WO2020105567A1 - Bandelette réactive d'immunochromatographie et kit de détection d'immunochromatographie - Google Patents

Bandelette réactive d'immunochromatographie et kit de détection d'immunochromatographie

Info

Publication number
WO2020105567A1
WO2020105567A1 PCT/JP2019/044997 JP2019044997W WO2020105567A1 WO 2020105567 A1 WO2020105567 A1 WO 2020105567A1 JP 2019044997 W JP2019044997 W JP 2019044997W WO 2020105567 A1 WO2020105567 A1 WO 2020105567A1
Authority
WO
WIPO (PCT)
Prior art keywords
conjugate
sample
immunochromatographic
pad
detection
Prior art date
Application number
PCT/JP2019/044997
Other languages
English (en)
Japanese (ja)
Inventor
佳菜子 伊藤
景吾 河野
駿 酒井
旭 中島
元喜 森田
Original Assignee
積水メディカル株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 積水メディカル株式会社 filed Critical 積水メディカル株式会社
Publication of WO2020105567A1 publication Critical patent/WO2020105567A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to an immunochromatographic test strip and an immunochromatographic detection kit including the test strip. More specifically, the present invention relates to a test piece for immunochromatography in which a conjugate is allowed to coexist with a specific sugar, and an immunochromatographic detection kit including the test piece.
  • a test piece for an immunochromatographic detection method (hereinafter sometimes simply referred to as an immunochromatographic test piece) generally includes a membrane made of a porous body having at least a sample supply section, a developing section, and a detection section.
  • a labeled antibody (conjugate) that forms a complex with an object to be detected is retained at the expansion start site of the expansion part so that it can be eluted, and the antibody is immobilized on a part of the downstream side of the expansion part to detect the detection part.
  • the detection target specifically binds to the labeled antibody at the developing unit to form a complex.
  • the complex binds to the immobilized antibody at the detection part while expanding the development part toward the downstream direction.
  • a sandwich type complex is formed by the labeled antibody, the detection target and the immobilized antibody, and the detection target can be qualitatively or quantitatively analyzed. ..
  • An example of the labeled substance that constitutes the labeled antibody is gold colloid particles, and qualitative detection is possible by a color reaction caused by the gold colloid particles.
  • the labeled antibody conjuggate
  • the labeled antibody is generally held in a dried state on a member called a conjugate pad, and the pad constitutes a part of the immunochromatographic test strip.
  • Patent Documents 1 to 3 are known as improved techniques relating to a detection reagent containing such a conjugate.
  • latex particles, a buffer solution, a protein, and saccharose are used as a latex composition for immunoassay for preventing spontaneous aggregation of latex particles having an antibody bound thereto and enabling highly sensitive immunoassay.
  • Trehalose, maltose, lactose, sorbitol, D-mannitol, and polyhydric alcohols are disclosed as compositions containing one or more anticoagulants.
  • the types of anti-aggregation agents are limited, and among them, only sucrose, trehalose and glycerin have been confirmed to have anti-aggregation effects by tests.
  • no consideration has been given to variations in measured values.
  • a water-soluble polysaccharide having a 1 wt% viscosity of 0.1 cP or more and 300 cP or less is used in the range of 0.001 wt% or more and 20.00 wt% or less.
  • a developing solution for immunochromatography containing a detection reagent labeled with a carrier such as gold colloid particles having an average particle diameter of 1 nm or more and 60 nm or less is disclosed, and CMC is mentioned as a water-soluble polysaccharide having such characteristics.
  • CMC is mentioned as a water-soluble polysaccharide having such characteristics.
  • Patent Document 3 as a solid-phased immunoreagent capable of maintaining high stability in a dry state for a long period of time, an antibody is immobilized on an insoluble carrier such as polystyrene beads and then immersed in a buffer solution containing a specific sugar.
  • Patent Document 4 discloses an immunochromatographic test strip containing as a constituent a conjugate pad in which a detection reagent containing a conjugate in which gold colloid is sensitized with an antibody is impregnated together with sucrose. However, there is no particular disclosure about the role of sucrose, and there is no suggestion about other sugars.
  • the present invention is to provide an immunochromatographic test strip capable of preventing the variability of the measured value, in which the conjugate retained in the immunochromatographic test strip is rapidly eluted by the passage of the sample liquid, the amount of the conjugate contributing to the reaction is little changed.
  • the conjugate was coexisted with glucose and / or maltose in the immunochromatographic test piece, whereby the conjugate was rapidly eluted by the passage of the sample liquid. It was found that the amount of conjugate elution was small, and as a result, it was possible to prevent variations in the detected values. Further, they have also found that coagulation of the conjugate itself can be suppressed by allowing the conjugate to coexist with glucose and / or maltose, and have completed the present invention. That is, the present invention has the following configurations.
  • An immunochromatographic test strip including at least a sample supply unit, a development unit, and a detection unit, An immunochromatographic test strip in which a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so as to be eluted together with glucose and / or maltose in the developing part.
  • the immunochromatographic test piece according to (1) which includes, as a development part, a conjugate pad in which the conjugate is dried and held so as to be eluted together with glucose and / or maltose.
  • An immunochromatographic detection kit comprising the immunochromatographic test strip according to any one of (1) to (5).
  • An immunochromatographic detection method for detecting an object to be detected in a sample using a test piece for immunochromatography The immunochromatographic detection method, which comprises a step of bringing a substance to be detected in a sample into contact with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose and / or maltose.
  • An immunochromatographic detection method for detecting an object to be detected in a sample using an immunochromatographic test strip the detection method including steps (A) to (C).
  • A Process test piece for supplying a specimen to the sample supply section of the following test piece;
  • An immunochromatographic test strip comprising at least a sample supply unit, a development unit and a detection unit, In the development part, a conjugate containing a specific binding substance labeled with a labeling substance is retained in a dry state so that it can be eluted with glucose and / or maltose.
  • C a step of forming a complex between the substance to be detected and a conjugate of (C) the step of detecting the complex in a detection part (9), wherein the conjugate can be eluted with glucose and / or maltose
  • a method for reducing variation in measured values in a method for detecting an object to be detected in a sample using a test piece for immunochromatography A method for reducing variation in the above-mentioned measured values, which comprises a step of bringing a substance to be detected in a sample into contact with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose and / or maltose.
  • A Process test piece for supplying a specimen to the sample supply section of the following test piece;
  • An immunochromatographic test strip comprising at least a sample supply section, a development section and a detection section,
  • a conjugate containing a specific binding substance labeled with a labeling substance is retained in a dry state so that it can be eluted with glucose and / or maltose.
  • C a step of forming a complex between the substance to be detected and a conjugate of (C) the step of detecting the complex in a detection part
  • the conjugate in the immunochromatographic test strip, by holding the conjugate in the presence of glucose and / or maltose, the dispersibility of the conjugate is secured and aggregation does not occur, and the conjugate is rapidly retained by the passage of the sample liquid. It is possible to provide an immunochromatographic test strip that is more eluted and has a reduced variation in dissolution rate. Further, since the conjugate is rapidly eluted into the developing portion, the reaction is carried out in the detecting portion within a predetermined reaction time, so that the delay of the test can be prevented.
  • the immunochromatographic test strip of the present invention is an immunochromatographic test strip including at least a sample supply unit, a developing unit, and a detection unit, and a specific binding substance (specific binding substance for detection) is immobilized on the detection unit. Has been converted.
  • the specific binding substance also referred to as a conjugate
  • labeled with a labeling substance capable of forming a complex with the detection target is present as described below so that it can pass through the development part and reach the detection part after contact with the sample.
  • modalities AC
  • a sample pad that serves as a sample supply unit a conjugate pad that holds a conjugate that forms a complex with a detection target together with glucose and / or maltose so as to be eluted, and that serves as a part of a development unit
  • An example is a test piece including a porous membrane on which a detection antibody is partially immobilized and which serves as a developing portion and a detecting portion. That is, a typical immunochromatographic test strip of the present invention has the following constitution.
  • a sample pad to which a specimen is supplied (2) A conjugate which is arranged between a sample pad and a membrane including a developing portion, and in which a labeling substance such as gold colloid is sensitized with a first specific binding substance Is placed downstream of the conjugate pad (3) that is retained so that it can be eluted with glucose and / or maltose, and a second specific binding substance that binds to the complex of the conjugate and the detection target is Porous Membrane Immobilized and Having Detecting Portion
  • the sample pad, the conjugate pad, and the porous membrane may each form separate carriers, or two may form one carrier.
  • the immunochromatographic test piece also includes an immunochromatographic test piece in which any one or more of an absorption pad and 3rd Pad is further arranged and mounted.
  • the test piece can also be usually placed on a solid support such as a plastic pressure-sensitive adhesive sheet.
  • a solid support such as a plastic pressure-sensitive adhesive sheet.
  • the method for detecting an object to be detected in a sample using the immunochromatographic test strip of the present invention has at least the following steps; A step of dropping a sample on the sample supply unit, A step of contacting the detection target in the sample with the conjugate in the presence of glucose and / or maltose, and a step of detecting a complex of the detection target and the conjugate in the sample at the detection unit.
  • sample pad is a part that serves as a sample supply unit that receives a specimen, absorbs a liquid sample in a state where the sample is molded into the pad, and allows the liquid and the components of the detection target to pass through. It includes any substance and form.
  • the sample pad of the present invention can be subjected to a hydrophilic treatment so that the development of the sample can be performed smoothly, and the treatment may be performed on at least the sample supply portion of the sample pad where the sample is dropped, It may be wider than the sample supply portion in the developing direction, and is preferably applied to the entire sample pad.
  • the hydrophilic treatment can be performed with sugars, surfactants and the like.
  • the sample pad of the present invention holds glucose and / or maltose in order to accelerate the elution of the conjugate, like the conjugate pad.
  • the sample pad of the present invention may contain a hemagglutination agent. In this case, it may be contained in at least a part of the sample pad, or may be contained in the entire sample pad. Specific examples of materials suitable for the sample pad include, but are not limited to, glass fiber, acrylic fiber, hydrophilic polyethylene material, dry paper, paper pulp, woven fabric, and the like. A glass fiber pad is preferably used.
  • the sample pad can also have the function of a conjugate pad described later. Further, the sample pad may contain a commonly used blocking reagent, if necessary, within a range that does not deviate from the object of the present invention and does not affect the reaction system.
  • the conjugate refers to a substance in which a specific binding substance for a target substance to be detected or a specific binding substance for control (or antigen) is immobilized on a labeling substance.
  • the labeling substance used in the present invention can form a conjugate by immobilizing (also referred to as sensitization) a specific binding substance, and detects a target substance (antigen) in a sample by contacting with a sample. Any method may be used as long as it can play a role as a labeling substance in the method, and examples thereof include metal colloid, polystyrene, polyethylene, porous glass, glass beads, and magnetic particles. preferable.
  • the particle diameter of the gold colloid particles is preferably 20 to 100 nm, more preferably 30 to 100 nm, and particularly preferably 30 to 70 nm.
  • the conjugate of the present invention can also block a region on the surface of gold colloid particles or the like to which a specific binding substance is not bound, with a blocking agent.
  • the conjugate is preferably present together with glucose and / or maltose so as to enhance dispersibility and allow rapid elution by allowing a sample, a developing solution, or the like to pass through.
  • the conjugate may be present as a conjugate pad, that is, may be present in a state of being impregnated with a dedicated pad (conjugate pad) other than the sample pad, 3rd Pad, and porous membrane ( Type A), it may be present as a detection reagent holding part in a part of the sample pad (type B). Furthermore, separately from the test strip, it may be present as a separate detection reagent so as to be mixed with the analyte (type C).
  • test piece of the type A in which the conjugate is present will be described.
  • the sample pad, the conjugate pad, 3rd Pad, and the porous membrane are arranged in this order from upstream to downstream in the flow direction of the sample, and are arranged such that at least a part of each overlaps with the upper and lower layers.
  • a test piece having such an arrangement example is shown in FIG.
  • the complex is then passed through a 3rd Pad placed in contact with the lower surface of the conjugate pad and developed into a porous membrane.
  • a conjugate pad holds glucose and / or maltose together with the conjugate in a dry state.
  • a specific binding substance or antigen that immunologically reacts with the detection target is immobilized on a part of the porous membrane, so that the complex binds and is immobilized by the immunoreaction here. Will be realized.
  • the immobilized complex is detected by means of detecting absorbance, reflected light, fluorescence, magnetism and the like derived from the labeling substance.
  • the labeling substance is a labeling substance having visibility such as colloidal gold or colored latex, it can be detected by visual observation.
  • the sample supply section is a section that supplies a sample containing a detection target
  • the detection reagent holding section is a section that contains a conjugate
  • the sample supply section is on the upstream side of the detection reagent holding section.
  • glucose and / or maltose are dried and held in such a detection reagent holding unit.
  • a test piece whose conjugate is of type C will be described.
  • the difference from the above type A test strip is that the conjugate pad does not exist as a test strip, but the conjugate does exist as an individual detection reagent.
  • a filter chip in which the conjugate is retained in the filter can be mentioned.
  • the conjugate retained in the filter and the target substance to be detected bind to each other to form a complex.
  • An object to be detected can be detected by supplying this to the same test piece as that of type A except that it has no conjugate pad.
  • glucose and / or maltose are dried and held together with the conjugate in such a filter chip.
  • the “detection reagent” is specifically a solution containing at least the conjugate.
  • the conjugate is preferably retained in a dry state together with glucose and / or maltose in a state capable of being eluted in the immunochromatographic test strip. Therefore, the detection reagent of the present invention contains glucose and / or maltose in addition to the conjugate.
  • the conjugate coexists with glucose and / or maltose, the aggregation of the conjugate is suppressed and dispersed even when applied to the conjugate pad, and the dispersibility is maintained even when dried.
  • the conjugate that is dried and retained is eluted together with glucose and / or maltose when the conjugate is eluted by passage of a sample solution or the like, the conjugate elution rate becomes constant, and as a result, the conjugate that elutes within a predetermined time The amount becomes constant, which brings about an effect that variation in measured values is reduced.
  • the concentration of glucose contained in the detection reagent As the concentration of glucose contained in the detection reagent, assuming that the standard area of the detection reagent holding part is 0.78 cm 2 and the detection reagent application amount is 52.2 ⁇ L, 0.5 to 10.0% is desirable, 1.0 to 5.0% is more desirable, and 2.0 to 3.0% is even more desirable. Needless to say, the concentration of glucose contained in the detection reagent can be adjusted in consideration of the amount of glucose retained per area of the detection reagent retaining unit when glucose is dried and retained in the detection reagent retaining unit.
  • the concentration of maltose contained in the detection reagent is preferably 0.5 to 10.0%, more preferably 1.0 to 5.0%, and more preferably 2.0 to 3.0% based on the same assumption as that of glucose. Is even more desirable. When the detection reagent contains both glucose and maltose, the total concentration is preferably within the above range.
  • the detection reagent is for the purpose of keeping the conjugate in a stable state and promoting the specific reaction of the specific binding substance immobilized on the conjugate with the detection target when mixed with the sample. Further, it may further contain stabilizers such as bovine serum albumin (BSA) and amino acids. Furthermore, the detection reagent may also contain known sensitizers and chelating agents such as EDTA and EGTA for the purpose of improving detection sensitivity, if necessary. In this specification, the term “detection” or “measurement” needs to be interpreted in the broadest sense including proof of the presence of a detection target and / or quantification, and is not limited to any meaning. must not.
  • the “conjugate pad” is a material obtained by impregnating a material suitable for the conjugate pad with a detection reagent that specifically reacts with an object to be detected and drying the material.
  • the conjugate pad has a function of forming a complex between the detection reagent and the detection target when the sample passes through the conjugate pad.
  • the conjugate pad may be arranged so as to contact the specific binding substance-immobilized membrane by itself. Alternatively, it is placed in contact with the sample pad, receives the sample that has passed through the sample pad by capillary flow, and then transfers the sample by capillary flow to a 3rd Pad that is in contact with a surface different from the contact surface with the sample pad. You may arrange so. It should be noted that selection of one or more sites of the sample pad and the conjugate pad and how to arrange the selected sites on the specific binding substance-immobilized membrane can be appropriately changed.
  • Suitable materials for the conjugate pad include, but are not limited to, paper, cellulose blends, nitrocellulose, polyesters, acrylonitrile copolymers, glass fibers or non-woven fibers such as rayon.
  • a glass fiber pad is preferably used.
  • the conjugate pad of the present invention can be obtained by immersing the coating solution containing glucose and / or maltose and the conjugate in the conjugate pad and drying. Thus, the conjugate will be kept dry in the conjugate pad with glucose and / or maltose.
  • a detection reagent solution containing glucose and / or maltose at a predetermined concentration is prepared, and the solution is horizontally moved while discharging the solution from the nozzle at a constant speed. It is possible to hold the solution so that it can be eluted by applying it to the conjugate pad in a line or the like and drying it using an apparatus having a mechanism capable of doing so.
  • a predetermined amount of the detection reagent solution having a predetermined concentration may be sampled with a pipette or the like and applied to a part or the whole surface of the conjugate pad. Further, the conjugate pad may be dipped in a container containing a detection reagent solution having a predetermined concentration and applied on the entire surface.
  • the conjugate pad is a "control reagent" for ensuring the reliability of the detection result by the immunochromatographic detection method, for example, a specific binding substance that does not react with a sample component labeled with a labeling substance. Or a highly antigenic protein such as KLH (Keyhole mussel hemocyanin) labeled with a labeling substance.
  • control reagents are components (substances) that are unlikely to be present in the sample and can be appropriately selected.
  • the lower limit of the amount of glucose coexisting with the conjugate in the detection reagent holding part is preferably 0.335 mg / cm 2 or more, and more preferably 0.669 mg / cm 2 or more. Even more preferably, it is 1.34 mg / cm 2 or more.
  • the upper limit is preferably 6.69 mg / cm 2 or less, more preferably 3.35 mg / cm 2 or less. Even more preferably, it is 2.01 mg / cm 2 or less.
  • the range of the retained amount is preferably 0.335 to 6.69 mg / cm 2 , and more preferably 0.669 to 3.35 mg / cm 2 . Even more preferably, it is 1.34 to 2.01 mg / cm 2 .
  • the concentration of glucose in the detection reagent may be adjusted by adjusting the glucose concentration in the detection reagent solution so as to have the preferable retention amount, and the area of the standard detection reagent retention unit. Is 0.78 cm 2 and the coating amount is 52.2 ⁇ L, the lower limit of the glucose concentration (C) in the detection reagent solution is 0.5% or more, and more preferably 1.0% or more from the following formula. And even more preferably 2.0 or more.
  • the upper limit is 10% or less, more preferably 5.0% or less, and even more preferably 3.0% or less.
  • the range of glucose concentration (C) in the detection reagent solution is 0.5% to 10%, more preferably 1.0 to 5.0%, and even more preferably 2.0 to It is 3.0%.
  • a (mg / cm 2 ) B ⁇ L ⁇ (C% / 100) / Dcm 2
  • Hemagglutination agent when whole blood is used as a sample, it is desirable to use a hemagglutination agent in addition to the above 3rd Pad.
  • a known polycationic hemagglutination agent can be used as the hemagglutination agent.
  • polybrene is preferred. Polybrene is a kind of a cationic polymer which has a chemical name of hexadimethrin bromide and is assigned CAS number 28728-55-4.
  • the hemagglutination agent such as polybrene
  • the hemagglutination agent in addition to the mode of adding it to the sample diluent or directly to the specimen, the hemagglutination agent should be contained in the sample supply section (sample pad) of the immunochromatographic test strip. You can According to such a use mode, the hemagglutination agent comes into contact with whole blood, and the red blood cells in the whole blood are aggregated.
  • Immobilization of specific binding substance on porous membrane Immobilization of a specific binding substance such as an antibody against an object to be detected in the immunochromatographic test strip of the present invention onto a porous membrane can be carried out by a generally known method.
  • the above-mentioned specific binding substance is prepared at a predetermined concentration, and the solution is applied to the porous membrane in a specific symbol shape such as a dot or +.
  • the protein or compound that can bind to the conjugate is immobilized at a position different from the specific binding substance that binds to the detection target, and the “control detection is performed.
  • control detection unit it is also possible to immobilize the specific binding substance that binds to the control reagent at a position different from the position of the specific binding substance that binds to the detection target to form a “control detection unit”.
  • a device having a mechanism capable of moving the above-mentioned specific binding substance to a predetermined concentration and horizontally moving it while discharging the liquid from the nozzle at a constant speed is used.
  • the concentration of the specific binding substance is preferably 0.1 to 5 mg / mL, more preferably 0.5 to 3 mg / mL.
  • the immobilized amount of the specific binding substance on the porous membrane can be optimized by adjusting the coating amount dropped on the porous membrane in the case of the flow-through type, and the above in the case of the lateral flow type. It can be optimized by adjusting the discharge speed from the nozzle of the device.
  • the term "flow-through type membrane assay” refers to a method in which a sample solution or the like is developed so as to pass perpendicularly to the porous membrane
  • the term “lateral flow type membrane assay” refers to It refers to the method of developing so that the sample liquid and the like move in parallel to the porous membrane.
  • the coating position on the porous membrane of the specific binding substance that binds to the target in the case of a lateral flow type, from the detection reagent holding unit, the detection reagent is developed by capillary action, It may be arranged so as to sequentially pass through the lines coated with the respective specific binding substances. It is preferable that the line coated with the specific binding substance that binds to the detection target is located upstream, and the line coated with the control specific binding substance is located downstream thereof. At this time, it is desirable that the distance between the lines be sufficiently large so that the signal of the labeling substance can be detected. Also in the case of the flow-through type, the application position of the specific binding substance that binds to the target may be arranged so that the signal of the labeling substance can be detected.
  • the specific binding substance solution to be applied to the above-mentioned porous membrane can be usually prepared using a predetermined buffer solution.
  • the buffer solution include a commonly used buffer solution such as a phosphate buffer solution, a Tris buffer solution, and a Good's buffer solution.
  • the pH of the buffer solution is preferably in the range of 6.0 to 9.5, and may be set appropriately according to the properties of the specific binding substance used.
  • a buffer solution having a pH of 8.0 can be used for the anti-cTnI antibody described below.
  • the buffer solution may further contain salts such as NaCl, stabilizers and preservatives such as sucrose, and preservatives such as proclin.
  • the salts include not only salts such as NaCl that are added for adjusting the ionic strength, but also salts such as sodium hydroxide that are added in the step of adjusting the pH of the buffer solution.
  • blocking can also be performed by further applying a commonly used blocking agent in the form of a solution or vapor to cover other than the specific binding substance immobilization site.
  • specific binding substance-immobilized porous membrane as described above is referred to as “specific binding substance-immobilized membrane”
  • the antibody-immobilized porous membrane is referred to as “antibody-immobilized”.
  • porous membrane In the present invention, as the porous membrane (hereinafter sometimes simply referred to as a membrane), any porous material can be used. Examples thereof include, but are not limited to, polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, and ceramics. Specific examples thereof include glass fiber filter papers and nitrocellulose membranes sold by Merck Millipore, Toyo Roshi Kaisha, GE Healthcare, and the like. In addition, by appropriately selecting the pore size and structure of the porous membrane made of this porous material, the speed at which the immune complex of the gold colloid-labeled specific binding substance (conjugate) and the detection target flows through the membrane can be determined. It is possible to control. By controlling the rate of flow through the membrane, the amount of the labeled specific binding substance that binds to the specific binding substance immobilized on the membrane can be adjusted. It is desirable to optimize the immunochromatographic test piece in consideration of the combination with other constituent materials.
  • 3rd Pad means that among the reaction components of the sample and the detection reagent, components unnecessary for the detection of the detection target are removed, and the components required for the reaction have a specific binding substance immobilized thereon.
  • the porous membrane can be arranged for the purpose of smoothly expanding it. For example, it is desirable that blood cells, insoluble blood cell lysates, and the like be removed as unnecessary components for detection.
  • the aggregates generated by the reaction of the antigen-specific binding substance among the aggregates generated by the reaction of the antigen-specific binding substance, the aggregates that have moved to the specific binding substance-immobilized membrane and have become large enough to prevent smooth development are removed in advance. It is also possible to have the additional effect of.
  • the 3rd Pad includes any substance and form through which a liquid, a component to be detected, and a detection reagent can pass. Specific examples include, but are not limited to, glass fiber, glass fiber, acrylic fiber, hydrophilic polyethylene material, polysulfone, dry paper, paper pulp, woven fabric, and the like.
  • 3rd Pad is sometimes called a blood cell separation membrane.
  • a blood cell separation membrane In the present invention, when whole blood is used as a sample, it is preferable to use a blood cell separation membrane in order to more reliably separate and remove blood cells that could not be completely removed by the sample pad alone.
  • the absorbent pad is a portion having liquid absorbency that controls the development of the sample by absorbing the sample that has moved / passed through the porous membrane.
  • the absorbent pad may be provided at the most downstream side of the strip structure, and in the flow through type, it may be provided, for example, below the specific binding substance-immobilized membrane.
  • the absorbent pad may be, for example, filter paper, but is not limited to this.
  • the immunochromatographic test strip of the present invention is suitable in consideration of the size of the test strip, the method and position of addition of the sample, the immobilization position of the specific binding substance on the specific binding substance immobilization membrane, the detection method of the signal, etc. It can be stored and installed in a container (housing) for use, and the state in which it is stored and installed is called a "detection device.”
  • a developing solution supply section for separately supplying a developing solution can be provided on the upstream side of the sample supply section.
  • a "porous membrane” is a “solid phase", a specific binding substance such as an antigen or an antibody is physically or chemically retained on a porous membrane, or the state of being retained is “fixed”, It may be expressed as “immobilization”, “immobilization”, “sensitization”, or “adsorption”.
  • the “specimen” containing the detection target refers to blood, urine, sputum, saliva, nasal discharge, nasal swab, pharyngeal swab, other body fluids, biological samples such as feces, and the like.
  • the biological sample may be used as a sample as it is, and a sample obtained by appropriately diluting with a diluting solution, extracting and / or extracting by filtration is also included in the sample of the present invention.
  • Blood samples include whole blood, red blood cells, plasma, serum and the like.
  • the blood sample also includes a plasma sample collected by a blood collection tube to which an anticoagulant such as EDTA or heparin is added at the time of blood collection.
  • sample diluent In the present invention, a diluent may be used when the sample needs to be diluted depending on the concentration of the detection target in the sample.
  • the diluent significantly inhibits the reaction between the target substance and the specific binding substance, or conversely significantly accelerates the reaction to cause over-aggregation of the labeling substance, resulting in poor expansion in capillary phenomenon, and Diluents of any composition may be used as long as signal detection of the reaction between the specific binding substance and the specific binding substance depending on the concentration of the binding substance is not impossible.
  • the sample diluting solution may also be called a sample extract or developing solution.
  • the detection target of the present invention is a substance present in a biological sample such as blood (whole blood), red blood cells, serum, plasma, urine, saliva or sputum, for example, CRP (C-reactive protein), IgA, IgG.
  • a biological sample such as blood (whole blood), red blood cells, serum, plasma, urine, saliva or sputum, for example, CRP (C-reactive protein), IgA, IgG.
  • Inflammation-related markers such as IgM, fibrin degradation products (eg D-dimer), soluble fibrin, TAT (thrombin-antithrombin complex), PIC (plasmin-plasmin inhibitor complex) and other coagulation / fibrinolytic markers, oxidized LDL, Circulation-related markers such as BNP (brain natriuretic peptide), H-FABP (heart-type fatty acid binding protein), cardiac troponin I (cTnI), metabolism-related markers such as adiponectin, CEA (carcinoembryonic antigen), AFP ( ⁇ -) Tumor markers such as fetoprotein), CA19-9, CA125, PSA (prostate specific antigen), HBV (hepatitis B virus), HCV (hepatitis C virus), chlamydia trachomatis, gonorrhea-related markers, allergen specific Examples include IgE (immunoglobulin E), hormones, drugs and the like.
  • IgE
  • specific binding substances for the substance to be measured carried on insoluble carrier particles include proteins, peptides, amino acids, lipids, carbohydrates, DNA, RNA, receptors, haptens, and the like.
  • insoluble carrier particles such as latex
  • proteins, peptides, amino acids, lipids, carbohydrates, DNA, RNA, receptors, haptens, and the like There are no particular restrictions on the origin of the molecular weight, such as high or low, natural or synthetic, and examples include antibodies or antigens that can be used in immunoassays that utilize immune reactions.
  • the antibody against the detection target used in the present invention is not limited by the method for producing it as long as it is an antibody that specifically reacts with the detection target, and may be a polyclonal antibody or a monoclonal antibody. May be. More preferably, it is a monoclonal antibody.
  • a hybridoma producing the antibody is a myeloma of the same species as the spleen cells of an animal immunized with the detection target as an immunogen according to the method of Kohler and Milstein (see Nature, Vol. 256, page 495 (1975)). It can be produced by cell fusion with cells (myeloma cells).
  • the antibody of the present invention in addition to the whole antibody molecule, it is also possible to use a functional fragment of an antibody having an antigen-antibody reaction activity.
  • a functional fragment of an antibody having an antigen-antibody reaction activity In addition to those obtained through the process of immunizing general animals (mouse, goat, sheep, etc.), the amino acid sequence of animal species different from the animal immunized with the immunogen (substance to be measured) by genetic recombination technology etc.
  • Antibodies chimeric specific binding substance, humanized antibody, fully humanized antibody, etc.
  • the functional fragment of the antibody include F (ab ′) 2 , Fab ′ and single chain antibody (scFv), which are fragments having antigen-antibody reaction activity.
  • Functional fragments of these antibodies can be produced by treating the antibodies obtained as described above with a proteolytic enzyme (eg, pepsin or papain).
  • a proteolytic enzyme eg, pepsin or papain.
  • the epitope of the second antibody is different from that of the first antibody when the epitope of the first antibody is monovalent, and is the second when the epitope of the first antibody is multivalent.
  • the epitope of the antibody may be the same as or different from that of the first antibody.
  • the detection kit utilizing the immunochromatography of the present invention is an immunochromatographic test strip having at least a sample supply unit, a developing unit and a detecting unit, and a part of the developing unit is labeled with a labeling substance. It is sufficient that the conjugate (detection reagent) containing the specific binding substance contains the immunochromatographic test strip retained so as to be eluted together with glucose and / or maltose.
  • the detection kit may further include reagents necessary for detection, a sample diluent, a test tube, a filtration filter, a swab for collecting a sample, an instruction manual, a housing for storing a test piece, and the like.
  • the immunochromatographic detection method for detecting a detection target substance in a sample according to the present invention using a test piece for immunochromatography is a specific binding of the detection target substance in the sample labeled with a labeling substance in the presence of glucose and / or maltose.
  • a method comprising contacting with a conjugate containing a volatile substance. More specifically, the method includes the following steps (A) to (C).
  • A Process test piece for supplying a specimen to the sample supply section of the following test piece;
  • An immunochromatographic test strip comprising at least a sample supply unit, a development unit and a detection unit, In the developing part, a conjugate containing a specific binding substance labeled with a labeling substance is retained in a dry state so that it can be eluted together with glucose and / or maltose.
  • C a step of forming a complex between the substance to be detected and a conjugate of (C) a step of detecting the complex in the detection section, wherein the detection of the complex includes qualitative detection by visual observation and optical detection. Both quantitative measurements by means are included.
  • the optical detecting means of the present invention in addition to the method of measuring the test pieces one by one, a method of using an apparatus for continuously and automatically measuring a plurality of test pieces and a plurality of test pieces Various modes are included, such as a method using a device for simultaneously measuring the. Specific examples of the present invention will be described below, but these are merely examples and the present invention is not limited thereto.
  • Example 1 Screening of saccharides applied to the conjugate pad of the present invention As a saccharide applied to a conjugate pad, a blank value is reduced as compared with conventionally used lactose, and sample measurement sensitivity is increased, Was screened under the condition.
  • Detection Device of the Present Invention 1) Preparation of Gold Colloid-Labeled Anti-cTnI Monoclonal Antibody (Anti-cTnI Antibody Conjugate) (i) Preparation of Gold Colloid Solution 7% (w) to 500 mL of purified water heated to 93 ° C. / V) 1 mL of an aqueous solution of triammonium citrate was added and mixed with stirring.
  • conjugate pad 3 OD / mL of anti-cTnI antibody conjugate, 0.75 OD / mL of KLH conjugate, 0.5% Lipidure BL-1301, 0.25 mg / mL Heteroblock, various saccharides described below, 2
  • a detection reagent was prepared by mixing 0.0% NPS and 20 mM MOPS (pH 7.2), and 52.2 ⁇ L of the solution was added per 0.78 cm 2 to a glass fiber pad (Merck Millipore) that was appropriately cut into a necessary size. It was permeated in a volume ratio. It was dried by heating in a dry oven at 70 ° C. for 45 minutes to obtain a conjugate pad.
  • the anti-cTnI monoclonal antibody was adjusted to 1 ⁇ L / cm using an immunochromatographic dispenser “XYZ3050” (BIO DOT). It was set and applied in a line to form a test line. An anti-KLH polyclonal antibody was similarly applied at intervals of about 4 mm from the position of the test line to form a control line. It was dried at 70 ° C. for 45 minutes in a dry oven to obtain an antibody-immobilized membrane.
  • sample pad 20 mM MOPS (pH 7.2) containing 0.5% lactose and 2% polybrene was cut into a glass fiber pad (Lydall) 1.5 times the volume of the pad. What was soaked in a volume and dried in a dry oven at 70 ° C. for 45 minutes was used as a sample pad.
  • an immunochromatographic test piece was produced by cutting into a structure in which the respective constituent elements were superposed.
  • the test strip may be stored and mounted in a dedicated plastic housing (having a sample addition window and a detection window, not shown in FIG. 9) to form a detection device.
  • FIG. 9 shows a schematic configuration diagram of the immunochromatographic test piece.
  • sucrose, trehalose, maltose, and glucose suppress blank values as compared with conventional lactose, and that the measurement sensitivity of the sample is high.
  • Test Example 2 CV reduction effect confirmation test (1) Among the sugars selected by the screening in Test Example 1, the CV reducing effect was confirmed for various concentrations of sucrose and glucose. For comparison, the CV reduction effect was similarly confirmed for the conventionally used lactose. 1. Test method The sample is a plasma sample containing 150 pg / mL cardiac troponin I (cTnI), and 1.0%, 2.4%, 4.0% sucrose or glucose as a saccharide for adjusting the detection reagent, 2.4% Was used, and the coloration amount (absorbance) of the test line was measured in the same manner as in Test Example 1 except that the n number was changed to 5, and the CV values (variations) thereof were calculated. CV stands for Coefficient of Variation and can be obtained by dividing the standard deviation by the average value. Results are shown in FIG.
  • Test Example 3 Simultaneous reproducibility and accelerated stability confirmation test after storage under severe conditions (1) A test piece in which glucose, lactose, and sucrose were added to the conjugate coating solution was stored at 37 ° C. for 10 days to be stored under severe conditions, and a test for confirming variation variation and stability of measured values was performed. 1. Test Method A test piece of the present invention was produced in the same manner as in Test Example 1 except that 2.4% lactose, 4.0% sucrose, and 2.4% glucose were used as saccharides for adjusting the detection reagent.
  • FIG. 4 shows the relative values of the absorbance measurement values before and after storage under severe conditions (the absorbance measurement value after “acceleration” with respect to the absorbance measurement value before “acceleration”).
  • Test Example 4 CV reduction effect confirmation test (2) Among the sugars selected by the screening in Test Example 1, glucose, maltose, and trehalose were confirmed to have a CV reducing effect. For comparison, lactose was similarly confirmed to have a CV reducing effect. 1. Test method A HIGH sample (a plasma sample containing 500 pg / mL cardiac troponin I (cTnI)) was used, and 2.4% glucose, maltose, trehalose, and lactose were used as saccharides for adjusting the detection reagent.
  • cTnI cardiac troponin I
  • Test Example 5 Accelerated stability confirmation test after storage under severe conditions (2) A test piece prepared by adding glucose, maltose, and trehalose to the conjugate coating solution was stored at 37 ° C. for 10 days to be stored under severe conditions, and a test for confirming stability was conducted. 1. Test method A test piece of the present invention was prepared in the same manner as in Test Example 1 except that 2.4% lactose, 2.4% glucose, 2.4% maltose, and 2.4% trehalose were used as saccharides for adjusting the detection reagent. did.
  • n The number of n was set to 5, and a HIGH sample (a plasma sample containing 1000 pg / mL cardiac troponin I (cTnI)) was used, and a nitrocellulose membrane (UniSart CN150 white backed, Sartorius Stedim Biotech, model number 1UN15WR100025NT) was used. Tests were performed in the same manner as in Test Example 3 except that the stability was evaluated. The relative values of the absorbance measurement values before and after storage under severe conditions are shown in FIG. 8 with respect to the “after acceleration” absorbance measurement values with respect to the “before acceleration” absorbance measurement values.
  • Test results According to this figure, when glucose was added to the conjugate coating solution, it was confirmed that the stability of the measured values was small and the stability was excellent compared to the case where maltose was added even after storage under severe conditions. It was From the results of Test Examples 1 and 4 described above, it was confirmed that when glucose or maltose was added to the conjugate coating solution, the simultaneous reproducibility was excellent as compared with the case where conventional lactose or another sugar was added. .. Moreover, when the results of Test Examples 1 to 5 are summed up, it can be reconfirmed that when glucose is added to the conjugate coating solution, both simultaneous reproducibility and stability are superior to conventional lactose and other sugars. It was
  • the conjugate since the conjugate is allowed to coexist with glucose and / or maltose to be retained, the dispersibility of the conjugate is ensured, and the conjugate is promptly passed by the passage of the sample liquid. It is possible to provide an immunochromatographic test strip which is eluted from the detection reagent holding part of (3) and has a reduced variation in the elution rate. Further, according to the immunochromatographic test strip of the present invention, the conjugate is rapidly eluted to the test strip developing portion, so that the detection portion surely develops color within a predetermined reaction time and prevents the delay of the test. You can

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention aborde le problème de la fourniture d'une bandelette réactive d'immunochromatographie dans laquelle est contenu un conjugué et qui est rapidement éluée en raison du passage d'un liquide de test de sorte à pouvoir réagir avec une substance à détecter, la quantité du conjugué contribuant à la faible variation de la réaction et des variations des valeurs mesurées pouvant être empêchées.　La présente invention concerne une bandelette réactive d'immunochromatographie ayant une membrane poreuse comprenant au moins une section de fourniture d'échantillon, une section de déploiement et une section de détection. Dans une partie de la section de déploiement, un conjugué contenant une substance de liaison spécifique marquée avec une substance de marquage est maintenu, conjointement avec du glucose et/ou du maltose, dans un état pouvant être élué et sec.
PCT/JP2019/044997 2018-11-19 2019-11-18 Bandelette réactive d'immunochromatographie et kit de détection d'immunochromatographie WO2020105567A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018-216522 2018-11-19
JP2018216522A JP2022046827A (ja) 2018-11-19 2018-11-19 イムノクロマト用試験片及びイムノクロマト検出キット

Publications (1)

Publication Number Publication Date
WO2020105567A1 true WO2020105567A1 (fr) 2020-05-28

Family

ID=70774503

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/044997 WO2020105567A1 (fr) 2018-11-19 2019-11-18 Bandelette réactive d'immunochromatographie et kit de détection d'immunochromatographie

Country Status (2)

Country Link
JP (1) JP2022046827A (fr)
WO (1) WO2020105567A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112816705A (zh) * 2020-12-31 2021-05-18 北京赛诺浦生物技术有限公司 人心肌钙蛋白i、人生长分化因子15以及人d二聚体的三联检测层析试纸条及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004233127A (ja) * 2003-01-29 2004-08-19 Tokuyama Corp 免疫学的測定方法および免疫クロマトグラフィー法測定キット。
JP2007315883A (ja) * 2006-05-25 2007-12-06 Denka Seiken Co Ltd 免疫測定用ラテックス組成物
JP2008203135A (ja) * 2007-02-21 2008-09-04 Denka Seiken Co Ltd 検査デバイスの標識体部の形成方法及びラテラルフロー免疫測定用検査デバイス
WO2013119763A1 (fr) * 2012-02-07 2013-08-15 Intuitive Biosciences, Inc. Peptides spécifiques du bacille de koch pour la détection d'une infection ou d'une immunisation chez des primates non humains
JP2013195403A (ja) * 2012-03-22 2013-09-30 Tanaka Kikinzoku Kogyo Kk イムノクロマトグラフィー検出方法
WO2013145767A1 (fr) * 2012-03-30 2013-10-03 田中貴金属工業株式会社 Kit de détection du virus de la grippe a
JP2015230221A (ja) * 2014-06-04 2015-12-21 田中貴金属工業株式会社 免疫学的測定試薬におけるプロゾーン現象の解消法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004233127A (ja) * 2003-01-29 2004-08-19 Tokuyama Corp 免疫学的測定方法および免疫クロマトグラフィー法測定キット。
JP2007315883A (ja) * 2006-05-25 2007-12-06 Denka Seiken Co Ltd 免疫測定用ラテックス組成物
JP2008203135A (ja) * 2007-02-21 2008-09-04 Denka Seiken Co Ltd 検査デバイスの標識体部の形成方法及びラテラルフロー免疫測定用検査デバイス
WO2013119763A1 (fr) * 2012-02-07 2013-08-15 Intuitive Biosciences, Inc. Peptides spécifiques du bacille de koch pour la détection d'une infection ou d'une immunisation chez des primates non humains
JP2013195403A (ja) * 2012-03-22 2013-09-30 Tanaka Kikinzoku Kogyo Kk イムノクロマトグラフィー検出方法
WO2013145767A1 (fr) * 2012-03-30 2013-10-03 田中貴金属工業株式会社 Kit de détection du virus de la grippe a
JP2015230221A (ja) * 2014-06-04 2015-12-21 田中貴金属工業株式会社 免疫学的測定試薬におけるプロゾーン現象の解消法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112816705A (zh) * 2020-12-31 2021-05-18 北京赛诺浦生物技术有限公司 人心肌钙蛋白i、人生长分化因子15以及人d二聚体的三联检测层析试纸条及其应用

Also Published As

Publication number Publication date
JP2022046827A (ja) 2022-03-24

Similar Documents

Publication Publication Date Title
US5081013A (en) Immunodiagnostic device and method
JP4846573B2 (ja) 基準としての天然分析物を有するラテラルフローアッセイ装置および方法
JP2010512537A (ja) 間接側方流動サンドイッチアッセイ
EP0564494B1 (fr) Procede d'essai et kit reactif associe
JP7356204B2 (ja) イムノクロマト用試験片
CN103477226A (zh) 能将不含试样的样品确定为不当操作样品的免疫层析检测方法及其使用的测试条
JP6426872B1 (ja) イムノクロマト用試験片及びイムノクロマト検出キット
JP6399632B2 (ja) 赤血球含有サンプル中の対象物を検出するためのイムノクロマトグラフィー用テストストリップ、および該テストストリップを使用するイムノクロマトグラフィー
US8101429B2 (en) Native analyte as a reference in lateral flow assays
EP3859334A1 (fr) Pièce d'essai pour immunochromatographie
WO2020105567A1 (fr) Bandelette réactive d'immunochromatographie et kit de détection d'immunochromatographie
EP3885767B1 (fr) Éprouvette pour immunochromatographie et kit de détection immunochromatographique
JP6464308B1 (ja) イムノクロマト用試験片
CN107209179B (zh) 用于检测含红细胞的样品中的对象物的免疫色谱用测试条、及使用该测试条的免疫色谱
JP2000258418A (ja) 免疫クロマトグラフィーを用いた測定方法およびそれに用いる検体分析用具
JP2020052028A (ja) イムノクロマト用試験片
JP7137168B2 (ja) イムノクロマト用試験片
JPH1068730A (ja) イムノクロマト法用試験用具
WO2021193680A1 (fr) Bandelette réactive et dispositif comprenant une bandelette réactive
JPH09184840A (ja) イムノクロマト法用試験用具
JP2017026591A (ja) 免疫学的検出方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19886125

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19886125

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP