case that contains an insoluble carrier having a reactive site, a silver-containing compound, and a reducing agent capable of reducing silver ions.
• the kit of the present invention may further include a pot having a breakable member, and the silver-containing compound and the reducing agent capable of reducing silver ions may be enclosed in the pot, respectively. In this case, the pot can be broken by an external force.
• FIG. 1 is a schematic perspective view of an immunochromatography kit 100
• FIG. 2 is a schematic exploded perspective view of the immunochromatography kit 100 of FIG. 1 and 2
• the immunochromatography kit 100 of this embodiment includes a test strip 1 including an insoluble carrier 2 having a test area for a substance to be measured, which develops a sample liquid, and a first pot 40 and a second pot 45 each having a surface with a sheet member and containing a first amplifying liquid 41 and a second amplifying liquid 46, respectively, for amplifying a detection signal in the test area, which are enclosed in a housing case 9.
• the housing case 9 includes a lower case 20 having a storage section 21 in which the test strip 1 is placed, an upper case 10 joined to the lower case 20 at its periphery, and an intermediate member 30 disposed between the upper case 10 and the lower case 20.
• the upper case 10 side is defined as the top
• the lower case 20 side is defined as the bottom.
• the intermediate member 30 has a pot accommodating section 32 that receives the first pot 40 and has an amplification liquid filling hole on the bottom surface for dripping the first amplification liquid 41 onto the insoluble carrier 2.
• a protruding breaking portion 34 that breaks the sheet member 43 is provided at a position facing the sheet member 43 of the first pot 40 in the pot accommodating section 32.
• the first pot 40 is placed above the pot accommodating section 32 so that the surface having the sheet member 43 is the lower surface, and a breaking portion 34 is provided on the bottom surface of the pot accommodating section 32 that faces the sheet member 43 (see FIG. 3).
• the intermediate member 30 also includes a flow path forming portion 35 extending downstream of the bottom surface of the pot accommodating portion 32.
• the flow path forming portion 35 is disposed above the testing region L1 , the confirmation region L2 , and the amplification indicator region L3 , and is formed of a transparent material so that these regions L1 to L3 can be visually confirmed.
• the upper case 10 has a first convex deformation portion 12 that deforms toward the first pot 40 when pressure is applied from the outside, causing the sheet member 43 of the first pot 40 to break at the breaking portion 34 of the intermediate member 30.
• the upper case 10 also has a second convex deformation portion 14 that deforms toward the second pot 45 when pressure is applied from the outside, causing the sheet member 48 of the second pot 45 to break.
• the upper case 10 is also provided with an opening 16 for dropping sample liquid, and the sample liquid is dropped onto the marker-retaining pad 3 of the test strip 1 through this opening 16.
• the upper case 10 also has an observation window 18 at a position corresponding to the flow path forming portion 35 of the intermediate member 30, for visually observing the three regions L1 to L3 .
• the lower case 20 is provided with an insoluble carrier storage section 21 in which the insoluble carrier 2 is placed, and an absorbent pad storage section 22 in which the absorbent pad 6 is placed, downstream of the insoluble carrier storage section 21, as storage sections in which the test strip 1 is placed.
• a second pot storage section 24 in which a second pot 45 is stored is provided upstream of the insoluble carrier storage section 21.
• the test strip 1 includes an insoluble carrier 2 for developing the sample liquid, a label holding pad 3 containing a label modified with a binding substance immobilized on the insoluble carrier 2, a liquid delivery pad 4 for delivering a second amplification liquid 46 to the insoluble carrier 2, which is arranged in contact with one end of the insoluble carrier 2, and an absorbent pad 6 arranged in contact with the other end of the insoluble carrier 2.
• the insoluble carrier 2 is fixed and supported on a back adhesive sheet 7.
• the insoluble carrier 2 has a test region L 1 , a confirmation region L 2 , and an amplification indicator region L 3 between the label holding pad 3 and the absorbent pad 6, in this order from the label holding pad 3 side.
• the insoluble carrier 2 having the test region 084L1 , the confirmation region L2 , and the amplification indicator region L3 formed therein may be referred to as a chromatographic carrier.
• the liquid supply pad 4 side is defined as the upstream side
• the absorbent pad 6 side is defined as the downstream side, as shown in Figure 3.
• the intermediate member 30 is positioned above the downstream end of the test strip 1, and the first pot 40 is placed in the pot storage section 32 of the intermediate member 30 with the sheet member 43 facing down.
• the second pot 45 is stored below the upstream end of the test strip 1 in the lower case 20 with the sheet member 48 facing up.
• a gap (clearance) D is formed between the back surface 36 of the flow path forming portion 35 of the intermediate member 30 and the insoluble carrier 2 of the test strip 1.
• This gap D is preferably in the range of 0.01 mm to 1 mm. If it is 0.01 mm or more, the amplification liquid etc. can be sufficiently infiltrated, and if it is 1 mm or less, capillary force is exerted and the first amplification liquid 41 can uniformly fill the gap between the insoluble carrier 2 and the intermediate member 30.
• the first pot 40 containing the first amplification liquid 41 is filled in a container 42 having an opening on one side and made of, for example, a resin material, and the opening of the container 42 is covered and sealed by a breakable sheet member 43.
• the second pot 45 containing the second amplification liquid 46 is filled in a container 47 having an opening on one side and made of, for example, a resin material, and the opening of the container 47 is covered and sealed by a breakable sheet member 48.
• a laminate film such as an aluminum foil or an aluminum laminate sheet is preferably used as the breakable sheet members 43, 48 in the first pot 40 and the second pot 45. Breaking here refers to a state in which the material cannot be restored after being broken.
• Example 1 (1) Preparation of Immunochromatography Kit (1-1) Preparation of Anti-NP Antibody-Modified Gold Colloid as a Labeling Substance Modified with a First Substance Capable of Binding to a Substance to be Measured 1 mL of 50 mmol/L KH2PO4 buffer (pH 8.0) was added to 9 mL of a solution containing a 50 nm diameter gold colloid (product number: EM.GC50, manufactured by BBI) to adjust the pH, and then 1 mL of a solution containing 20 ⁇ g/mL anti-NP monoclonal antibody (a monoclonal antibody that recognizes the nucleocapsid protein of SARS-CoV-2: clone ID "4H2G1" manufactured by GenScript) was added and stirred for 10 minutes.
• KH2PO4 buffer pH 8.0
• This coating solution was uniformly applied at 1 mL per glass fiber pad (Glass Fiber Conjugate Pad, manufactured by Millipore) cut to 5 mm x 300 mm, and dried under reduced pressure for 24 hours to obtain an anti-NP antibody-modified gold colloid holding pad.
• Glass Fiber Conjugate Pad manufactured by Millipore
• a 15 mm line of anti-NP monoclonal antibody (monoclonal antibody that recognizes the nucleocapsid protein of SARS-CoV-2: clone ID "3F9C12" manufactured by GenScript) solution was applied to the 60 mm short side of the nitrocellulose membrane from the downstream side to form a test area.
• a 0.5 mg/mL anti-mouse IgG antibody solution was applied to the 60 mm short side from the downstream side to form a test area.
• a 30 mmol/L bromocresol green (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) solution was applied to the 60 mm short side from the downstream side to form a test area.
• the nitrocellulose membrane was dried in a hot air dryer at 50°C for 30 minutes. After drying was completed, the nitrocellulose membrane dried as described above was immersed in a tray containing 500 mL of blocking solution (50 mmol/L borate buffer (pH 8.5) containing 0.5% by mass casein (derived from milk, product number 030-01505, Fujifilm Wako Pure Chemical Industries, Ltd.)) and left to stand for 30 minutes. The nitrocellulose membrane was then removed and immersed in 500 mL of washing and stabilizing solution (50 mmol/L Tris-HCl (pH 7.5) buffer containing 0.5% by mass sucrose and 0.05% by mass sodium cholate) prepared in a separate tray and left to stand for 30 minutes.
• blocking solution 50 mmol/L borate buffer (pH 8.5) containing 0.5% by mass casein (derived from milk, product number 030-01505, Fujifilm Wako Pure Chemical Industries, Ltd.)
• 500 mL of washing and stabilizing solution 50 mmol/L Tris-HCl
• the nitrocellulose membrane was then removed from the solution and dried in an environment of 25°C for 24 hours.
• the area where the anti-NP antibody is immobilized corresponds to the test area containing a second substance that binds to the substance to be measured
• the area where the anti-mouse IgG antibody is immobilized corresponds to the confirmation area containing a substance that can bind to the first substance
• the area where the bromocresol green is immobilized corresponds to the amplification indicator area containing a substance that reacts with the first amplification solution.
• a liquid delivery pad (a glass fiber pad cut to 25 mm x 300 mm (Glass Fiber Conjugate Pad, manufactured by Millipore)) was attached to the upstream side of the chromatographic carrier so that the liquid delivery pad and the chromatographic carrier overlapped by 7 mm.
• the member thus produced was cut parallel to the direction perpendicular to the 300 mm long side with a guillotine cutter (CM4000, manufactured by Nippon Techno Cluster Co., Ltd.) to a width of 5 mm, to produce 60 test strips (excluding the absorbent pad).
• CM4000 manufactured by Nippon Techno Cluster Co., Ltd.
• this solution was mixed with a solution in which 5.9 mL of nitric acid (10 wt%), 0.1 g of dodecylamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 123-00246), and 0.1 g of surfactant C 12 H 25 -C 6 H 4 -O-(CH 2 CH 2 O) 50 H were dissolved in 47.6 g of water in advance, and this was used as the silver ion solution, which is the first amplification solution to be sealed in the first pot.
• nitric acid 10 wt%
• dodecylamine manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 123-00246
• surfactant C 12 H 25 -C 6 H 4 -O-(CH 2 CH 2 O) 50 H were dissolved in 47.6 g of water in advance, and this was used as the silver ion solution, which is the first amplification solution to be sealed in the first pot.
• the lower case 20, upper case 10, intermediate member 30, first pot 40, and second pot 45 constituting the immunochromatography kit 100 as shown in Figures 1 and 2 were each fabricated by injection molding using polypropylene as the material.
• the upper case was fabricated by injection molding using polypropylene containing 50 mass% of Tafthren (registered trademark), an olefin-based elastomer manufactured by Sumitomo Chemical Co., Ltd.
• the upper case 10 had two deformable portions (a first convex deformation portion and a second convex deformation portion), and these two deformation portions were not separated from the upper case 10, and were fabricated by injection molding as part of the upper case 10 at all boundaries.
• the first convex deformation portion 12 shown in Figures 1 and 2 has two protrusions, and the second convex deformation portion 14 has one protrusion.
• the second convex deformation portion 14 was pressed down to break the aluminum foil of the sheet member 48 sealing the second amplification liquid 46 enclosed in the second pot 45, and the liquid delivery pad 4 was immersed in the second pot 45, so that the second amplification liquid 46 was supplied to the insoluble carrier 2 by utilizing the capillary phenomenon.
• the first convex deformation portion 12 was pressed down to move the first pot 40 toward the breaking portion 34 of the first pot housing portion 32 of the intermediate member 30, whereby the aluminum foil of the sheet member 43 sealing the first pot 40 was broken by the breaking portion 34, and the silver ion solution, which was the first amplification liquid 41, was supplied to the insoluble carrier 2 from the opening of the intermediate member 30, thereby carrying out a silver amplification reaction.
• the silver amplification reaction was completed in several tens of seconds.
• the color intensity in the test area and background area was measured using LAS4000 (GE), the ⁇ intensity was calculated by subtracting the background area intensity from the test area intensity, and the detection ability was classified into the following five levels. - (No difference in density) ⁇ (slight concentration difference) + (clear concentration difference) ++ (significant difference in density) +++ (even more significant concentration difference)
• Example 2 was evaluated in the same manner as in Example 1, except that in the preparation of the surfactant-containing extract described in Example 1 (2-2), Brij58 was used as the surfactant.
• Example 3 was evaluated in the same manner as in Example 1, except that in the preparation of the surfactant-containing extract described in Example 1 (2-2), Brij S20 was used as the surfactant.
• Example 4 was evaluated in the same manner as in Example 1, except that in the preparation of the surfactant-containing extract described in Example 1 (2-2), Brij35 was added to give concentrations of 0.0001% by mass, 0.001% by mass, 0.01% by mass, 0.1% by mass, 1% by mass, or 10% by mass.
• Example 5 was evaluated in the same manner as in Example 1, except that in the preparation of the surfactant-containing extract described in Example 1 (2-2), Brij58 was added to give concentrations of 0.0001 mass%, 0.001 mass%, 0.01 mass%, 0.1 mass%, 1 mass%, or 10 mass%.
• Comparative Example 1 was evaluated in the same manner as in Example 1, except that in the preparation of the surfactant-containing extract described in Example 1 (2-2), no surfactant was added, and the color intensity in the test area and the background area was measured without carrying out the silver amplification reaction described in Example 1 (2-3).
• Comparative Example 2 was evaluated in the same manner as in Example 1, except that in the preparation of the surfactant-containing extract solution described in Example 1 (2-2), Tween 80 was used as the surfactant, and the color intensity in the test area and the background area was measured without performing the silver amplification reaction described in Example 1 (2-3).
• Comparative Example 3 was evaluated in the same manner as in Example 1, except that Triton X-100 was used as the surfactant in the preparation of the surfactant-containing extract solution described in Example 1 (2-2), and that the color intensity in the test area and the background area was measured without carrying out the silver amplification reaction described in Example 1 (2-3).
• Comparative Example 4 was evaluated in the same manner as in Example 1, except that the color intensity in the test area and the background area was measured without carrying out the silver amplification reaction described in Example 1 (2-3).
• Comparative Example 5 was evaluated in the same manner as in Example 1, except that no surfactant was added in the preparation of the surfactant-containing extract described in Example 1 (2-2).
• Comparative Example 6 was evaluated in the same manner as in Example 1, except that in the preparation of the surfactant-containing extract described in Example 1 (2-2), Tween 80 was used as the surfactant.
• Comparative Example 7 was evaluated in the same manner as in Example 1, except that in the preparation of the surfactant-containing extract described in Example 1 (2-2), Triton X-100 was used as the surfactant.

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