WO2017002882A1 - クロマト分析装置およびクロマト分析方法 - Google Patents
クロマト分析装置およびクロマト分析方法 Download PDFInfo
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- WO2017002882A1 WO2017002882A1 PCT/JP2016/069343 JP2016069343W WO2017002882A1 WO 2017002882 A1 WO2017002882 A1 WO 2017002882A1 JP 2016069343 W JP2016069343 W JP 2016069343W WO 2017002882 A1 WO2017002882 A1 WO 2017002882A1
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- 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
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- 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
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- 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
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- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
- G01N2333/135—Respiratory syncytial virus
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- 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
- G01N33/56983—Viruses
Definitions
- the present invention relates to a chromatographic analyzer and a chromatographic analysis method.
- immunochromatographic immunoassays that do not require pretreatment of specimens have been used as simple in-vitro diagnostic kits or portable diagnostic devices that detect antigens in sample solutions using the specific reactivity of antibodies.
- pathogen testing kits such as viruses and bacteria are familiar immunochromatographic analyzers widely used in general hospitals and clinics.
- the simplest structure of a conventional immunochromatography analyzer is that a sample addition unit, a labeling substance holding unit, a chromatographic medium unit carrying a detection unit, and an absorption unit that absorbs liquid that has passed through the detection unit are interconnected. Structure.
- Such an immunochromatography analyzer is currently required to have high sensitivity in order to detect a trace amount of antigen such as influenza virus.
- Patent Document 1 proposes a technique in which a metal ion masking agent coexists in a reaction solution.
- Patent Document 2 discloses a technique for forming a control line for preventing diffusion of a biomaterial-containing solution due to capillary action at a predetermined position, dropping the biomaterial-containing solution in the vicinity of the control line, and localizing the control line. Is disclosed.
- the conventional immunochromatography analyzer cannot increase the sensitivity to the required level while suppressing the non-specific reaction.
- an object of the present invention is to provide a chromatographic analysis apparatus and a chromatographic analysis method capable of increasing the detection sensitivity of a substance to be detected while suppressing non-specific reactions.
- the present invention is as follows. 1.
- a chromatographic analyzer including at least a chromatographic medium portion on which a detection unit for detecting a target substance contained in a specimen is supported,
- the chromatographic medium part has a configuration in which a membrane is provided on a support,
- the chromatographic analyzer wherein the membrane has an average film thickness of 110 ⁇ m to 130 ⁇ m, and the development flow rate of the membrane is 30 to 45 seconds / 40 mm.
- 2. The chromatographic analyzer according to 1, wherein a sample addition unit for adding the sample, a labeling substance holding unit for holding a labeling substance for recognizing a target substance contained in the sample, and the chromatographic medium part are sequentially provided. 3. 3.
- a chromatographic analysis method wherein the following steps (1) to (4) are sequentially performed using the chromatographic analyzer according to the above item 2.
- a step of adding the sample to the sample addition unit (2)
- a step of recognizing a substance to be detected contained in the sample by a labeling substance held in the labeling substance holding unit (3)
- a sample and a labeling substance Step of developing in a chromatographic medium part as a mobile phase (4) Step of detecting a substance to be detected in the developed mobile phase by a detection part 8.
- the average film thickness and the development flow rate of the membrane in the chromatographic medium part are set in a specific range, the adverse effect of the substance that causes the nonspecific reaction in the detection part can be prevented to the maximum. And the detection sensitivity can be increased at the same time.
- FIG. 1 is a cross-sectional view for explaining an example of the structure of a chromatographic analyzer.
- FIG. 2 is a cross-sectional view for explaining an example of the structure of the support and membrane of the chromatographic medium part.
- FIG. 3 is a scanning electron microscope (SEM) photograph of a cross section of the chromatographic medium part of Example 6.
- the chromatographic analyzer and the chromatographic analysis method of the present invention are not particularly limited as long as they use specific binding based on the affinity of biomolecules.
- an immunochromatographic analyzer and method utilizing the binding of an antigen and an antibody a nucleic acid chromatographic analyzer and method utilizing a nucleic acid hybridization, etc., as well as a binding of a sugar and a lectin, a hormone and a receptor
- a chromatographic analysis apparatus and method utilizing the binding of an enzyme and an inhibitor.
- the immunochromatography analyzer of the present invention includes at least a chromatographic medium part on which a detection part for detecting a substance to be detected contained in a specimen is supported, and the membrane in the chromatographic medium part has an average described below. Although it will not restrict
- the immunochromatographic analyzer of the present invention comprises a sample adding part (also called a sample pad) (1), a labeling substance holding part (also called a conjugate pad) (2), and a chromatographic medium part (3).
- the detection unit (4), the absorption unit (5) and the backing sheet (6) are sequentially included.
- the sample addition part (1) is a part to which a sample as a specimen is added in the immunochromatography analyzer.
- the sample addition unit (1) may be any material as long as it is a material used in a normal immunochromatography analyzer. That is, in the sample addition part (1), although a sample is absorbed rapidly, it can be comprised with the porous sheet
- the porous sheet include cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth.
- the labeling substance holding part (2) contains a labeling substance (marker substance), which will be described later, bound in advance to an antibody that binds to the substance to be detected. When the substance to be detected moves in the labeling substance holding part, it binds to the antibody and is labeled.
- the labeling substance holding part (2) is made of, for example, a glass fiber nonwoven fabric or a cellulose film.
- the chromatographic medium part (3) is a developed part of the chromatograph.
- the chromatographic medium part (3) is an inert film made of a microporous material that exhibits capillary action.
- the chromatographic medium part (3) has a structure in which a membrane (34) is provided on a support (32).
- the average film thickness of the membrane (34) is 110 ⁇ m to
- the development flow rate of the membrane (34) is 30 to 45 seconds / 40 mm.
- the membrane (34) When the average film thickness of the membrane (34) is less than 110 ⁇ m, the membrane (34) is detached from the support (32) or easily damaged, which is not practical. On the other hand, when the average film thickness of the membrane (34) exceeds 130 ⁇ m, non-specific reaction with respect to a highly viscous specimen tends to occur.
- the thickness of the membrane (34) can be determined, for example, from the scanning electron micrograph (for example, FIG. 3) of the cross section of the chromatographic medium part (3) comprising the support (32) and the membrane (34). ) And the membrane (34) can be identified, and the thickness of the membrane (34) can be measured.
- each film thickness can be measured with a film thickness measuring instrument such as a meter or a dial gauge, and the film thickness of the membrane (34) can be calculated from the difference in film thickness before and after dissolution.
- a plurality of different locations in the chromatographic medium part (3) are measured (preferably 9 to 10 or more locations), and the average value is calculated as the average film thickness of the membrane (34).
- the average film thickness of the membrane (34) is preferably 110 ⁇ m to 127 ⁇ m, more preferably 110 ⁇ m to 120 ⁇ m, and more preferably 114 ⁇ m to 117 ⁇ m in order to suppress non-specific reactions with higher sensitivity. Things are optimal. Further, if the thickness of the membrane (34) is in the range of 110 ⁇ m to 132 ⁇ m, the effect of the present invention can be sufficiently exerted, and a thickness of 110 ⁇ m to 122 ⁇ m is more preferably used.
- a more preferable development flow rate of the membrane (34) is 35 to 45 seconds / 40 mm.
- deployment flow velocity in this invention means the value obtained by measuring the time which developed 40 mm of water on the membrane in the perpendicular direction.
- the average film thickness of the membrane (34) becomes thin, the average pore diameter of the membrane (34) becomes small and the development flow rate becomes slow. Conversely, as the average film thickness of the membrane (34) increases, the development flow rate increases. In the present invention, the average film thickness of the membrane (34) is made thinner and the development flow rate is set faster than in the conventional chromatographic analyzer. As the average film thickness of the membrane (34) is reduced, the contact probability between the detected substance contained in the specimen and the antibody in the detection part (4) is increased, so that high sensitivity can be achieved and the development flow rate is increased. Thus, the adverse effect of the substance that causes the non-specific reaction in the detection unit (4) can be prevented to the maximum.
- nitrocellulose membrane a membrane made of cellulose acetate
- cellulose acetate membrane a membrane made of cellulose acetate
- Cellulose membranes, nylon membranes and porous plastic cloth polyethylene, polypropylene can also be used.
- nitrocellulose membrane it is only necessary to mainly contain nitrocellulose, and a membrane mainly composed of nitrocellulose such as a pure product or a nitrocellulose mixed product can be used.
- the nitrocellulose membrane exhibits a capillary phenomenon, but can further contain a substance that promotes the capillary phenomenon.
- a substance that lowers the surface tension of the film surface and brings about hydrophilicity is preferable.
- a substance having an amphipathic action such as sugars, amino acid derivatives, fatty acid esters, various synthetic surfactants or alcohols, which has no effect on the movement of the substance to be detected on the immunochromatograph and is a labeling substance
- Substances that do not affect the color development for example, gold particles are preferred.
- the chromatographic medium part (3) has the membrane (34) on the support (32).
- the configuration is provided.
- the support (32) examples include a support made of a water-impermeable plastic and the like, and examples thereof include a film-like support made of polyethylene terephthalate, polyethylene, and polyurethane.
- the support (32) preferably has a color that is not similar to the color caused by the labeling substance, and is usually colorless or white. Is preferred.
- the thickness of the support (32) is, for example, 50 ⁇ m to 130 ⁇ m, preferably 80 ⁇ m to 120 ⁇ m.
- the form and size of the chromatographic medium part (3) represented by the nitrocellulose membrane and cellulose acetate membrane as described above are not particularly limited, and are appropriate in terms of actual operation and observation of reaction results. If it is.
- the detection part (4) is formed on the chromatographic medium part (3), that is, an antibody that specifically binds to the substance to be detected is immobilized at an arbitrary position.
- antibodies include polyclonal antibodies and monoclonal antibodies.
- Monoclonal antibodies and polyclonal antibodies or fragments thereof are known and available, and can be prepared by known methods.
- the antibody can be immobilized at an arbitrary position as an immobilization reagent to form a detection part (4) as a reaction site.
- the immobilization reagent is immobilized on the chromatographic medium part (3) by directly immobilizing the immobilization reagent on the chromatographic medium part (3) by physical or chemical means, and the immobilization reagent is finely divided.
- a direct immobilization method physical adsorption may be used, or covalent bonding may be used. In the case of a nitrocellulose membrane, physical adsorption can be performed. In the covalent bond, cyanogen bromide, glutaraldehyde, carbodiimide or the like is generally used to activate the chromatographic medium part (3), but any method can be used.
- an immobilization reagent is bound to insoluble fine particles and then immobilization to the chromatographic medium part (3).
- the particle size of the insoluble fine particles can be selected so as to be captured by the chromatographic medium part (3) but cannot move, and is preferably fine particles having an average particle size of about 5 ⁇ m or less.
- organic polymers such as organic polymer latex particles obtained by emulsion polymerization methods such as polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate or acrolein-ethylene glycol dimethacrylate copolymer.
- Examples include fine particles of substances, fine particles such as gelatin, bentonite, agarose or cross-linked dextran, inorganic oxides such as silica, silica-alumina or alumina, or inorganic particles obtained by introducing a functional group to an inorganic oxide by silane coupling treatment, etc. .
- direct immobilization is preferable from the viewpoint of ease of sensitivity adjustment.
- Various methods can be used to immobilize the immobilization reagent on the chromatographic medium part (3).
- various techniques such as a microsyringe, a pen with a control pump, or ink jet printing can be used.
- the form of the reaction site is not particularly limited, but it may be fixed as a circular spot, a line extending perpendicularly to the development direction of the chromatographic medium, a numeral, a character, or a symbol such as +,-.
- the chromatographic medium part (3) may be subjected to a blocking treatment by a known method as necessary in order to further prevent the accuracy of the analysis from being lowered due to a nonspecific reaction.
- a blocking treatment by a known method as necessary in order to further prevent the accuracy of the analysis from being lowered due to a nonspecific reaction.
- proteins such as bovine serum albumin, skim milk, casein or gelatin are preferably used for the blocking treatment.
- one or a combination of two or more surfactants such as Tween 20, Triton X-100, or SDS may be washed.
- the absorption part (5) is installed at the end of the chromatographic medium part (3) as necessary to absorb the liquid such as the specimen and the developing solution that has passed through the detection part (4).
- the absorption part (5) contains, for example, a glass fiber, pulp, cellulose fiber or the like, or a non-woven fabric containing a hydrophilic drug having a polymer such as an acrylic acid polymer or an ethylene oxide group. Glass fiber is particularly preferable. When the absorption part (5) is made of glass fiber, the return of the sample liquid can be greatly reduced.
- the backing sheet (6) is a base material. By applying an adhesive on one side or sticking an adhesive tape, one side has adhesiveness, and the sample addition part (1), labeling substance holding part (2), chromatographic medium part ( 3) A part or all of the detection unit (4) and the absorption unit (5) are provided in close contact with each other.
- the backing sheet (6) is not particularly limited as long as the backing sheet (6) becomes impermeable and impermeable to the sample solution by the adhesive.
- the immunochromatographic analysis method of the present invention includes at least a step of detecting a substance to be detected contained in a sample by the detection unit (4) supported on the chromatographic medium unit (3) using the above-described immunochromatography analysis device. Although not particularly limited, it is preferable to sequentially perform the following steps (1) to (4).
- a step of adding the sample to the sample addition unit (2) A step of recognizing a substance to be detected contained in the sample by a labeling substance held in the labeling substance holding unit (3) A sample and a labeling substance Step of developing the chromatographic medium portion as a mobile phase (4) Step of detecting a substance to be detected in the developed mobile phase by the detection portion
- Step of adding specimen to sample addition section first, the concentration is such that the specimen moves smoothly in the immunochromatographic medium section (3) without reducing the measurement accuracy. It is preferable to prepare or dilute the sample with a sample diluent. Secondly, a predetermined amount (usually 0.1 to 2 ml) of the specimen-containing liquid is dropped onto the sample addition part (1). When the specimen-containing liquid is dropped, the specimen-containing liquid starts to move in the sample addition part (1).
- the sample diluent can also be used as a developing solution, but usually water is used as a solvent, and a buffer solution, a salt, and a nonionic surfactant, and further, for example, an antigen-antibody reaction is promoted. Alternatively, one or more of proteins, polymer compounds (PVP, etc.), ionic surfactants or polyanions, antibacterial agents, chelating agents, etc. for suppressing nonspecific reactions may be added.
- a sample and developing solution mixed in advance can be supplied and dropped onto the sample addition unit, or developed, or after the sample is first supplied and dropped onto the sample addition unit. Alternatively, the developing solution may be supplied and dropped on the sample addition unit to be developed.
- specimen containing the substance to be detected examples include biological samples, that is, nasal discharge, sputum, saliva, nasal wipe, pharyngeal wipe, stool, whole blood, serum, plasma, urine, spinal fluid, amniotic fluid, nipple discharge, In addition to tears, sweat, exudates from the skin, extracts from tissues, cells and stool, and the like, extracts from milk, eggs, wheat, beans, beef, pork, chicken, and foods containing them, and the like.
- the specimen is preferably at least one selected from nasal discharge, sputum, saliva, nasal wipe, pharyngeal wipe, and stool. That is, a so-called highly viscous specimen is preferable.
- a high-viscosity specimen contains a viscous substance such as mucin, and it is known that such a viscous substance tends to cause a nonspecific reaction.
- the average film thickness of the membrane (34) is set to 110 ⁇ m to 130 ⁇ m and the development flow rate of the membrane (34) is set to a range of 30 to 45 seconds / 40 mm as described above.
- the substance to be detected include carcinoembryonic antigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-9, ⁇ -fetoprotein (AFP), immunosuppressive acidic protein (IPA), CA15- 3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), syphilis antibody, Examples include, but are not limited to, influenza virus, RS virus, human hemoglobin, chlamydia antigen, group A ⁇ -streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin or glycated albumin.
- fecal occult blood troponin I, troponin T, CK-MB, CRP, influenza virus, RS virus, human hemoglobin, chlamydia antigen, group A ⁇ streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin or Glycated albumin is used as a substance to be detected.
- the labeling substance labels the antibody.
- Enzymes and the like are generally used for labeling the detection reagent in the immunochromatographic analysis method, but it is preferable to use an insoluble carrier as the labeling substance because it is suitable for visually determining the presence of the substance to be detected.
- a labeled detection reagent can be prepared by sensitizing the antibody to an insoluble carrier.
- the means for sensitizing the antibody to the insoluble carrier may be in accordance with a known method.
- the insoluble carrier as a labeling substance, metal particles such as gold, silver or platinum, metal oxide particles such as iron oxide, non-metallic particles such as sulfur and latex particles made of synthetic polymers, or other insoluble carriers are used. be able to.
- the insoluble carrier is a labeling substance suitable for visually determining the presence of the substance to be detected, and is preferably colored in order to facilitate the visual determination.
- the metal particles and the metal oxide particles themselves exhibit a specific natural color corresponding to the particle diameter, and the color can be used as a label.
- the average particle diameter of the gold particles is, for example, 10 nm to 250 nm, preferably 35 nm to 120 nm.
- the average particle diameter was measured by measuring the equivalent area diameter of 100 particles randomly using a projection photograph taken with a transmission electron microscope (TEM: manufactured by JEOL Ltd., JEM-2010). It can be calculated from the value.
- Step (3) is a step in which the substance to be detected is recognized after the substance to be detected is recognized by the labeling substance in the labeling substance holding part in step (2). In this step, the substance is passed through the chromatographic medium as a mobile phase.
- Step (4) is a step in which the substance to be detected in the specimen that has passed as a mobile phase on the chromatographic medium part is a specific antigen / antibody. This is a step in which the detection part is colored by specific reaction and binding so as to be sandwiched between the antibody and the labeling reagent that are held or immobilized on the detection part by the target binding reaction.
- the labeling reagent dissolved in the moisture of the sample does not cause a specific binding reaction even when it passes through the detection part on the chromatographic medium part, so that the detection part is not colored.
- the immunochromatography analyzer and the immunochromatography analysis method have been described as examples.
- the present invention is not limited to the above-described form, and the effect is achieved as long as the specific binding based on the affinity of the biomolecule is used. Can be enjoyed.
- sample addition part The nonwoven fabric (Millipore company_made: 300 mm x 30 mm) which consists of glass fiber was used as a sample addition part.
- labeling substance holding part 0.5 ml of colloidal gold suspension (manufactured by Tanaka Kikinzoku Kogyo Co., Ltd .: LC 40 nm) diluted with phosphate buffer (pH 7.4) to a concentration of 0.05 mg / ml 0.1 ml of the mouse-derived anti-RS virus monoclonal antibody was added and allowed to stand at room temperature for 10 minutes.
- a phosphate buffer pH 7.4 containing 1% by weight of bovine serum albumin (BSA) was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then, after sufficiently stirring, centrifugation was performed at 8000 ⁇ g for 15 minutes to remove the supernatant, and 0.1 ml of a phosphate buffer solution (pH 7.4) containing 1% by mass of BSA was added.
- the labeling substance solution was prepared by the above procedure.
- the labeling substance holding part was produced by drying with a dryer.
- the film thickness is measured at three arbitrary positions from the observation, and the minimum film thickness, the maximum film thickness, and the average film thickness (average film thickness) of the chromatographic medium are measured from the total nine film thicknesses measured.
- 150 ⁇ L of a solution obtained by diluting a mouse-derived anti-RS virus monoclonal antibody with a phosphate buffer solution (pH 7.4) containing 5% by mass of isopropyl alcohol so as to have a concentration of 1.0 mg / ml was dried on the membrane. It is applied to the upper detection site (detection line) with a width of 1 mm, and has a wide affinity for gold nanoparticle labeling substances, etc.
- Sample diluent 1% by mass of nonionic surfactant (manufactured by NOF Corporation, trade name: MN811 and Nacalai Tesque, trade name: NP-40, 1: 1 mixture), 80 mM potassium chloride, 20 mM
- the average film thickness of the membrane is in the range of 110 ⁇ m to 130 ⁇ m, and the development flow rate of the membrane is in the range of 30 to 45 seconds / 40 mm. It was found that the non-specific reaction did not occur even in the analysis used. Sensitivity is also good. On the other hand, in Comparative Examples 1 and 2 in Table 2, the development flow rate is within the range of the present invention, but since the average film thickness of the membrane exceeds the upper limit specified in the present invention, nonspecific reaction is not caused. occured. In Comparative Example 3, the average film thickness of the membrane was within the range of the present invention, but the development flow rate exceeded the upper limit defined by the present invention, so the sensitivity was low compared to the Examples.
- Sample pad 2 Labeling substance holding part 3 Chromatographic medium part 32 Support body 34 Membrane 4 Detection part 5 Absorption part 6 Backing sheet
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Abstract
Description
1.検体に含まれる被検出物質を検出する検出部が担持されたクロマトグラフ媒体部を少なくとも含むクロマト分析装置において、
前記クロマトグラフ媒体部が、支持体上にメンブレンを設けた構成を有し、
前記メンブレンの平均膜厚が、110μm~130μmであり、かつ
前記メンブレンの展開流速が、30~45秒/40mmであるクロマト分析装置。
2.前記検体を添加する試料添加部と、前記検体に含まれる被検出物質を認識する標識物質を保持する標識物質保持部と、前記クロマトグラフ媒体部とを順次含む前記1に記載のクロマト分析装置。
3.前記メンブレンが、ニトロセルロースメンブレンである前記1または2に記載のクロマト分析装置。
4.前記検体が、鼻汁、喀痰、唾液、鼻腔拭い液、咽頭拭い液、便から選択される少なくとも1種である前記1~3のいずれか1に記載のクロマト分析装置。
5.免疫クロマト分析装置である前記1~4のいずれか1に記載のクロマト分析装置。
6.前記1~5のいずれか1に記載のクロマト分析装置を用い、前記クロマトグラフ媒体部に担持された検出部によって、前記検体に含まれる被検出物質を検出する工程を少なくとも有するクロマト分析方法。
7.前記2に記載のクロマト分析装置を用い、下記工程(1)~(4)を順次実施する、クロマト分析方法。
(1)前記検体を試料添加部に添加する工程
(2)前記標識物質保持部に保持されている標識物質により前記検体に含まれる被検出物質を認識させる工程
(3)前記検体および標識物質を移動相としてクロマトグラフ媒体部に展開させる工程
(4)展開された移動相中の被検出物質を検出部で検出する工程
8.前記メンブレンが、ニトロセルロースメンブレンである前記7に記載のクロマト分析方法。
9.前記検体が、鼻汁、喀痰、唾液、鼻腔拭い液、咽頭拭い液、便から選択される少なくとも1種である前記6~8のいずれか1に記載のクロマト分析方法。
10.前記クロマト分析装置が、免疫クロマト分析装置である前記6~9のいずれか1に記載のクロマト分析方法。
なお、本発明における展開流速は、メンブレン上に水を垂直方向に40mm展開させた時間を測定することにより得られた値を意味する。
(2)前記標識物質保持部に保持されている標識物質により前記検体に含まれる被検出物質を認識させる工程
(3)前記検体および標識物質を移動相としてクロマトグラフ媒体部に展開させる工程
(4)展開された移動相中の被検出物質を検出部で検出する工程
各工程について以下に説明する。
工程(1)では、第1に、検体を、測定精度を低下させることなく、免疫クロマトグラフ媒体部(3)中をスムーズに移動する程度の濃度に検体希釈液で調整または希釈して検体含有液とするのが好ましい。第2に、該検体含有液を試料添加部(1)上に、所定量(通常、0.1~2ml)滴下する。検体含有液が滴下されると、検体含有液は試料添加部(1)中で移動を開始する。
工程(2)は、工程(1)において試料添加部に添加された検体含有液を、標識物質保持部(2)へと移動させ、標識物質保持部に保持されている標識物質により検体中の被検出物質を認識させる工程である。
工程(3)は、工程(2)において被検出物質が標識物質保持部において標識物質に認識された後、検体および標識物質を、クロマトグラフ媒体部上を移動相として通過させる工程である。
工程(4)は、クロマトグラフ媒体部上を移動相として通過した検体中の被検出物質が、抗原・抗体の特異的結合反応により、検出部に保持、即ち、担持固定されている抗体と標識試薬とによってサンドイッチ状に挟まれるように特異的に反応結合して、検出部が着色する工程である。
被検出物質が存在しない場合には、試料の水分に溶解した標識試薬は、クロマトグラフ媒体部上の検出部を通過しても特異的結合反応が起こらないので、検出部が着色しない。
試料添加部としてグラスファイバーからなる不織布(ミリポア社製:300mm×30mm)を用いた。
(2)標識物質保持部の作製
金コロイド懸濁液(田中貴金属工業社製:LC40nm)0.5mlに、リン酸緩衝液(pH7.4)で0.05mg/mlの濃度になるように希釈したマウス由来抗RSウイルスモノクローナル抗体を0.1ml加え、室温で10分間静置した。
次いで、1質量%の牛血清アルブミン(BSA)を含むリン酸緩衝液(pH7.4)を0.1ml加え、更に室温で10分間静置した。その後、十分撹拌した後、8000×gで15分間遠心分離を行い、上清を除去した後、1質量%のBSAを含むリン酸緩衝液(pH7.4)を0.1ml加えた。以上の手順で標識物質溶液を作製した。
上記作製した標識物質溶液300μLに300μLの10質量%トレハロース水溶液と1.8mLの蒸留水を加えたものを15mm×300mmのグラスファイバーパッド(ミリポア社製)に均一になるように添加した後、真空乾燥機にて乾燥させ、標識物質保持部を作製した。
ポリエチレンテレフタラート製の厚さ100μmの支持体上に、ニトロセルロースからなりかつ下記表1及び表2に示す厚さおよび展開流速を有するメンブレン(300mm×25mm)を積層し、クロマトグラフ媒体部とした。表1及び表2に示すメンブレンの厚さは、後の免疫クロマト分析装置の作成後(裁断後)、免疫クロマト分析装置内のクロマトグラフ媒体部の走査型電子顕微鏡写真による任意の3箇所の断面観察から夫々任意の3箇所の膜厚測定を行い、測定された合計9箇所の膜厚からクロマトグラフ媒体の最小値の膜厚、最大値の膜厚および平均値の膜厚(平均膜厚)を算出した。
次に、5質量%のイソプロピルアルコールを含むリン酸緩衝液(pH7.4)で1.0mg/mlの濃度になるようにマウス由来抗RSウイルスモノクローナル抗体を希釈した溶液150μLを、乾燥されたメンブレン上の検出部位(検出ライン)に1mmの幅で塗布し、金ナノ粒子標識試薬の展開の有無や展開速度を確認するために検出部位の下流に、金ナノ粒子標識物質などと広く親和性を有するヤギ由来抗血清をリン酸緩衝液(pH7.4)で希釈した液をコントロール部位(コントロールライン)に塗布した。その後、50℃で30分間乾燥させ、室温で一晩乾燥させ、クロマトグラフ媒体部上に検出部を作製した。
次に、バッキングシートから成る基材に、試料添加部、標識物質保持部、検出部が担持されたクロマトグラフ媒体部、展開した試料や標識物質を吸収するための吸収部としてグラスファイバー製の不織布を順次貼り合わせた。そして、裁断機で幅が5mmとなるように裁断し、免疫クロマト分析装置とした。なお、標識物質保持部の試料展開方向の長さを8mmとした。
1質量%の非イオン界面活性剤(日油株式会社製、商品名:MN811とナカライテスク社製、商品名NP-40の1:1混合物)、80mMの塩化カリウム、20mMのグアニジン塩酸塩、0.4重量%のポリビニルピロリドン(平均分子量36万)を含む50mMのHEPES緩衝液(pH7.5)を調製し、検体を希釈処理するための試薬とした。
RSウイルス陰性の臨床実検体鼻汁を前記検体希釈液で希釈し10%の濃度とし検体とした。この検体の120μlを免疫クロマト分析装置の試料添加部上に載せて展開させ、15分経過後に、検出部の着色の度合いを目視で確認し、以下の評価基準によって非特異反応の有無を評価した。
++:はっきりしたラインが確認された。
+:薄いラインが確認された。
-:ラインは確認されなかった。
これに対し、表2における比較例1~2は、展開流速は本発明の範囲内であるが、メンブレンの平均膜厚がいずれも本発明で規定する上限を超えているので、非特異反応が生じた。比較例3は、メンブレンの平均膜厚は本発明の範囲内であるが、展開流速が本発明で規定する上限を超えているので、感度が実施例と比較して低かった。
なお、前記検体希釈液のみを検体として上記試験を繰り返した場合は、感度および非特異反応ともに、いずれも「-」評価であった。
更に、実施例1~6について、夫々繰り返し50回測定した場合、実施例4~6では非特異反応は検出されなかったが、実施例1~3では、1~2回の非特異反応が検出された。
2 標識物質保持部
3 クロマトグラフ媒体部
32 支持体
34 メンブレン
4 検出部
5 吸収部
6 バッキングシート
Claims (10)
- 検体に含まれる被検出物質を検出する検出部が担持されたクロマトグラフ媒体部を少なくとも含むクロマト分析装置において、
前記クロマトグラフ媒体部が、支持体上にメンブレンを設けた構成を有し、
前記メンブレンの平均膜厚が、110μm~130μmであり、かつ
前記メンブレンの展開流速が、30~45秒/40mmであるクロマト分析装置。 - 前記検体を添加する試料添加部と、前記検体に含まれる被検出物質を認識する標識物質を保持する標識物質保持部と、前記クロマトグラフ媒体部とを順次含む請求項1に記載のクロマト分析装置。
- 前記メンブレンが、ニトロセルロースメンブレンである請求項1または2に記載のクロマト分析装置。
- 前記検体が、鼻汁、喀痰、唾液、鼻腔拭い液、咽頭拭い液、便から選択される少なくとも1種である請求項1~3のいずれか1項に記載のクロマト分析装置。
- 免疫クロマト分析装置である請求項1~4のいずれか1項に記載のクロマト分析装置。
- 請求項1~5のいずれか1項に記載のクロマト分析装置を用い、前記クロマトグラフ媒体部に担持された検出部によって、前記検体に含まれる被検出物質を検出する工程を少なくとも有するクロマト分析方法。
- 請求項2に記載のクロマト分析装置を用い、下記工程(1)~(4)を順次実施する、クロマト分析方法。
(1)前記検体を試料添加部に添加する工程
(2)前記標識物質保持部に保持されている標識物質により前記検体に含まれる被検出物質を認識させる工程
(3)前記検体および標識物質を移動相としてクロマトグラフ媒体部に展開させる工程
(4)展開された移動相中の被検出物質を検出部で検出する工程 - 前記メンブレンが、ニトロセルロースメンブレンである請求項7に記載のクロマト分析方法。
- 前記検体が、鼻汁、喀痰、唾液、鼻腔拭い液、咽頭拭い液、便から選択される少なくとも1種である請求項6~8のいずれか1項に記載のクロマト分析方法。
- 前記クロマト分析装置が、免疫クロマト分析装置である請求項6~9のいずれか1項に記載のクロマト分析方法。
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CN107709994A (zh) | 2018-02-16 |
KR20180014758A (ko) | 2018-02-09 |
US20180284115A1 (en) | 2018-10-04 |
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