WO2017213227A1 - 糖鎖抗原を抽出し測定するためのイムノクロマト試験片及び検体添加用デバイス、並びにそれを用いたイムノクロマト法 - Google Patents
糖鎖抗原を抽出し測定するためのイムノクロマト試験片及び検体添加用デバイス、並びにそれを用いたイムノクロマト法 Download PDFInfo
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- WO2017213227A1 WO2017213227A1 PCT/JP2017/021339 JP2017021339W WO2017213227A1 WO 2017213227 A1 WO2017213227 A1 WO 2017213227A1 JP 2017021339 W JP2017021339 W JP 2017021339W WO 2017213227 A1 WO2017213227 A1 WO 2017213227A1
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- chain antigen
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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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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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
- G01N33/558—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
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/10—Devices for withdrawing samples in the liquid or fluent state
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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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
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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
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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
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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
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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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
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to an immunochromatographic test strip for extracting and measuring a sugar chain antigen, a device for sample addition, and an immunochromatography method using the same. .
- a nitrite extraction method is known as a method for extracting sugar chain antigens. This method is mainly used for extraction of sugar chain antigens of microorganisms belonging to the genus Streptococcus such as group A ⁇ -hemolytic streptococci and oral streptococci.
- This nitrous acid extraction method is a method in which bacterial cells and a nitrous acid aqueous solution are mixed to expose sugar chain antigens.
- a general nitrous acid extraction method is used for a specimen containing microorganisms such as a sodium nitrite aqueous solution, acetic acid, hydrochloric acid, etc. It is carried out by mixing a nitrous acid extract that has produced nitrous acid by mixing with an acidic solution and reacting the microorganism with the nitrous acid for a sufficient time. Since the above extract is strongly acidic, it is generally analyzed after adding a basic solution such as Tris or sodium hydroxide after the reaction to neutralize the extract.
- a rapid diagnostic agent based on general immunochromatography can be measured quickly and easily by suspending a specimen in a specimen suspension and then supplying the suspension to an immunochromatographic test piece.
- a nitrite solution and an acidic solution are mixed in advance immediately before the measurement to prepare a nitrite extract and mixed with the specimen. That is, after reacting with group A ⁇ -hemolytic streptococci and nitrous acid, neutralization is performed with a basic aqueous solution. The neutralized extract is supplied to the immunochromatographic test piece.
- nitrite extract used in this method cannot be stored for a long time, it is necessary to mix and prepare the nitrite solution and the acid solution immediately before the test. Moreover, since this nitrous acid extract is too low in pH and interferes with the antigen-antibody reaction, it must be neutralized to a certain pH.
- the steps of mixing the two liquids and the step of further neutralization are increased, the number of reagents is increased as compared with a rapid diagnostic agent based on a general immunochromatography method, and the operation is likely to be complicated. Therefore, the existing rapid diagnostic reagent is devised so that the sample treatment can be performed only by the operation of mixing the nitrite solution and the acidic solution by previously including the neutralizing reagent in the immunochromatographic test strip.
- there are still many operation steps compared to the rapid diagnostic reagent based on the conventional immunochromatography method it is possible to extract correctly without mixing the two liquids, such as an error in the amount of the liquid to be mixed or a difference in the mixed solution. There was a problem that it could not be measured accurately because it was not operated.
- sodium nitrite is designated as a deleterious substance by the Poisonous and Deleterious Substances Control Law, it belongs to the dangerous goods category 1 in the Fire Service Act, and is further designated as a hazardous substance in Article 2 of the Enforcement Order by the Water Pollution Control Law It is a difficult chemical to handle.
- a manufacturing process in which a high concentration sodium nitrite aqueous solution is applied to the member of the immunochromatographic test piece and then dried is necessary.
- the object of the present invention is to simplify the operation of nitrite extraction by performing the glycan antigen extraction step in a filtration step in order to extract the glycan antigen more efficiently and to compare with the conventional nitrite extraction method. Even so, it is to provide an immunochromatographic test piece and a specimen addition device capable of specifically measuring sugar chain antigens accurately and without any difference in performance, and an immunochromatography method using the same.
- the present inventors extract a sugar chain antigen using nitrite and an acidic reagent so that the number of steps required for the extraction is not increased when extracting the sugar chain antigen and measuring using an immunochromatographic test strip.
- the sample containing the target substance may be filtered in advance using a sample addition device including a filter, etc., and the sugar chain antigen is extracted from this device. By doing this, it was found that measurement can be performed quickly and easily.
- an investigation was made to include an acidic reagent in a dried state in the porous material and to include the porous material in the device.
- liquid acid reagents such as hydrochloric acid and acetic acid volatilize and disappear on the porous material in the dry state, so that they did not settle on the porous material and sufficient nitrous acid extraction was not performed. Therefore, by using a solid acidic reagent such as tartaric acid, malic acid, malonic acid, maleic acid, citric acid, etc., the acidic reagent is fixed without volatilizing even on a dry porous material. Compared with the method of neutralization after treating with a liquid nitrite extract mixed with two liquid acidic reagents, it is possible to measure quickly and easily, and the performance can be measured without inferiority.
- the inventors have found that by using tartaric acid among acidic reagents, antigens can be sufficiently extracted, storage stability is stable, and non-specific reactions are unlikely to occur, and the present invention has been completed.
- the present invention is an immunochromatographic test method for measuring a sugar chain antigen, wherein the step of contacting tartaric acid as a solid acidic reagent after mixing a specimen and a nitrite solution is performed in the filtration step. Provide a method.
- the present invention is as follows.
- An immunochromatographic test method for measuring a sugar chain antigen wherein a step of mixing a sample and a nitrite solution, contacting with tartaric acid, and extracting a sugar chain antigen in the sample is performed in a filtration step.
- the filtration step is performed in a specimen addition device including a porous material impregnated with tartaric acid and a filter.
- the neutralization step after the sample and the nitrite solution are mixed, the neutralization step after the step of bringing the tartaric acid into contact is performed at a site separated from the immunochromatographic test piece.
- the sugar chain antigen is a sugar chain antigen of protozoa, fungus, bacteria, mycoplasma, rickettsia, chlamydia or virus, and the sugar chain antigen in the sample is obtained by the immunochromatography method according to any one of [1] to [5] How to measure.
- the sample addition device further includes a porous material impregnated with a neutralizing reagent.
- the sugar chain antigen is a sugar chain antigen of a protozoan, fungus, bacterium, mycoplasma, rickettsia, chlamydia, or virus.
- the immunochromatographic test strip and the device for sample addition of the present invention compared with the conventional method of neutralizing after treatment with a nitrous acid extract previously mixed with two liquids of nitrite and a liquid acidic reagent, it is quicker and simpler. In addition, it is possible to measure without inferior performance.
- the present invention relates to a method that simplifies the nitrous acid extraction treatment of a sugar chain antigen in a filtration step, and enables a sugar chain antigen as a substance to be detected to be measured quickly and accurately.
- a sample collected from the pharynx or nasal cavity of a patient infected with a virus or bacteria is suspended in a nitrite solution as described later.
- the nitrite suspension (sample) is filtered by a filtration means, and treated with an acidic reagent during filtration to extract sugar chain antigens.
- the filtrate is added dropwise to an immunochromatographic test piece, and the presence or absence of a detection target in the sample is measured by detecting the presence or absence of a sugar chain antigen.
- This procedure is an example and is not limited to this procedure.
- the filtration step of the present invention is performed by a specimen addition device.
- the specimen addition device is an accessory part outside the immunochromatographic test piece, and can also be used as a container for performing a nitrous acid treatment of the collected specimen.
- the nitrite treatment of the specimen for sugar chain antigen extraction is performed by producing nitrite by mixing the specimen with nitrite and further mixing an acidic reagent.
- the sample addition device may contain a nitrite solution in advance.
- the specimen may be mixed with the nitrous acid solution in advance, and the mixed solution may be placed in the specimen addition device.
- Such devices include vials, syringes, tubes and the like.
- the device includes not only a container but also a filtering means for filtering the specimen when the specimen is added and supplied to the immunochromatographic test strip.
- the sample addition device includes a container portion for storing the sample and the nitrite solution, and a portion for adding and supplying the sample in the container to the immunochromatographic test strip.
- the latter part to which the specimen is added and supplied has a part having a nozzle (opening) for discharging the specimen from the container, and this part can also serve as a lid for the container part.
- the portion where the latter specimen is added and supplied to the immunochromatographic test strip is also referred to as a nozzle portion or a lid portion.
- the specimen addition device may be composed of two parts, a nozzle portion and a container portion, each of which includes a filter housing including a filtration filter, as will be described later.
- the filter housing has an opening through which the sample passes through the filter and an opening through which the sample that has passed through the filter is discharged, and the filter exists between both openings.
- a syringe or the like that can pass the sample through the filter under pressure may be used.
- the acidic reagent necessary for nitrous acid treatment may be preliminarily contained in the sample addition device, or may be added when the sample is subjected to nitrous acid treatment.
- the acidic reagent a solid acidic reagent is used.
- the solid acidic reagent used in the present invention is solid at room temperature, has a property of not volatilizing at high temperature, and is not colored on an immunochromatographic test piece, specifically, white or dry heat in a dry state. Reagents that are not easily colored by oxidation are preferred.
- solid acidic reagent having these characteristics examples include tartaric acid, malonic acid, malic acid, maleic acid, citric acid and the like.
- the antigen has all the above characteristics and is equivalent to the liquid acidic reagent.
- the only solid acidic reagent having extractability is tartaric acid.
- Tartaric acid is impregnated into a porous material and dried.
- a porous material commonly used filter paper, glass fiber, nonwoven fabric, and the like can be used.
- the amount of tartaric acid used in the present invention is not particularly limited, but is usually about 0.01 ⁇ g to 1 mg, preferably 0.1 ⁇ g to 0.1 mg per immunochromat test piece. Degree. However, it is preferable to select an optimum amount that can provide an effect depending on the composition of the sample suspension and the amount of the drop.
- FIG. 1 shows an example of a sample addition device containing filtration means and tartaric acid, but the sample addition device of the present invention is not limited to that shown in FIG. 1, and can contain a sample, and the sample is an immunochromatographic test piece. Any device can be used as long as it can be added to the specimen addition site.
- the lower part is a container part that contains a sample
- the upper part is a lid part (also referred to as a nozzle part) of a container having a specimen addition nozzle, and a filter is placed on the lid part. Can be set.
- a filtration filter which is a filtration means, and a porous material containing tartaric acid are separated without contact, and a porous material containing tartaric acid is present upstream of the filtration filter.
- the porous material containing tartaric acid may be fixed inside the device, for example, inside the container by an appropriate fixing means, or may be put without fixing. Alternatively, it may be added to the device when performing nitrous acid treatment.
- the upstream refers to the upstream with respect to the flow of the sample fluid accommodated in the device when the sample is added using the sample addition device, and the portion further away from the sample addition nozzle in the device is referred to as the upstream, The part close to the specimen addition nozzle is called downstream.
- the sample is placed in the nitrite solution in the device, and mixed by inversion, and the sample, the nitrite solution, and the tartaric acid in the porous material are contact-mixed to perform the nitrite treatment of the sample.
- the specimen and the nitrous acid solution can be mixed in advance, and the mixture can be added to the container containing tartaric acid in the device, and the specimen, the nitrite solution, and the tartaric acid in the porous material can be contact-mixed.
- the resulting mixture is added to the immunochromatographic test strip from the nozzle portion through a filter.
- a porous material containing tartaric acid can be incorporated as one layer of a filtration filter composed of one or more layers of filters.
- a nitrite solution is contained in a container at the bottom of the device, and the specimen is first mixed with the nitrite solution.
- the mixed specimen is added to the immunochromatographic test piece, the specimen passes through the porous material containing tartaric acid, and the tartaric acid impregnated at this time is dissolved in the specimen.
- the specimen is treated with nitrous acid.
- the mixture is added to the immunochromatographic test strip from the nozzle portion through a filter.
- the filtration filter and the porous material containing tartaric acid are brought into contact with each other, and the porous material containing tartaric acid is provided on the downstream side of the filter.
- a nitrite solution is contained in a container at the bottom of the device, and the specimen is first mixed with the nitrite solution.
- the mixed specimen is added to the immunochromatographic test strip, the mixed specimen passes through the filter, passes through the porous material containing tartaric acid, and the impregnated tartaric acid is dissolved in the specimen.
- the specimen is treated with nitrous acid.
- the mixture is added to the immunochromatographic test strip from the nozzle portion through a filter.
- FIG. 1D shows that a filtration filter as a filtering means and a porous material containing tartaric acid are separated without contacting each other, and a porous material containing tartaric acid is present downstream of the filtration filter.
- an appropriate space may be provided between the filter and the nozzle, and a porous material containing tartaric acid may be included in the space.
- a nitrite solution is contained in a container at the bottom of the device, and the specimen is first mixed with the nitrite solution.
- the mixed specimen When the mixed specimen is added to the immunochromatographic test piece, the specimen passes through the filter, and the tartaric acid and the specimen are contacted and mixed in the space where the porous material containing tartaric acid is present, so that the specimen is treated with nitrous acid. . Since the specimen is temporarily stored in the space, the contact time between the specimen and the porous material containing tartaric acid can be extended, and sufficient contact between the specimen and tartaric acid is achieved. At this time, it is desirable to provide an air hole that communicates the space portion with the outside of the device. For this purpose, as shown in FIG. 1D, the nozzle portion and the portion incorporating the filter may be separated.
- the nozzle portion When the mixture of the sample and tartaric acid is added to the immunochromatographic test piece, the nozzle portion may be pressed against the filter-incorporated portion, and the sample is dropped from the nozzle at the same time as the air hole is blocked (FIG. 1E).
- the filtering means contact mixing and filtration of the sample and tartaric acid can be performed almost simultaneously in one step.
- a filtering means incorporating tartaric acid to the sample addition device, contact mixing of the sample with tartaric acid, filtration of impurities, and addition of the mixture of the sample and tartaric acid to the immunochromatographic test piece in one step. It can be done almost simultaneously.
- “can be performed in one step” is one operation of adding a sample, contact mixing and filtration of the sample, nitrite solution and tartaric acid, or contact mixing of the sample, nitrite solution and tartaric acid, This means that the filtration of impurities and the addition of a mixture of specimen, nitrite solution and tartaric acid to the immunochromatographic test strip can be performed continuously, and it is not necessary to perform these simultaneously. In addition, it is necessary to incorporate tartaric acid so that it does not come off before use.
- the size of the sample addition device is not limited, but for example, the height is several centimeters and the diameter of the thickest part is about several centimeters.
- the size of the filter, the porous material containing tartaric acid, and the porous material containing the neutralizing reagent are not limited, but in the case of a circular shape, for example, the diameter is about several mm to several cm.
- the immunochromatographic test strip comprises a support having a detection region on which an antibody (antibody 1) that captures a substance to be detected (antigen etc.) is immobilized, a labeled region having a movable labeled antibody (antibody 2), and a sample drop
- an antibody that captures a substance to be detected (antigen etc.) is immobilized
- a labeled region having a movable labeled antibody (antibody 2)
- sample drop A sample pad to be added, an absorption band for absorbing the developed specimen liquid, a backing sheet for bonding these members together, and the like are provided.
- the immunochromatographic test piece of the present invention may be contained in a storage container, and the storage container can prevent deterioration due to, for example, ultraviolet rays or moisture in the air. Further, when using a sample sample having a contamination property or an infectivity property, it is possible to prevent the tester who performs the assay from the containment vessel from being contaminated or infected.
- a sample sample having a contamination property or an infectivity property it is possible to prevent the tester who performs the assay from the containment vessel from being contaminated or infected.
- an appropriately sized resin case may be used as a storage container, and the apparatus of the present invention may be stored in the case. Further, the surface of the test piece on which the antigen or antibody is immobilized may be covered with a resin film or the like (top laminate).
- the containment vessel and the test piece stored in the containment vessel may be collectively referred to as an immunochromatography device.
- the number of detection regions and the type of labeled antibody included in the labeled body region are not limited to one, and two or more antigens can be tested in the same test by using antibodies corresponding to a plurality of substances to be detected. It can be measured with a piece.
- the support is a material having the ability to immobilize an antibody for capturing a substance (antigen) to be detected, and has a performance that does not prevent the liquid from passing in the horizontal direction.
- it is a porous thin film having a capillary action, and is a material capable of transporting a liquid and components dispersed therein by absorption.
- the material forming the support is not particularly limited, and examples thereof include cellulose, nitrocellulose, cellulose acetate, polyvinylidene difluoride (PVDF), glass fiber, nylon, and polyketone. Of these, a thin film using nitrocellulose is more preferred.
- a membrane on which an antibody is immobilized is called an antibody-immobilized membrane.
- the labeled body region is composed of a porous substrate containing a labeled antibody, and the glass substrate or nonwoven fabric that is generally used can be used as the material of the substrate.
- the substrate is preferably in the form of a pad having a thickness of about 0.3 mm to 0.6 mm in order to impregnate a large amount of labeled antibody.
- the porous substrate impregnated with the labeled antibody and dried is also referred to as a dry pad.
- enzymes such as alkaline phosphatase and horseradish peroxidase, metal colloids such as gold colloid, silica particles, cellulose particles, colored polystyrene particles, and colored latex particles are often used.
- metal colloids such as gold colloid, silica particles, cellulose particles, colored polystyrene particles, and colored latex particles
- coloring occurs due to aggregation of these labeling reagents, and this coloring is measured.
- the particles on which the antibody is immobilized are called antibody-immobilized particles.
- the detection region refers to a partial region of the support on which an antibody that captures a substance to be detected (antigen) is immobilized.
- the detection region is provided with at least one region on which an antibody for capturing an antigen is immobilized.
- the detection region may be included in the support, and the antibody may be immobilized on the support.
- the sample pad is a part for dropping the specimen and is a porous material.
- the sample pad is the most upstream site of the immunochromatographic test strip.
- As the material commonly used filter paper, glass fiber, non-woven fabric and the like can be used.
- it is preferably a pad shape having a thickness of about 0.3 mm to 1 mm. Samples include samples prepared using samples, such as samples obtained by floating samples in other solutions.
- the absorption band is a member for absorbing components that are supplied to the support and are not involved in the reaction in the detection region.
- As the material filter paper with high water retention, sponge, etc. made of general natural polymer compounds, synthetic polymer compounds, etc. can be used, but those having high water absorption are preferable for promoting the development of the specimen. .
- the backing sheet is a member for affixing and fixing all the above-mentioned materials, that is, the support, the sample pad, the marker region, and the absorption band with a partial overlap.
- the backing sheet is not necessarily required as long as these materials are arranged and fixed at an optimal interval, but it is generally preferable to use the backing sheet for convenience in manufacturing or use.
- a control display region may further exist.
- the control display area is a site indicating that the test has been performed correctly.
- the control display area exists downstream of the detection area, and when the specimen sample passes through the detection area and reaches the control display area, a signal is emitted by coloring or the like.
- a substance that binds to the antibody to which the labeling carrier is bound may be immobilized, or a reagent such as a pH indicator that changes color when the sample arrives is immobilized. Also good.
- the antibody to which the labeling carrier is bound is a mouse monoclonal antibody, an anti-mouse IgG antibody may be used.
- the size of the immunochromatographic test piece is not limited, but is, for example, about several centimeters to several tens of centimeters in length and several millimeters to several centimeters in length.
- the specimen passes through the porous flow path formed by a series of connections of the sample pad, the label body region, the support, the detection region, and the absorption band. Therefore, in this embodiment, all of these become the specimen movement area.
- the specimen movement region defined in this specification does not matter whether the specimen is inside or the interface.
- glycerin antigens are extracted by generating nitrous acid using a nitrite solution and tartaric acid. Since the extracted sugar chain antigen solution is acidic, the antigen-antibody reaction is inhibited as it is, so that it is necessary to neutralize the tartaric acid solution.
- a neutralizing reagent is used for neutralizing the tartaric acid solution.
- the neutralizing reagent may be included on the immunochromatographic test strip or may be included in the above-described sample addition device. That is, the neutralization step is performed on the immunochromatographic test piece or at a site separated from the immunochromatographic test piece, such as in a sample addition device.
- the neutralization reagent may be impregnated into the sample pad, the support may be impregnated, or a porous material such as a nonwoven fabric other than the support may be impregnated to obtain the neutralization reagent-impregnated porous material obtained.
- the material may be arranged upstream of the marker region.
- the region on the immunochromatographic test piece impregnated with the neutralizing reagent is called a neutralizing reagent region or a basic reagent region.
- the immunochromatographic test piece having a neutralizing reagent region has a sample pad, a neutralizing reagent region, a labeled region, a detection region, and an absorption band from the upstream on the support.
- the sample pad, neutralizing reagent region, labeling region, detection region and absorption band may or may not be in contact with each other. Further, it is not always necessary to impregnate the neutralizing reagent region and the label body region into separate porous materials, and a plurality of or all of the regions may be impregnated into the same porous material.
- the porous material is impregnated with the neutralization reagent and dried.
- the porous material commonly used filter paper, glass fiber, nonwoven fabric, and the like can be used. What is necessary is just to contain the porous material containing the dried neutralization reagent so that it may be located downstream of the porous material containing tartaric acid.
- a porous material containing a neutralizing reagent may be provided in a portion where the filter exists. In the case of the specimen addition device shown in FIG.
- a porous material containing a neutralizing reagent may be provided in a portion where a filter downstream of the porous material containing tartaric acid exists.
- a porous material containing tartaric acid is provided near the most downstream of the sample addition device.
- the neutralizing reagent used in the present invention is solid at normal temperature and does not volatilize at high temperature.
- Preferred neutralizing reagents used in the present invention include tris (trishydroxylmethylaminomethane), sodium hydroxide, dipotassium hydrogen phosphate, trisodium citrate, and a good buffer having a buffer capacity in the alkaline region. .
- the amount of the neutralizing reagent used in the present invention is not particularly limited, but is usually about 0.01 ⁇ g to 1 mg per porous material or per test strip of the immunochromatographic test strip. Preferably, it is about 0.1 ⁇ g to 0.1 mg. However, it is preferable to select an optimal amount that is effective depending on the type of the neutralizing reagent to be used, the composition of the specimen suspension, the amount of addition, and the like.
- the neutralizing reagent is dissolved once, the solution is applied to the sample pad or the porous material, and then dried.
- FIG. 2 and 3 are diagrams showing a preferred embodiment of a typical immunochromatographic test piece.
- the immunochromatographic test piece shown in FIG. 2 is called a first example, and the immunochromatographic test piece shown in FIG. 3 is called a second example.
- an immunochromatographic test piece is not limited to what is shown in FIG.2 and FIG.3. 2 and 3, 9 indicates a support, 10 indicates a marker region, 11 indicates a detection region, 12 and 13 indicate sample pads, 14 indicates an absorption band, and 15 indicates a backing sheet. Moreover, you may affix a top laminate on the whole test piece.
- FIG. 2A and 3A are top views, and FIG. 2B and FIG. 3B are cut sectional views.
- a support 9, an absorption band 14, a marker region 10, sample pads 12 and 13, etc., each having a single detection region 11 formed on a backing sheet 15 made of resin or the like are laminated. Yes.
- one end of the absorption band 14 and one end of the support 9, the other end of the support 9 and one end of the marker region 10, The other end and one end of the sample pad 13 are overlapped, and the upstream portion of the sample pad 13 is impregnated with tartaric acid, and the downstream portion of the sample pad is impregnated with the neutralizing reagent after a while.
- the neutralization reagent region 13 exists upstream of the label body region 10, and these are overlapped to form a continuous lateral flow channel.
- the neutralizing reagent region 13 also serves as a sample pad. That is, a neutralizing reagent region exists on the sample pad. There may also be a sample pad upstream of the neutralizing reagent region.
- a method for measuring sugar chain antigens using the specimen addition device of FIG. 1A or FIG. 1B and the immunochromatographic test piece having the neutralization region shown in FIG. 2 will be described.
- the sample addition device of FIG. 1A start by contacting the sample or the sample prepared using the sample with the nitrite solution, floating the sample in the nitrite solution, and adding the sample to the sample addition device. Is done. At this time, 5 to 100 ⁇ L of the sample and 0.01 to 2 mL of 0.1M to 8M nitrite may be mixed and added to the sample addition device. Examples of nitrites include sodium nitrite and potassium nitrite.
- a nitrite solution is placed in advance in the container portion of the specimen addition device, and the specimen is added thereto. Put 0.1 to 2mL of 0.1M to 8M nitrite in the container of the sample addition device, and add 5 to 100 ⁇ L of sample to it.
- the specimen in the specimen addition device When the specimen in the specimen addition device is dropped from the nozzle onto the sample pad 12 of the immunochromatographic test piece, the specimen passes through the filter and is mixed with the tartaric acid contained in the porous material. Nitrite mixed with the sample reacts with tartaric acid to generate free nitrous acid, and the sugar chain antigen is extracted from the sample by the action of the nitrous acid. The specimen containing the extracted sugar chain antigen is added to the immunochromatographic test strip. If the sample addition device contains a neutralizing reagent, the sample is neutralized before being added to the immunochromatographic test strip. If the neutralizing reagent is contained on the immunochromatographic test strip, the sample is placed on the immunochromatographic test strip. To be summed.
- a specimen containing a sugar chain antigen which is a substance to be detected, provided to the sample pad 12 is developed into the sample pad 12 and the neutralizing reagent region 13 by capillary action.
- the marker region 10, the support 9, and the absorption band 14 are sequentially developed in the horizontal direction.
- the extracted sugar chain antigen moves to the neutralizing reagent region 13 together with the acidic developing solution, and the pH of the acidic developing solution containing the sugar chain antigen is neutralized and adjusted to the neutral region in the neutralizing reagent region 13. .
- the sugar chain antigen develops and moves further downstream under neutral conditions.
- the labeled antibody is released into the liquid and developed on the support 9 as the specimen sample is developed.
- a sugar chain antigen is present in the specimen sample, the sugar chain antigen is specifically captured by the capture antibody in the detection region 11 of the support 9, and the sugar chain antigen forms a complex by a specific reaction with the labeled antibody. To do.
- an antibody sandwich via the sugar chain antigen is established in the detection region, and the labeled antibody-sugar chain antigen complex can be measured in the detection region 11.
- the sample is preliminarily measured before the measurement using the immunochromatographic test strip and the sample addition device. It is not necessary to extract the sugar chain antigen in the sample, and the sugar chain antigen in the sample can be measured in one step.
- a biological sample to be a specimen is not particularly limited, and examples thereof include body fluids such as serum, plasma, blood, urine, feces, saliva, tissue fluid, spinal fluid, wiping fluid, and the like, or dilutions thereof.
- the sample addition device used in the method of the present invention includes a filtering means, a pharyngeal or nasal swab, pharyngeal or nasal rinse, nasal suction, saliva, Rectal wiping liquid, stool, stool suspension, corneal wiping liquid and the like are preferably used.
- the substance to be detected is a sugar chain antigen that can be measured by an immunoassay, that is, an assay utilizing an antigen-antibody reaction.
- an immunoassay that is, an assay utilizing an antigen-antibody reaction.
- the antigen include polysaccharides that are sugar chain antigens present on the cell walls of bacteria extracted by nitrite extraction treatment. Protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses and the like containing these substances can also be measured.
- the subject's biological sample contains sugar chain antigens derived from protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, virus, etc.
- sugar chain antigens derived from protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, virus, etc.
- a sugar chain antigen it can be determined that the subject is suffering from an infection caused by protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, and the like.
- group A ⁇ -hemolytic streptococci Streptococcus pyogenes
- Escherichia coli Legionella, Campylobacter, etc.
- Example 1 Effects of the conventional method and the method of the present invention Immobilization of anti-Streptococcus pyogenes (Group A ⁇ -hemolytic streptococci) antibody on nitrocellulose membrane
- Anti-Streptococcus pyogenes Group A ⁇ -hemolytic streptococci
- nitrocellulose membrane Prepare anti-Streptococcus pyogenes antibody diluted with purified water to 1.0 mg / mL and anti-rabbit IgG antibody.
- An anti-Streptococcus pyogenes antibody was applied to the sample pad side of the nitrocellulose membrane lined with a film, and an anti-rabbit IgG antibody was applied to the absorption band side in a linear form. Thereafter, the nitrocellulose membrane was dried at 45 ° C. for 30 minutes to obtain an anti-Streptococcus pyogenes antibody-immobilized membrane.
- This membrane is referred to as “antibody-immobilized membrane” in this
- Anti-Streptococcus pyogenes antibody was diluted with purified water to 1.0 mg / mL, and colored polystyrene particles were added to this to 0.1%. After stirring, carbodiimide was added. Add to 1% and stir further. The supernatant was removed by centrifugation and resuspended in 50 mM Tris (pH 9.0) and 3% BSA to obtain a 0.04% anti-Streptococcus pyogenes antibody-bound colored polystyrene particle suspension. This particle is referred to as “antibody-immobilized particle” in this example.
- test piece using a device having a nozzle in which the acidic reagent-impregnated non-woven fabric prepared in 5 was incorporated into a sample filtration filter and a neutralizing reagent (basic reagent) -impregnated non-woven fabric as a sample pad was designated as “the present test device And “invention test piece”.
- the structure of the “test device of the present invention” is as shown in FIG. 1A, and the structure of the “test specimen of the present invention” is as shown in FIG.
- Similar test pieces were prepared using non-coated nonwoven fabrics in place of the acidic reagent and neutralizing reagent (basic reagent) impregnated nonwoven fabrics prepared in 4 and 5 and used as conventional examples.
- test piece used as a nozzle and a sample pad in which a non-woven fabric coated with nothing is incorporated into a sample filtration filter is referred to as a “conventional method test nozzle” or a “conventional method test piece”.
- the test piece is equipped with a neutralizing reagent (basic reagent) impregnated nonwoven fabric, a dry pad (labeled body region), an antibody-immobilized membrane (detection region), and an absorption band from the upstream along the flow of the specimen. It is.
- Sample Streptococcus pyogenes was cultured, and the culture solution was prepared with physiological saline to 1.0 ⁇ 10 7 CFU / mL and 0.25 ⁇ 10 7 CFU / mL.
- physiological saline was used as a negative sample.
- malonic acid, malic acid, maleic acid, citric acid, and tartaric acid are present as powders at room temperature and are not volatile. Therefore, the coating amount is also applied in the drying process when producing an acidic reagent-impregnated nonwoven fabric. Since the nitrous acid extraction treatment can be performed without inferiority compared with the conventional method, the sensitivity was not significantly reduced. Furthermore, it was possible to measure with fewer operations compared with the conventional method. However, malonic acid and malic acid reacted with nitrous acid to color the immunochromatographic specimen, and maleic acid and citric acid caused a nonspecific reaction. Among them, tartaric acid was not colored on the immunochromatographic test piece, and sensitivity and specificity were not inferior compared with the conventional method.
- Example 2 Examination of application amount of acidic reagent The application amount of the acidic reagent in Example 1 was changed, and the test results (Table 2) were compared and examined.
Abstract
Description
[1] 糖鎖抗原を測定するイムノクロマト試験法であって、検体と亜硝酸塩溶液を混合した後、酒石酸を接触させ、検体中の糖鎖抗原を抽出する工程が濾過工程で行われる方法。
[2] 濾過工程が、酒石酸を含浸させた多孔質材料及びフィルターを含む検体添加用デバイス中で行われる、[1]の方法。
[3] 検体と亜硝酸塩溶液を混合した後、酒石酸を接触させる工程の後の中和工程がイムノクロマト試験片と分離した部位で行われる、[1]又は[2]の方法。
[4] 検体と亜硝酸塩溶液を混合した後、酒石酸を接触させる工程の後の中和工程が、イムノクロマト試験片上で行われる、[1]又は[2]の方法。
[5] 中和試薬が、トリスヒドロキシルメチルアミノメタン又は水酸化ナトリウムである、[1]~[4]のいずれかのイムノクロマト法により検体中の糖鎖抗原を測定する方法。
[6] 糖鎖抗原が、原生動物、真菌、細菌、マイコプラズマ、リケッチア、クラミジア又はウイルスの糖鎖抗原である、[1]~[5]のいずれかのイムノクロマト法により検体中の糖鎖抗原を測定する方法。
[7] 糖鎖抗原を測定するイムノクロマト試験片と酒石酸を含浸させた多孔質材料及びフィルターを含む検体添加用デバイスを有するキット。
[8] 検体添加用デバイスにさらに中和試薬を含浸させた多孔質材料が含まれる、[7]のキット。
[9] 中和試薬が、トリスヒドロキシルメチルアミノメタン又は水酸化ナトリウムである、[7]又は[8]のキット。
[10] 糖鎖抗原が、原生動物、真菌、細菌、マイコプラズマ、リケッチア、クラミジア又はウイルスの糖鎖抗原である、[7]~[9]のいずれかのキット。
(1)ウイルスや細菌等に感染した患者の咽頭あるいは鼻腔等から採取した検体を後述するような亜硝酸塩溶液に浮遊させる。
(2)この亜硝酸塩浮遊液(試料)を、濾過手段で濾過し、濾過の際に酸性試薬で処理し糖鎖抗原を抽出する。
(3)この濾過液を、イムノクロマト試験片に滴加して、糖鎖抗原の有無を検出することにより、検体中の被検出物の有無を測定する。
中和試薬を含浸させたイムノクロマト試験片上の領域を中和試薬領域又は塩基性試薬領域と呼ぶ。
1.抗Streptococcus pyogenes(A群β溶血性レンサ球菌)抗体のニトロセルロースメンブレンへの固定化
抗Streptococcus pyogenes抗体を1.0mg/mLになるように精製水で希釈した液及び抗ウサギIgG抗体を準備し、PETフィルムで裏打ちされたニトロセルロースメンブレンのサンプルパッド側に抗Streptococcus pyogenes抗体、吸収帯側に抗ウサギIgG抗体をそれぞれ線状に塗布した。その後、ニトロセルロースメンブレンを45℃、30分間乾燥させ、抗Streptococcus pyogenes抗体固定化メンブレンを得た。このメンブレンを本実施例において、「抗体固定化メンブレン」と呼ぶ。
抗Streptococcus pyogenes抗体を1.0mg/mLになるように精製水で希釈し、これに着色ポリスチレン粒子を0.1%になるように加え、攪拌後、カルボジイミドを1%になるように加え、さらに攪拌する。遠心操作により上清を除き、50mM Tris(pH9.0)、3%BSAに再浮遊し、0.04%抗Streptococcus pyogenes抗体結合着色ポリスチレン粒子浮遊液を得た。この粒子を、本実施例において、「抗体固定化粒子」と呼ぶ。
2で作製した抗体固定化粒子浮遊液を不織布に所定量を塗布し、45℃、30分間乾燥させた。得られた不織布を、本実施例において、「乾燥パッド」と呼ぶ。
中和試薬(塩基性試薬)として、2Mトリス(Trizma Base)を15μL/cmで不織布に塗布した。塗布後に直ちに45℃、1時間、乾燥して、中和試薬含浸不織布を得た。
酸性試薬として、1M塩酸、1M酢酸、1Mマロン酸、1Mリンゴ酸、1Mマレイン酸、1Mクエン酸、1M酒石酸を3.75μLずつ不織布に塗布した。塗布後に直ちに45℃、1時間、乾燥して、酸性試薬含浸不織布を得た。
1で作製した抗体固定化メンブレン、3で作製した乾燥パッド、4で作製した中和試薬(塩基性試薬)含浸不織布を他部材(バッキングシート、吸収帯)と貼り合せて5mm幅に切断し、Streptococcus pyogenes試験片とした。また5で作製した酸性試薬含浸不織布を試料ろ過フィルターへ組み込んだノズルを有するデバイスと中和試薬(塩基性試薬)含浸不織布をサンプルパッドとして用いた試験片を本実施例において、「本発明試験デバイス」及び「本発明試験片」と呼ぶ。「本発明試験デバイス」の構造は図1A、「本発明試験片」の構造は図2に示すとおりである。4、5で作製した酸性試薬及び中和試薬(塩基性試薬)含浸不織布の替わりに何も塗布していない不織布を用いて同様の試験片を作製し、従来例として使用した。何も塗布していない不織布を試料ろ過フィルターへ組み込んだノズル及びサンプルパッドとして用いた試験片を「従来法試験ノズル」「従来法試験片」と呼ぶ。なお、試験片は、検体の流れに沿って、上流から、中和試薬(塩基性試薬)含浸不織布、乾燥パッド(標識体領域)、抗体固定化メンブレン(検出領域)、吸収帯を具備するものである。
Streptococcus pyogenesを培養し、培養液を生理食塩水で菌数1.0×107CFU/mLと0.25×107CFU/mLに調製した。
検体20μLを亜硝酸ナトリウム溶液(2M NaNO2)180μLに浮遊し、そのうち75μLを本発明試験片に滴加した。また、従来法として亜硝酸ナトリウムと塩酸を混合した亜硝酸抽出液に検体を浮遊した後、トリス溶液で中和した検体浮遊液を従来法試験片に50μL滴加した。5分後に抗Streptococcus pyogenes抗体を固定化した所定位置上の着色ポリスチレン粒子の堆積の有無とその程度を目視判定にて行った。その線状の堆積の程度が強いものを順に+++、++、+とし、判定が難しい場合を±、堆積がみられなかったものを-とした。
2 検体収容容器部分
3 ノズル
4 ノズル部位
5 フィルター(一部が中和試薬を含ませた多孔質材料のときがある)
6 酒石酸を含ませた多孔質材料
7 酒石酸と検体が一時的に貯留し混合される空間
8 空気抜き穴
9 支持体(検出領域を含む)
10 標識体領域
11 検出領域
12 サンプルパッド
13 中和試薬領域
14 吸収帯
15 バッキングシート
Claims (10)
- 糖鎖抗原を測定するイムノクロマト試験法であって、検体と亜硝酸塩溶液を混合した後、酒石酸を接触させ、検体中の糖鎖抗原を抽出する工程が濾過工程で行われる方法。
- 濾過工程が、酒石酸を含浸させた多孔質材料及びフィルターを含む検体添加用デバイス中で行われる、請求項1記載の方法。
- 検体と亜硝酸塩溶液を混合した後、酒石酸を接触させる工程の後の中和工程がイムクロマト試験片と分離した部位で行われる、請求項1又は2に記載の方法。
- 検体と亜硝酸塩溶液を混合した後、酒石酸を接触させる工程の後の中和工程が、イムノクロマト試験片上で行われる、請求項1又は2に記載の方法。
- 中和試薬が、トリスヒドロキシルメチルアミノメタン又は水酸化ナトリウムである、請求項1~4のいずれか1項に記載のイムノクロマト法により検体中の糖鎖抗原を測定する方法。
- 糖鎖抗原が、原生動物、真菌、細菌、マイコプラズマ、リケッチア、クラミジア又はウイルスの糖鎖抗原である、請求項1~5のいずれか1項に記載のイムノクロマト法により検体中の糖鎖抗原を測定する方法。
- 糖鎖抗原を測定するイムノクロマト試験片と酒石酸を含浸させた多孔質材料及びフィルターを含む検体添加用デバイスを有するキット。
- 検体添加用デバイスにさらに中和試薬を含浸させた多孔質材料が含まれる、請求項7記載のキット。
- 中和試薬が、トリスヒドロキシルメチルアミノメタン又は水酸化ナトリウムである、請求項7又は8に記載のキット。
- 糖鎖抗原が、原生動物、真菌、細菌、マイコプラズマ、リケッチア、クラミジア又はウイルスの糖鎖抗原である、請求項7~9のいずれか1項に記載のキット。
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US16/308,145 US20190145864A1 (en) | 2016-06-09 | 2017-06-08 | Immunochromatographic test piece and specimen adding device for extracting and measuring sugar chain antigen, and immunochromatography method using same |
MYPI2018002367A MY199249A (en) | 2016-06-09 | 2017-06-08 | Immunochromatographic test piece and specimen adding device for extracting and measuring sugar chain antigen, and immunochromatography method using same |
JP2018521778A JP7018876B2 (ja) | 2016-06-09 | 2017-06-08 | 糖鎖抗原を抽出し測定するためのイムノクロマト試験片及び検体添加用デバイス、並びにそれを用いたイムノクロマト法 |
CN201780034669.4A CN109313185B (zh) | 2016-06-09 | 2017-06-08 | 用于提取并测量糖链抗原的免疫色谱试验片及检体添加用设备和使用其的免疫色谱法 |
EP17810400.6A EP3470841A4 (en) | 2016-06-09 | 2017-06-08 | IMMUNCHROMATOGRAPHY TEST PIECE AND DEVICE FOR ADDING SAMPLES FOR THE EXTRACTION AND MEASUREMENT OF SUGAR CHAIN ANTIQUE AND IMMUNCHROMATOGRAPHICAL METHOD THEREFOR |
KR1020187038250A KR102317128B1 (ko) | 2016-06-09 | 2017-06-08 | 당사슬 항원을 추출하고 측정하기 위한 면역크로마토그래피 시험편 및 검체 첨가용 디바이스, 및 그것을 이용한 면역크로마토그래피법 |
EP23159698.2A EP4220167A1 (en) | 2016-06-09 | 2017-06-08 | Immunochromatography test piece and specimen adding device for extracting and measuring sugar chain antigen |
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WO2023095843A1 (ja) * | 2021-11-24 | 2023-06-01 | 旭化成株式会社 | イムノクロマト装置及びその製造方法、並びにそれを用いた対象細菌の検出方法 |
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CN109313185B (zh) * | 2016-06-09 | 2022-07-26 | 电化株式会社 | 用于提取并测量糖链抗原的免疫色谱试验片及检体添加用设备和使用其的免疫色谱法 |
JP6832758B2 (ja) * | 2017-03-14 | 2021-02-24 | デンカ株式会社 | 非特異反応を防止し得る、糖鎖抗原を抽出し測定するためのイムノクロマト試験片 |
JP6815232B2 (ja) * | 2017-03-14 | 2021-01-20 | デンカ株式会社 | 糖鎖抗原を抽出し測定するためのイムノクロマトデバイス |
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MY199249A (en) | 2023-10-23 |
CN109313185A (zh) | 2019-02-05 |
EP4220167A1 (en) | 2023-08-02 |
KR20190017826A (ko) | 2019-02-20 |
EP3470841A1 (en) | 2019-04-17 |
JP7018876B2 (ja) | 2022-02-14 |
JP2021193395A (ja) | 2021-12-23 |
CN109313185B (zh) | 2022-07-26 |
US20190145864A1 (en) | 2019-05-16 |
KR102317128B1 (ko) | 2021-10-22 |
EP3470841A4 (en) | 2019-12-11 |
JPWO2017213227A1 (ja) | 2019-04-04 |
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