WO2017126593A1 - クロマトグラフ媒体 - Google Patents
クロマトグラフ媒体 Download PDFInfo
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- WO2017126593A1 WO2017126593A1 PCT/JP2017/001698 JP2017001698W WO2017126593A1 WO 2017126593 A1 WO2017126593 A1 WO 2017126593A1 JP 2017001698 W JP2017001698 W JP 2017001698W WO 2017126593 A1 WO2017126593 A1 WO 2017126593A1
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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
- 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
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
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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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
Definitions
- the present invention relates to a chromatographic medium, an immunochromatographic apparatus, an immunochromatographic kit, and an immunochromatographic analysis method.
- an immunoassay based on an immunochromatography method that does not require pretreatment of a specimen is a simple in vitro diagnostic kit or portable diagnostic device that detects an antigen in a sample solution using, for example, specific reactivity of an antibody.
- pathogen testing kits such as viruses and bacteria are familiar immunochromatographic devices widely used in general hospitals and clinics.
- the simplest structure of a conventional immunochromatography apparatus is that a sample addition unit, a labeling substance holding unit, a chromatographic medium having a detection unit (chromatographic medium unit), and an absorption unit that absorbs liquid that has passed through the detection unit are mutually connected. It is a structure connected to.
- a protein such as an antibody that binds to the detection target substance is fixed to the detection part of the chromatographic medium.
- This protein is denatured depending on temperature conditions and humidity conditions, and the detection sensitivity of the detection target is lowered. Therefore, there is a problem that the immunochromatograph apparatus cannot be stably stored for a long time. Therefore, various studies have been made on means for suppressing denaturation of proteins contained in the detection part of the chromatographic medium and enhancing storage stability.
- Patent Document 1 discloses an immunochromatography apparatus that uses a sugar or a sugar derivative in a solid phase such as a detection unit of an immunochromatography apparatus to keep the humidity in the apparatus moderate and has storage stability. Yes. Further, Non-Patent Document 1 discloses that the storage stability of an antibody is improved by adding an amino acid such as glycine or lysine or a sugar such as sucrose or trehalose to the membrane of a chromatographic medium, thereby increasing the storage stability of the antibody. An immunochromatographic apparatus in which the decrease is suppressed is disclosed.
- An object of the present invention is to provide a chromatographic medium having sufficiently improved storage stability.
- the inventors of the present invention in the immunochromatography apparatus used in the immunochromatography method, by including both a specific saccharide and a basic amino acid in the detection part of the chromatographic medium, it is found that it is possible to sufficiently suppress the denaturation of the protein that is the detection target immobilized on the detection unit, sufficiently increase the storage stability, prevent the decrease in the activity of the detection substance, and further improve the activity of the detection substance. We have also found out that it is possible to complete the present invention.
- a chromatographic medium comprising: 2.
- the polysaccharide contained in the detection unit is at least one polysaccharide selected from the group consisting of raffinose, nigerotriose, maltotriose, melezitose, maltotriurose, kestose, nystose, nigerotetraose, stachyose and maltotetraose. 2.
- An immunochromatography apparatus comprising the chromatographic medium described in any one of 1 to 9 above.
- An immunochromatography kit comprising the immunochromatography apparatus according to 10 and a specimen diluent. 12
- a step of adding a specimen together with a specimen diluent to a sample addition section (2) A step of recognizing a substance to be detected contained in the specimen by a labeling substance held in a labeling substance holding section (3) The specimen and label The step of developing a substance as a mobile phase on a chromatographic medium (4) The step of detecting a substance to be detected in the developed mobile phase by a detection unit
- a chromatographic medium that sufficiently suppresses denaturation of a protein to be detected fixed to a detection unit, has sufficiently high storage stability, and can prevent a decrease in activity of a detection substance. Can do. In addition, the activity of the detection substance can be improved.
- FIG. 1 is a cross-sectional view for explaining the structure of an immunochromatography apparatus.
- FIG. 2 is a graph showing the relative activity of the color value of the detection unit in Examples 1 and 2 and Comparative Examples 1 and 2.
- FIG. 3 is a graph showing the relative activity of the color value of the detection part after 2 days of humidification in Examples 3 to 6 and Comparative Examples 3 to 10.
- fixed means that the antibody or the like is arranged on a carrier such as a membrane so that the antibody or the like does not move
- hold means that the antibody or the like is in a carrier such as a membrane or the like. It means that the surface is movably arranged.
- the chromatographic medium of the present invention has a detection part to which a detection substance made of protein is fixed, and the detection part contains a polysaccharide of trisaccharide or more and a basic amino acid.
- the detection unit is formed on the chromatographic medium. That is, a protein that is a detection substance that binds to a detection target substance is immobilized at an arbitrary position.
- the detection substance in the detection part has a moisture resistance by containing a polysaccharide of trisaccharide or more in the detection part, and as a result, protein denaturation is suppressed. , The storage stability is increased, and the decrease in the activity of the detection substance can be prevented.
- polysaccharides higher effects can be obtained as compared with monosaccharides and disaccharides by being a saccharide of trisaccharide or more having many hydroxyl groups capable of hydrogen bonding in one molecule.
- the chromatographic medium of the present invention is not sure about the mechanism of action that can increase the storage stability of the detection substance by including a polysaccharide of trisaccharide or higher in the detection part, the polysaccharide of trisaccharide or higher
- the polysaccharide of trisaccharide or higher By maintaining the hydrogen bonding network of the protein of the detection substance, when the bound water is dissociated from the protein of the detection substance by drying, the three-dimensional structure of the protein is prevented from being destroyed, and the protein is prevented from denaturing and thus stable storage. It is thought that this is because the property can be increased and the decrease in activity can be prevented.
- the polysaccharide is not particularly limited as long as it is a trisaccharide or more. Examples thereof include the following trisaccharides or tetrasaccharides. Examples of the trisaccharide include raffinose, nigerotriose, maltotriose, melezitose, maltotriurose, and kestose. Examples of the tetrasaccharide include nystose, nigerotetraose, stachyose and maltotetraose.
- trisaccharide or tetrasaccharide is preferable.
- at least one polysaccharide selected from the group consisting of raffinose, nigerotriose, maltotriose, melezitose, maltotriulose, kestose, nystose, nigerotetraose, stachyose and maltotetraose is preferable. More preferred is a trisaccharide, and among the trisaccharides, raffinose is still more preferred.
- the detection part of the chromatographic medium of the present invention may contain one kind of the above sugars or a mixture of two or more kinds.
- the content of polysaccharide in the detection part is, for example, 5 to 50 nmol, preferably 10 to 40 nmol, more preferably 20 to 30 nmol per chromatographic medium. By setting it in the above range, it is possible to sufficiently enhance storage stability and prevent a decrease in activity, and to further suppress reaction inhibition between the substance to be detected and the detection substance.
- the chromatographic medium of the present invention contains the basic amino acid together with the polysaccharide in the detection part, so that the protein as the detection substance in the detection part has moisture resistance, and as a result, denaturation of the protein. Can be suppressed, storage stability can be improved, and a decrease in the activity of the detection substance can be prevented. Moreover, a higher effect can be acquired compared with another amino acid by being a basic amino acid.
- the action mechanism that can enhance the storage stability of the detection substance by containing a basic amino acid in the detection part is not clear, but the basic amino acid is the detection substance. This is considered to be because it interacts weakly with the protein, suppresses the aggregation and aggregation of the proteins, and prevents the activity of the detection substance from decreasing.
- the type of basic amino acid is not particularly limited, and examples thereof include arginine, lysine, ornithine, and histidine. From the viewpoint of enhancing the storage stability and preventing the decrease in activity, lysine and arginine are preferably used, and lysine is optimally used.
- one kind of the above basic amino acids may be contained, or two or more kinds thereof may be mixed and contained.
- the content of basic amino acid in the detection part is, for example, 5 to 50 nmol, preferably 10 to 40 nmol, more preferably 15 to 30 nmol per chromatographic medium. It is because the fall of the activity of a detection substance can be prevented more by setting it as the said range.
- the detection part contains both a polysaccharide of tri- or higher sugars and a basic amino acid, and the storage stability of the detection substance is remarkably improved as compared with the case where each of them is contained alone. To do.
- the detection part contains both a polysaccharide of tri- or higher sugars and a basic amino acid, so that the polysaccharide forms a hydrogen bond network and maintains a correct three-dimensional structure, and is basic. It is presumed that the activity is maintained for a long time by suppressing the aggregation of antibodies due to weak interaction between amino acids and antibodies.
- the basic amino acid can moderately loosen the association between the polysaccharide and the detection substance without losing the effect of the storage stability due to the polysaccharide when detecting the detection substance with the detection substance, This is presumably because a decrease in reaction activity with the detection substance can also be suppressed.
- the ratio of the polysaccharide to the basic amino acid to be contained in the detection unit is usually 1:10 to 10: 1, preferably 1: 4 to 4: 1, more preferably in terms of mass (mole). 4: 5 to 5: 4.
- the synergistic effect obtained by including both a polysaccharide and a basic amino acid becomes more effective.
- the protein that is a detection substance immobilized on the detection part of the chromatographic medium of the present invention is not particularly limited as long as it is a protein that specifically binds to the detection target, and examples thereof include antibodies, antigens, and hormones.
- antibodies include polyclonal antibodies or monoclonal antibodies and fragments thereof.
- monoclonal antibody and the polyclonal antibody or a fragment thereof known and available antibodies can be used, and they can be prepared by a known method.
- the amount of antibody immobilized per chromatographic medium is, for example, 0.1 to 1 ⁇ g, preferably 0.2 to 0.6 ⁇ g, and more preferably 0.3 to 0.4 ⁇ g.
- the chromatographic medium of the present invention can be used as a development site for an immunochromatographic apparatus.
- the chromatographic medium is an inert film made of a microporous material that exhibits capillary action.
- a membrane made of nitrocellulose hereinafter referred to as a nitrocellulose membrane
- a cellulose acetate membrane hereinafter sometimes referred to as “cellulose acetate membrane”
- a nitrocellulose membrane is more preferable.
- Cellulose membranes, nylon membranes and porous plastic cloth polyethylene, polypropylene
- 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 may further contain a substance that promotes capillary action.
- a substance that lowers the surface tension of the film surface and brings about hydrophilicity is preferable.
- various synthetic surfactants or substances that have amphiphilic action such as alcohol, have no effect on the movement of the substance to be detected on the chromatographic medium, and affect the color development of marker substances such as colloidal gold. Substances that do not reach are preferred.
- Nitrocellulose membrane is porous and exhibits capillary action.
- the index of the capillary phenomenon can be confirmed by measuring the water absorption rate (water absorption time: capillary flow time).
- the water absorption rate affects detection sensitivity and inspection time.
- the form and size of the chromatographic medium represented by the above nitrocellulose membrane and cellulose acetate membrane are not particularly limited, and may be appropriate in terms of actual operation and observation of reaction results. .
- the chromatographic medium of the present invention can be subjected to a blocking process by a known method, if necessary, in order to prevent a decrease in analysis accuracy due to nonspecific adsorption.
- a protein such as bovine serum albumin, skim milk, casein or gelatin is preferably used for the blocking treatment.
- blocking treatment for example, polyethylene glycol sorbitan monolaurate (for example, Tween 20: manufactured by Wako Pure Chemical Industries), polyoxyethylene octyl phenyl ether (for example, Triton X-100: manufactured by Wako Pure Chemical Industries) or You may wash
- surfactants such as sodium dodecyl sulfate
- the detection part in the chromatographic medium of the present invention is formed on the chromatographic medium. That is, the protein that specifically binds to the substance to be detected is immobilized at an arbitrary position.
- the specimen containing the substance to be detected is not particularly limited.
- biological samples that is, whole blood, serum, plasma, urine, saliva, sputum, nasal cavity or throat swab, cerebrospinal fluid, amniotic fluid, nipple discharge, tears
- extracts from milk, eggs, wheat, beans, beef, pork, chicken and foods containing them can be used.
- 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 , Influenza virus, human hemoglobin, chlamydia antigen, group A ⁇ -streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, or glycoproteins such as glycated albumin and glycated hemoglobin. Is not to be done.
- glycoprotein is preferable, glycated hemoglobin is more preferable, and HbA1c is particularly preferable.
- the HbA1c antibody needs to recognize a slight difference between a state in which glucose is bound to hemoglobin and a state in which it does not bind, and its steric structure must be strictly maintained. Therefore, when the detected substance is HbA1c, This is because the effects of the present invention function particularly effectively.
- the immunochromatography apparatus of the present invention in addition to the above-mentioned chromatographic medium and detection unit, it is usually composed of a sample addition unit, a labeling substance holding unit, an absorption unit, and a backing sheet.
- a sample addition unit a labeling substance holding unit
- an absorption unit a labeling substance holding unit
- a backing sheet a backing sheet.
- the order in which each part is provided is, in the order in which the sample is developed, the sample addition unit (1), the labeling substance holding unit (2), the chromatographic medium (also referred to as chromatographic medium unit) (3), and the detection unit (4). It is an absorption part (5).
- the sample addition part (1) is a part to which a specimen sample is added in the immunochromatography apparatus.
- the sample is rapidly absorbed, but it can be constituted by a porous sheet having such a property that the sample moves quickly.
- 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, 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 (3) and the detection unit (4) use the above-described chromatographic medium of the present invention.
- 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 absorbent portion (5) is made of, for example, glass fiber, pulp, cellulose fiber, or the like, or a non-woven fabric such as a polymer such as an acrylic acid polymer, a hydrophilic drug having an ethylene oxide group or the like. What was contained can be used. Glass fiber is 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) and part or all of the absorption part (5) is provided in close contact.
- 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 present invention can be combined with a sample diluent and the above immunochromatograph apparatus to form an immunochromatograph kit.
- 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. Or you may add 1 type, or 2 or more types, such as a protein for suppressing a nonspecific reaction, high molecular compounds (PVP etc.), an ionic surfactant, or a polyanion, or an antibacterial agent, a chelating agent.
- PVP high molecular compounds
- a sample and a developing solution mixed in advance can be supplied and added to the sample addition section, or developed, or after the sample is first supplied and added to the sample addition section.
- the developing solution may be supplied and added onto the sample addition unit to be developed.
- the following steps (1) to (4) are sequentially performed, and a substance to be detected contained in a specimen is detected using the immunochromatographic kit.
- a step of adding a specimen together with a specimen diluent to a sample addition section (2)
- a step of recognizing a substance to be detected contained in the specimen by a labeling substance held in a labeling substance holding section (3)
- the specimen and label Step of developing a substance as a mobile phase on a chromatographic medium (4) Step of detecting a detection target substance in the developed mobile phase by a detection unit Each step will be described below.
- Step (1) Step of adding a specimen together with a specimen diluent to the sample addition section
- the specimen is adjusted to a concentration that allows the specimen to move smoothly through the chromatographic medium without reducing measurement accuracy. It is preferable to prepare or dilute with a specimen diluent to prepare a specimen-containing liquid.
- a predetermined amount usually 0.1 to 2 ml
- the specimen-containing liquid starts to move in the sample addition section (1).
- the labeling substance labels the antibody.
- An enzyme or the like is generally used for labeling the detection reagent in the immunochromatography method. However, since it is suitable for visually determining the presence of the substance to be detected, it is preferable to use an insoluble carrier as the labeling substance.
- 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.
- metal particles such as gold, silver or platinum, metal oxide particles such as iron oxide, non-metal particles such as sulfur and latex particles composed of synthetic polymers, or other materials should be used. Can do.
- the insoluble carrier is a labeling substance suitable for visually determining the presence of the substance to be detected, and is preferably colored to facilitate 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.
- An insoluble carrier as a labeling substance is particularly preferable because gold particles are easy to detect and difficult to agglutinate and cause nonspecific color development.
- 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 with a transmission electron microscope (TEM: manufactured by JEOL Ltd., JEM-2010), and the projected area circle equivalent diameter of 100 particles was measured randomly using a photograph taken. It can be calculated from the average value.
- step (3) Step of developing specimen and labeling substance on chromatographic medium as mobile phase
- step (3) after the substance to be detected is recognized by the labeling substance in the labeling substance holding part in step (2), the specimen and labeling are performed.
- step (4) A step of detecting a substance to be detected in the developed mobile phase by the detection unit
- the substance to be detected in the specimen that has passed through the chromatographic medium as a mobile phase is a protein that is a detection substance.
- the detection part is colored by specifically reacting and binding so as to be sandwiched between the protein immobilized on the detection part and the labeling reagent.
- 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, so that the detection part is not colored.
- the detection unit contains a polysaccharide of trisaccharide or more and a basic amino acid, the denaturation of the protein to be detected fixed to the detection unit is sufficiently suppressed, the storage stability is sufficiently high, and the detection substance It is possible to prevent a decrease in activity.
- Example 1 Preparation of immunochromatographic kit (1) Preparation of chromatographic medium and detection part A sheet made of nitrocellulose (Millipore, trade name: HF75, 300 mm x 25 mm) was used as a membrane used for the chromatographic medium. Next, 150 ⁇ L of a solution obtained by diluting the anti-HbA1c monoclonal antibody (first antibody) to a concentration of 1.0 mg / ml with a phosphate buffer solution (pH 7.4) containing 5% by mass of isopropyl alcohol is dried. 300 mm was applied to the detection part (detection line) on the membrane with a width of 1 mm.
- a phosphate buffer solution pH 7.4
- a solution having a raffinose concentration of 8 mmol / mL (20 nmol per chromatographic medium) and 10 mmol / mL (concentration of 25 nmol per chromatographic medium) was prepared.
- a goat-derived antibody having a wide affinity with a gold nanoparticle labeling substance or animal meat protein as a detection target is provided downstream of the detection unit.
- a solution obtained by diluting serum with a phosphate buffer (pH 7.4) was applied to a control site (control line). Then, it was dried at 50 ° C. for 30 minutes and dried overnight at room temperature to prepare a chromatographic medium and a detection unit.
- the labeling substance solution was prepared by the above procedure. After adding 300 ⁇ L of a 10 mass% trehalose aqueous solution and 1.8 mL of distilled water to 300 ⁇ L of the prepared labeling substance solution to a 15 mm ⁇ 300 mm glass fiber pad (Millipore), vacuum is added.
- the labeling substance holding part was produced by drying with a dryer.
- an immunochromatography apparatus as shown in FIG. 1 was produced using the produced chromatographic medium, detection section, sample addition section, and labeling substance holding section.
- a non-woven fabric made of glass fiber was sequentially bonded to a substrate made of a backing sheet as a chromatographic medium having a sample addition part, a labeling substance holding part and a detection part, and an absorbent part for absorbing the developed sample and labeling substance.
- the length of the labeling substance holding part and the incubation part in the sample development direction was 8 mm.
- Sample dilution 1% by weight of a nonionic surfactant (manufactured by NOF Corporation, trade name: MN811 and Nacalai Tesque, trade name: 1: 1 mixture of NP-40), 80 mM potassium chloride, A 50 mM HEPES buffer solution (pH 7.5) containing 20 mM guanidine hydrochloride and 0.4% by weight polyvinylpyrrolidone (average molecular weight 360,000) was prepared and used as a reagent for subjecting the specimen to dilution treatment.
- a nonionic surfactant manufactured by NOF Corporation, trade name: MN811 and Nacalai Tesque, trade name: 1: 1 mixture of NP-40
- a 50 mM HEPES buffer solution pH 7.5
- 20 mM guanidine hydrochloride 20 mM guanidine hydrochloride
- polyvinylpyrrolidone average molecular weight 360,000
- the immunochromatograph apparatus prepared above was placed in a humidifier and left for the time shown in Table 1 at a humidity of 95% and a temperature of 37 ° C. Thereafter, blood was collected by puncturing the fingertips of healthy adult males and diabetic male patients, and the blood samples obtained by measuring the concentrations of HbA0 and HbA1c by the latex agglutination method had a HbA1c concentration of 6.5%.
- the color development value was measured using a sample (negative sample) consisting of only HbA0 with a HbA1c concentration of 0% corresponding to each Example / Comparative Example, but all showed measured values of less than 5 mAbs and were false. No positive was confirmed.
- Example 2 Example 1 was repeated except that “anti-influenza antibody” was used in place of the “anti-HbA1c monoclonal antibody” of the first antibody and the second antibody, and the specimen sample was changed to an influenza antigen. The degree of coloring (color value) of the detection part was measured with a densitometer. The results are shown in Table 1 and FIG.
- Example 1 Example 1 was repeated except that “sucrose” was used in place of “lysine and raffinose” in the detection part.
- sucrose concentration of 10 mmol / mL (concentration of 20 nmol per chromatographic medium) is used, and the degree of coloring (color value) of the detection part is measured with a densitometer. did.
- the results are shown in Table 2 and FIG.
- Example 2 was repeated except that “sucrose” was used in place of “lysine and raffinose” in the detector.
- sucrose concentration of 10 mmol / mL (concentration of 20 nmol per chromatographic medium) is used, and the degree of coloring (color value) of the detection part is measured with a densitometer. did. The results are shown in Table 2 and FIG.
- Examples 1 and 2 contain the basic amino acid lysine and the trisaccharide raffinose in the detection part. Therefore, when left for a certain period of time in an environment of 95% humidity and a temperature of 37 ° C. The decrease in the activity of the detection substance was suppressed. In Example 1 in which HbA1c was used as the substance to be detected, the result that the activity of the detection substance was improved was obtained. On the other hand, in Table 2 and FIG. 2, in Comparative Examples 1 and 2 containing only sucrose, which is a disaccharide, the activity of the detection substance decreased.
- Example 3 The immunochromatography apparatus used in Example 1 was left for 2 days in a humidified environment of temperature: 24-26 ° C. and humidity: 40-70%.
- the specimen used is the same HbA1c as in Example 1, and the degree of coloration (color value) of the detection part in the immunochromatography apparatus before being left and the degree of coloration (coloration value) of the detection part in the immunochromatography apparatus after being left for two days was measured with a densitometer.
- the results are shown in Table 3 and FIG.
- “relative activity” indicates the ratio of the color value measured in the immunochromatography apparatus after standing for 2 days, when the color development value measured in the immunochromatography apparatus before standing as 100%.
- Example 4 In Example 3, Example 3 was repeated except that the amounts of lysine and raffinose applied to the detection part of the immunochromatograph apparatus were changed as shown in Table 3. The results are shown in Table 3 and FIG.
- Example 5 In Example 3, Example 3 was repeated except that the amounts of arginine and raffinose shown in Table 3 were applied to the detection part of the immunochromatograph apparatus. The results are shown in Table 3 and FIG.
- Example 6 In Example 3, Example 3 was repeated except that lysine and maltotetraose in the amounts shown in Table 3 were applied to the detection part of the immunochromatograph apparatus. The results are shown in Table 3 and FIG.
- Example 3 Example 3 was repeated except that the amount of glycine shown in Table 3 was applied to the detection part of the immunochromatograph apparatus. The results are shown in Table 3 and FIG.
- Example 4 Example 3 was repeated except that the amount of arginine shown in Table 3 was applied to the detection part of the immunochromatograph apparatus. The results are shown in Table 3 and FIG.
- Example 5 Example 3 was repeated except that the amount of lysine shown in Table 3 was applied to the detection part of the immunochromatography apparatus. The results are shown in Table 3 and FIG.
- Example 6 Example 3 was repeated except that the amount of mannitol shown in Table 3 was applied to the detection part of the immunochromatography apparatus. The results are shown in Table 3 and FIG.
- Example 7 Example 3 was repeated except that the amount of sucrose shown in Table 3 was applied to the detection part of the immunochromatography apparatus. The results are shown in Table 3 and FIG.
- Example 8 In Example 3, Example 3 was repeated except that the amount of raffinose shown in Table 3 was applied to the detection part of the immunochromatograph apparatus. The results are shown in Table 3 and FIG.
- Example 9 Example 3 was repeated except that the amount of maltotetraose shown in Table 3 was applied to the detection part of the immunochromatography apparatus. The results are shown in Table 3 and FIG.
- Example 10 Example 3 was repeated except that the amounts of glycine and raffinose shown in Table 3 were applied to the detection part of the immunochromatograph apparatus. The results are shown in Table 3 and FIG.
- Examples 3 to 6 contained basic amino acids lysine and arginine, and polysaccharides (raffinose, maltotetraose) higher than trisaccharide in the detection part, and were left for 2 days. In this case, the decrease in the activity of the detection substance was suppressed. In particular, in Examples 3 and 4 containing both lysine and raffinose in the detection part, the decrease in the activity of the detection substance was significantly suppressed.
- Comparative Example 3 containing only the neutral amino acid glycine
- Comparative Examples 4 and 5 containing only the basic amino acids arginine and lysine
- Comparative Examples 8 and 9 containing only the trisaccharide raffinose and the tetrasaccharide maltotetraose
- the activity of the detection substance was significantly reduced.
- Comparative Example 10 containing the neutral amino acid glycine and the trisaccharide raffinose
- the activity of the detection substance was significantly reduced. From the above results, it was found that the decrease in the activity of the detection substance was significantly suppressed by including both the basic amino acid and the polysaccharide of trisaccharide or higher in the detection part.
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Abstract
Description
また、非特許文献1には、クロマトグラフ媒体のメンブレンに、グリシンやリシン等のアミノ酸や、スクロースやトレハロース等の糖をそれぞれ単独で含有させることによって、抗体の保存安定性を高め、検出感度の低下を抑制させたイムノクロマトグラフ装置が開示されている。
1.検体中の被検出物質を検出するイムノクロマトグラフィー法に用いられ、タンパク質からなる検出物質が固定された検出部を有するクロマトグラフ媒体であって、前記検出部に三糖以上の多糖類と塩基性アミノ酸を含むクロマトグラフ媒体。
2.検出部に含まれる前記多糖類が、ラフィノース、ニゲロトリオース、マルトトリオース、メレジトース、マルトトリウロース、ケストース、ニストース、ニゲロテトラオース、スタキオース及びマルトテトラオースからなる群より選ばれる少なくとも1つの多糖類である前記1に記載のクロマトグラフ媒体。
3.検出部に前記多糖類を5~50nmol含む前記1または2に記載のクロマトグラフ媒体。
4.検出部に前記塩基性アミノ酸を5~50nmol含む前記1~3のいずれか1に記載のクロマトグラフ媒体。
5.検出部に含まれる前記多糖類と前記塩基性アミノ酸のモル比が、1:10~10:1である前記1~4のいずれか1に記載のクロマトグラフ媒体。
6.検出部が、アルギニン、リジン、オルニチン、及びヒスチジンからなる群より選ばれる少なくとも1の塩基性アミノ酸を含む前記1~5のいずれか1に記載のクロマトグラフ媒体。
7.検出部がリジンを含む前記1~6のいずれか1に記載のクロマトグラフ媒体。
8.被検出物質が糖蛋白質である前記1~7のいずれか1に記載のクロマトグラフ媒体。
9.前記糖蛋白質がHbA1cである前記8に記載のクロマトグラフ媒体。
10.前記1~9のいずれか1に記載のクロマトグラフ媒体を含むイムノクロマトグラフ装置。
11.前記10に記載のイムノクロマトグラフ装置及び検体希釈液を含むイムノクロマトグラフキット。
12.前記11に記載のイムノクロマトグラフキットを用い、下記工程(1)~(4)を順次実施するイムノクロマト分析方法。
(1)検体を検体希釈液とともに試料添加部に添加する工程
(2)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
なお、本明細書において、「固定」とは、抗体等が移動しないように膜等の担体に配置されていることを意味し、「保持」とは、抗体等が膜等の担体の中または表面を移動可能に配置されることを意味する。
三糖としては、例えば、ラフィノース、ニゲロトリオース、マルトトリオース、メレジトース、マルトトリウロース、及びケストース等が挙げられる。
四糖としては、例えば、ニストース、ニゲロテトラオース、スタキオース及びマルトテトラオース等が挙げられる。
(1)検体を検体希釈液とともに試料添加部に添加する工程
(2)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、クロマトグラフ媒体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、上記検体含有液を試料添加部(1)上に、所定量(通常、0.1~2ml)添加する。検体含有液が添加されると、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、上記工程(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上を移動相として通過した検体中の被検出物質が、検出物質であるタンパク質との特異的結合反応により、検出部に固定されているタンパク質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。
本発明では、検出部に三糖以上の多糖類と塩基性アミノ酸を含むため、検出部に固定された検出対象であるタンパク質の変性を十分に抑制し、保存安定性が十分に高く、検出物質の活性低下を防ぐことができる。
1.イムノクロマトグラフキットの作製
(1)クロマトグラフ媒体および検出部の作製
クロマトグラフ媒体に用いるメンブレンとしてニトロセルロースからなるシート(ミリポア社製、商品名:HF75、300mm×25mm)を用いた。
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抗HbA1cモノクローナル抗体(第一抗体)を希釈した溶液150μLを、乾燥させたメンブレン上の検出部(検出ライン)に1mmの幅で300mm塗布した。なお該溶液は、8mmol/mL(クロマトグラフ媒体1つにつき20nmol)のリジン及び10mmol/mL(クロマトグラフ媒体1つにつき25nmolとなる濃度)のラフィノース濃度を有する溶液を準備した。
また、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、検出部の下流に、金ナノ粒子標識物質や被検出物質である動物肉タンパク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および検出部を作製した。
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。
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抗HbA1cモノクローナル抗体(第二抗体)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。
次に、上記作製したクロマトグラフ媒体および検出部、試料添加部、標識物質保持部を用いて図1に示すようなイムノクロマトグラフ装置を作製した。バッキングシートからなる基材に、試料添加部、標識物質保持部、検出部を有するクロマトグラフ媒体、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、イムノクロマトグラフ装置とした。なお、標識物質保持部およびインキュベーション部の試料展開方向の長さをそれぞれ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。
上記作製したイムノクロマトグラフ装置を加湿器に入れ、湿度を95%、温度を37℃の状態で表1に記載する時間放置した。その後、健康成人男性および糖尿病男性患者それぞれの指先を穿刺することにより、血液を採取し、ラテックス凝集法によりHbA0及びHbA1cの濃度を測定した夫々の血液試料を、HbA1cの濃度が、6.5%となるように混合し作製したHbA1c検体を上記検体希釈液で100倍に希釈し作製した検体試料150μLをイムノクロマトグラフ装置の試料添加部上に載せて展開させ、検出部の着色の度合い(発色値)をデンシトメーター(浜松ホトニクス社製、以下同じ)により測定した。結果を表1及び図2に示す。表中、相対活性とは、加湿器に入れないもの(経過時間0)で測定した発色値を100%としたときの発色値の割合を示したものである。なお、各実施例・比較例に対応する、HbA1cの濃度が0%でありHbA0のみからなる検体(陰性検体)を用いて、発色値を測定したが、すべて5mAbs未満の測定値を示し、偽陽性は確認されなかった。
第一抗体および第二抗体の「抗HbA1cモノクローナル抗体」の代わりに、「抗インフルエンザ抗体」を使用し、検体試料をインフルエンザ抗原に変更したこと以外は、実施例1を繰り返した。検出部の着色の度合い(発色値)をデンシトメーターにより測定した。結果を表1及び図2に示す。
検出部において、「リジン及びラフィノース」の代わりに、「スクロース」を使用したこと以外は、実施例1を繰り返した。なお、検出部の作製においては、10mmol/mL(クロマトグラフ媒体1つにつき20nmolとなる濃度)のスクロース濃度を有する溶液を使用し、検出部の着色の度合い(発色値)をデンシトメーターにより測定した。結果を表2及び図2に示す。
検出部において、「リジン及びラフィノース」の代わりに、「スクロース」を使用したこと以外は、実施例2を繰り返した。なお、検出部の作製においては、10mmol/mL(クロマトグラフ媒体1つにつき20nmolとなる濃度)のスクロース濃度を有する溶液を使用し、検出部の着色の度合い(発色値)をデンシトメーターにより測定した。結果を表2及び図2に示す。
一方、表2及び図2において、二糖であるスクロースのみを含有する比較例1及び2においては、検出物質の活性が低下した。
実施例1において使用したイムノクロマトグラフ装置を、温度:24-26℃、湿度:40-70%の加湿環境下で二日間放置した。検体は、実施例1と同じHbA1cを用い、放置前のイムノクロマトグラフ装置における検出部の着色の度合い(発色値)と、二日間放置後のイムノクロマトグラフ装置における検出部の着色の度合い(発色値)をそれぞれデンシトメーターにより測定した。その結果を表3及び図3に示す。表3中、相対活性とは、放置前のイムノクロマトグラフ装置において測定した発色値を100%としたときの、二日間放置後のイムノクロマトグラフ装置において測定した発色値の割合を示したものである。
実施例3において、イムノクロマトグラフ装置の検出部に塗布するリジンとラフィノースの量を、表3に示すように変更したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のアルギニンとラフィノースを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のリジンとマルトテトラオースを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のグリシンを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のアルギニンを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のリジンを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のマンニトールを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のスクロースを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のラフィノースを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のマルトテトラオースを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
実施例3において、イムノクロマトグラフ装置の検出部に対し、表3に示す量のグリシンとラフィノースを塗布したことを除いては、実施例3を繰り返した。その結果を表3及び図3に示す。
一方、中性アミノ酸であるグリシンのみを含有する比較例3、塩基性アミノ酸であるアルギニンやリジンのみを含有する比較例4及び5、単糖であるマンニトールや二糖であるスクロースのみを含有する比較例6及び7、三糖のラフィノースや四糖のマルトテトラオースのみを含有する比較例8及び9においては、検出物質の活性が顕著に低下した。また、中性アミノ酸のグリシンと三糖のラフィノースを含有する比較例10においても、検出物質の活性が顕著に低下した。
以上の結果より、検出部に塩基性アミノ酸及び三糖以上の多糖類を両方含有させることによって、検出物質の活性低下が大幅に抑制されることがわかった。
2 標識物質保持部
3 クロマトグラフ媒体
4 検出部
5 吸収部
6 バッキングシート
Claims (12)
- 検体中の被検出物質を検出するイムノクロマトグラフィー法に用いられ、タンパク質からなる検出物質が固定された検出部を有するクロマトグラフ媒体であって、前記検出部に三糖以上の多糖類と塩基性アミノ酸を含むクロマトグラフ媒体。
- 検出部に含まれる前記多糖類が、ラフィノース、ニゲロトリオース、マルトトリオース、メレジトース、マルトトリウロース、ケストース、ニストース、ニゲロテトラオース、スタキオース及びマルトテトラオースからなる群より選ばれる少なくとも1つの多糖類である請求項1に記載のクロマトグラフ媒体。
- 検出部に前記多糖類を5~50nmol含む請求項1または2に記載のクロマトグラフ媒体。
- 検出部に前記塩基性アミノ酸を5~50nmol含む請求項1~3のいずれか1項に記載のクロマトグラフ媒体。
- 検出部に含まれる前記多糖類と前記塩基性アミノ酸のモル比が、1:10~10:1である請求項1~4のいずれか1項に記載のクロマトグラフ媒体。
- 検出部が、アルギニン、リジン、オルニチン、及びヒスチジンからなる群より選ばれる少なくとも1の塩基性アミノ酸を含む請求項1~5のいずれか1項に記載のクロマトグラフ媒体。
- 検出部がリジンを含む請求項1~6のいずれか1項に記載のクロマトグラフ媒体。
- 被検出物質が糖蛋白質である請求項1~7のいずれか1項に記載のクロマトグラフ媒体。
- 前記糖蛋白質がHbA1cである請求項8に記載のクロマトグラフ媒体。
- 請求項1~9のいずれか1項に記載のクロマトグラフ媒体を含むイムノクロマトグラフ装置。
- 請求項10に記載のイムノクロマトグラフ装置及び検体希釈液を含むイムノクロマトグラフキット。
- 請求項11に記載のイムノクロマトグラフキットを用い、下記工程(1)~(4)を順次実施するイムノクロマト分析方法。
(1)検体を検体希釈液とともに試料添加部に添加する工程
(2)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
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KR1020187020654A KR20180088479A (ko) | 2016-01-22 | 2017-01-19 | 크로마토그래프 매체 |
EP17741470.3A EP3407065A4 (en) | 2016-01-22 | 2017-01-19 | Chromatograph medium |
US16/071,153 US20190353651A1 (en) | 2016-01-22 | 2017-01-19 | Chromatographic medium |
CN201780007291.9A CN108496082A (zh) | 2016-01-22 | 2017-01-19 | 层析介质 |
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JP2016010795A JP6738608B2 (ja) | 2016-01-22 | 2016-01-22 | クロマトグラフ媒体 |
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EP (1) | EP3407065A4 (ja) |
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KR (1) | KR20180088479A (ja) |
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WO2019138899A1 (ja) * | 2018-01-11 | 2019-07-18 | 東洋紡株式会社 | 測定試料希釈液およびキットおよび測定方法 |
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JP7352831B2 (ja) * | 2018-01-09 | 2023-09-29 | 東洋紡株式会社 | イムノクロマト試験片および測定キットおよび測定方法 |
WO2020085289A1 (ja) * | 2018-10-23 | 2020-04-30 | 積水メディカル株式会社 | イムノクロマト用試験片 |
JP7498556B2 (ja) * | 2019-12-12 | 2024-06-12 | デンカ株式会社 | 免疫測定方法及び免疫測定器具 |
CN117358321B (zh) * | 2023-12-04 | 2024-03-19 | 赛普(杭州)过滤科技有限公司 | 一种层析介质及其制备方法 |
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- 2017-01-19 KR KR1020187020654A patent/KR20180088479A/ko not_active Application Discontinuation
- 2017-01-19 CN CN201780007291.9A patent/CN108496082A/zh active Pending
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WO2019138899A1 (ja) * | 2018-01-11 | 2019-07-18 | 東洋紡株式会社 | 測定試料希釈液およびキットおよび測定方法 |
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JPWO2019138899A1 (ja) * | 2018-01-11 | 2021-01-07 | 東洋紡株式会社 | 測定試料希釈液およびキットおよび測定方法 |
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KR20180088479A (ko) | 2018-08-03 |
TW201732292A (zh) | 2017-09-16 |
JP6738608B2 (ja) | 2020-08-12 |
EP3407065A1 (en) | 2018-11-28 |
CN108496082A (zh) | 2018-09-04 |
EP3407065A4 (en) | 2018-11-28 |
US20190353651A1 (en) | 2019-11-21 |
TWI639002B (zh) | 2018-10-21 |
JP2017129533A (ja) | 2017-07-27 |
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