WO2016031892A1 - Bande de test d'immunochromatographie - Google Patents

Bande de test d'immunochromatographie Download PDF

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Publication number
WO2016031892A1
WO2016031892A1 PCT/JP2015/074138 JP2015074138W WO2016031892A1 WO 2016031892 A1 WO2016031892 A1 WO 2016031892A1 JP 2015074138 W JP2015074138 W JP 2015074138W WO 2016031892 A1 WO2016031892 A1 WO 2016031892A1
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WIPO (PCT)
Prior art keywords
pad
sample
conjugate
substance
detected
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PCT/JP2015/074138
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English (en)
Japanese (ja)
Inventor
いつみ 高野
公良 西谷
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積水メディカル株式会社
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Priority to JP2015560112A priority Critical patent/JP5936797B1/ja
Publication of WO2016031892A1 publication Critical patent/WO2016031892A1/fr

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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

Definitions

  • the present invention relates to an immunochromatographic test strip and a detection method using the same.
  • the present invention relates to a test strip suitable for detecting a substance to be detected in a sample derived from stool and a detection method using the test strip. Furthermore, it is related with the suppression method of a nonspecific reaction in the detection using the test strip for immunochromatography.
  • Detection methods using immunochromatography are widely used because detection with high specificity and sensitivity is possible.
  • samples applied for detection using immunochromatography include urine, feces, saliva, blood, serum, and the like, which are suitable as clinical laboratory samples and are widely used as diagnostically useful samples.
  • In order to detect a substance to be detected contained in these samples using immunochromatography it is necessary to exclude various detection interfering substances contained in the sample. As an interfering substance, a color reaction is carried out in spite of the fact that there is no substance that causes clogging of the insoluble membrane for development, which hinders the development of the sample itself by immunochromatography, or there is no substance to be detected. There are substances that cause so-called non-specific reactions as shown.
  • Patent Document 1 discloses a method in which a buffer containing a specific polymer is used as a developing solvent for immunochromatography in order to suppress nonspecific reactions.
  • Patent Document 2 discloses an immunochromatographic detection device in which serum albumin and a linear water-soluble polymer are coexisting with a conjugate in order to suppress nonspecific reactions.
  • gelatin, casein, bovine serum albumin and the like are generally known as reagents for blocking non-specific reactions in antigen-antibody reactions, and various methods for coexisting them in the immunochromatographic reaction system are also known. As described above, various methods for eliminating the influence of detection interference substances are known. In the detection method using immunochromatography, a method for suppressing these non-specific reactions when a sample derived from stool is applied. So far there has been no investigation.
  • the present invention relates to a method for suppressing nonspecific reactions peculiar to stool in a detection method using immunochromatography for detecting a substance to be detected in a sample by developing a sample derived from stool in an insoluble membrane, and
  • An object is to provide an immunochromatographic test strip to be used.
  • the present inventors have used a test strip in which a porous third pad made of polysulfone or cellulose acetate is arranged upstream of an insoluble membrane, so that a fecal sample can be used.
  • a test strip in which a porous third pad made of polysulfone or cellulose acetate is arranged upstream of an insoluble membrane, so that a fecal sample can be used.
  • the present inventors have found that the non-specific reaction can be suppressed and detected accurately, and the present invention has been completed.
  • These materials have been used as blood cell separation membranes in immunochromatography for developing whole blood as a sample, but have been completely used in immunochromatography for developing fecal samples. And the possibility has not been studied.
  • the present inventors have found that a nonspecific reaction can be unexpectedly suppressed by allowing a complex of a stool-derived sample and a conjugate to pass through using such a material suitable for blood cell separation, and the present invention. It came to complete. That is, the present invention has the following configuration.
  • An immunochromatographic test strip for detecting a complex of a detected substance and a conjugate in a sample by spreading a sample derived from stool in an insoluble membrane, wherein the conjugate is immunized against the detected substance.
  • the test strip wherein an antibody or antigen that reacts chemically is immobilized on a label and has the following constitutions (1) to (3).
  • Sample pad having a sample supply part
  • Porous third pad made of polysulfone or cellulose acetate disposed downstream of the sample pad and upstream of the insoluble membrane
  • Immunity against a substance to be detected Insoluble membrane ⁇ 2> having a detection part for detecting a complex of a substance to be detected and a conjugate in a sample, to which a chemically reactive antibody or antigen is immobilized
  • the porous third pad has an average pore diameter of 1 to 100 ⁇ m.
  • a detection method using immunochromatography that detects a complex of a substance to be detected and a conjugate in a sample by developing a sample derived from stool in an insoluble membrane, and includes the following steps (a) to (c): Comprising the steps of: (A) A sample is brought into contact with a conjugate in which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized on a label to form a complex of the substance to be detected and the conjugate in the sample.
  • a non-specific reaction suppression method in detection using immunochromatography for detecting a complex of a substance to be detected and a conjugate in a sample by developing a sample derived from stool in an insoluble membrane comprising the following steps (a ) To (c). (A) A sample is brought into contact with a conjugate in which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized on a label to form a complex of the substance to be detected and the conjugate in the sample.
  • the immunochromatographic test strip including a third pad made of a specific material according to the present invention can detect a substance to be detected in a stool-derived sample without causing a non-specific reaction peculiar to stool. Therefore, the substance to be detected in stool can be accurately detected using immunochromatography.
  • test strip which has arrange
  • stool is used as a sample.
  • the stool may be used as a sample as it is, or may be diluted with a diluent as appropriate. Moreover, you may use as a sample what was diluted and filtered suitably.
  • the substance to be detected of the present invention may be any substance as long as it is contained in feces as a sample and can be detected using an antigen-antibody reaction, and examples thereof include viruses, parasites, and proteins.
  • infectious gastroenteritis is mainly caused by viruses and parasites.
  • viruses that can be detected include norovirus, adenovirus, rotavirus, sapovirus, enterovirus, etc.
  • insects include cryptospiridium, amoeba dysentery, giardia and the like.
  • influenza virus as a virus to be detected, and proteins to be detected include human hemoglobin, hepatitis B virus antibody, hepatitis C virus antibody, human immunodeficiency virus antibody, and the like.
  • the immunochromatographic test strip of the present invention is a test strip for detecting a complex of a substance to be detected and a conjugate in a sample by developing a sample derived from stool in an insoluble membrane, and has the following configuration ( 1) to (3).
  • Sample pad having a sample supply part (2) Porous third pad made of polysulfone or cellulose acetate disposed downstream of the sample pad and upstream of the insoluble membrane (3) Immunity against a substance to be detected
  • An insoluble membrane having a detection part for detecting a complex of a substance to be detected and a conjugate in a sample, to which a chemically reactive antibody or antigen is immobilized
  • the conjugate is an antibody or antigen that immunologically reacts with a substance to be detected and is immobilized on a label.
  • the conjugate is present as a conjugate pad, a sample pad, a third pad, It may exist in a state where it is impregnated in another pad other than the insoluble membrane (type A), may exist as a conjugate part in a part of the sample pad (type B), and further, a test strip Alternatively, it may exist as a separate conjugate reagent to be mixed with the specimen (type C).
  • test strip of the type A in which the conjugate is present will be described.
  • the sample pad, the conjugate pad, the third pad, and the insoluble membrane are arranged in this order from upstream to downstream in the sample flow direction, and are arranged so that at least a part of the upper and lower layers overlap each other.
  • a test strip of such an arrangement is shown in FIG.
  • the composite is then passed through a porous third pad placed in contact with the bottom surface of the conjugate pad and developed into an insoluble membrane. Since an insoluble membrane is immobilized with an antibody or antigen that immunologically reacts with a substance to be detected, a part of the insoluble membrane is bound and immobilized by an immune reaction. Become.
  • the immobilized complex is detected by a means for detecting absorbance or reflected light derived from the conjugate.
  • test strip having a conjugate type of type B will be described.
  • the difference from the type A test strip is that the sample pad and the conjugate pad are integrated, that is, the sample supply part and the conjugate part are formed in a part of the sample pad.
  • the sample supply part is a part for supplying a sample containing a substance to be detected
  • the conjugate part is a part containing a conjugate
  • the sample supply part is on the upstream side of the conjugate part.
  • a test strip having a conjugate type of type C will be described.
  • the difference from the above type A test strip is that there is no conjugate pad and the conjugate is present as a separate conjugate reagent.
  • a filter chip in which a conjugate is incorporated in a filter can be mentioned.
  • the conjugate and the substance to be detected are bonded to each other by passing the specimen diluent, thereby forming a complex (aggregate).
  • the substance to be detected can be detected.
  • the sample pad used in the present invention is a portion that receives a sample, and includes any substance and form that can absorb a liquid sample while being molded into the pad and allow the liquid and the detection target to pass through.
  • materials suitable for the sample pad include, but are not limited to, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dry paper, paper pulp, and fabric.
  • a fiberglass pad is used.
  • the sample pad can also have the function of a conjugate pad described later.
  • the sample pad can contain a blocking reagent that is usually used for the purpose of preventing / suppressing nonspecific reaction (adsorption) in the antibody-immobilized membrane.
  • the third pad may be any one that can permeate the complex of the substance to be detected and the conjugate in the sample.
  • the purpose of the present invention is to capture a nonspecific reaction substance derived from stool. Therefore, the third pad needs to be a porous member made of polysulfone or cellulose acetate. Although the reason is not clear, the third pad made of these materials allows the non-specific reaction substance due to the stool sample to be captured and the non-specific reaction can be reduced, although the complex of the conjugate and the substance to be detected is passed. .
  • a third pad made of a material other than polysulfone and cellulose acetate did not show the effect of reducing non-specific reactions.
  • the average pore diameter of the porous third pad of the present invention is 1 ⁇ m or more as a lower limit, preferably 5 ⁇ m or more, more preferably 10 ⁇ m or more, further preferably 15 ⁇ m or more, particularly preferably 20 ⁇ m or more, and preferably 25 ⁇ m or more. Most preferred.
  • 100 micrometers or less are illustrated, 80 micrometers or less are preferable, 60 micrometers or less are more preferable, 55 micrometers or less are further more preferable, 50 micrometers or less are especially preferable, and 45 micrometers or less are the most preferable.
  • the range of the average pore diameter is exemplified by 1 to 100 ⁇ m which is a combination of the above lower limit and upper limit, preferably 5 to 80 ⁇ m, more preferably 10 to 60 ⁇ m, further preferably 15 to 55 ⁇ m, and particularly preferably 20 to 50 ⁇ m. 25 to 45 ⁇ m is most preferable. This is because if it is less than 1 ⁇ m, clogging is caused and the flow of the specimen itself becomes slow, and if it is more than 100 ⁇ m, it is considered that the function of capturing the non-specific reaction substance is lowered.
  • the said average hole diameter is calculated
  • the insoluble membrane used in the present invention has at least one detection unit on which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized. Immobilization of an antibody or antigen that reacts immunologically with a substance to be detected on an insoluble membrane carrier can be carried out by a conventionally known method.
  • a lateral flow type immunochromatographic reagent it has a mechanism capable of preparing a liquid containing the above-mentioned antibody or antigen at a predetermined concentration and moving it horizontally while discharging the liquid from the nozzle at a constant speed.
  • the solution can be immobilized by applying the solution to the insoluble membrane carrier in a line using a device or the like and drying.
  • the concentration of antibody or antigen in the above solution is preferably 0.1 to 5 mg / mL, more preferably 0.5 to 2 mg / mL.
  • the amount of antibody or antigen immobilized on an insoluble membrane carrier can be optimized by adjusting the ejection speed from the nozzle of the above apparatus in the case of the lateral flow type, and is preferably 0.5 to 2 ⁇ L / cm. It is.
  • the measurement method using the lateral flow type immunochromatography reagent is a measurement method in which the sample is developed so as to move in a parallel direction with respect to the insoluble membrane carrier by capillary action.
  • a solution containing the antibody or antigen described above at a predetermined concentration can be prepared by adding the antibody or antigen to a buffer solution.
  • the buffer examples include normal buffers such as phosphate buffer, Tris buffer, and Good's buffer.
  • the pH of the buffer is preferably in the range of 6.0 to 9.5, more preferably 6.5 to 8.5, and even more preferably 7.0 to 8.0.
  • the buffer may further contain salts such as sodium chloride, stabilizers such as sucrose, preservatives, preservatives such as procrine, and the like.
  • the salts include those added for the purpose of adjusting the pH of the buffer solution, such as sodium hydroxide, in addition to those included for adjusting the ionic strength, such as sodium chloride.
  • blocking can also be carried out by coating a region other than the region where the antibody or antigen is immobilized with a commonly used blocking agent in the form of a solution or vapor.
  • a control capture reagent conventionally used in immunochromatographic reagents may be immobilized on the insoluble membrane.
  • the control capture reagent is a reagent for ensuring the reliability of the assay, and captures the control reagent contained in the conjugate pad.
  • an anti-KLH antibody or the like corresponds to the control capture reagent.
  • the position at which the control capture reagent is immobilized can be appropriately selected to suit the design of the assay system.
  • a known membrane conventionally used as an insoluble membrane carrier for an immunochromatographic reagent can be used.
  • membranes composed of fibers made of polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, ceramics, and the like are preferred.
  • glass fiber filter paper and cellulose filter paper commercially available from Sartorius, Millipore, Toyo Roshi, Whatman and the like. Of these, Sartorius and UniSart® CN140 are preferred.
  • by appropriately selecting the pore size and structure of the insoluble membrane carrier it is possible to control the speed at which the complex of the conjugate and the substance to be detected in the sample flows through the insoluble membrane carrier.
  • the immunochromatographic test strip is preferably disposed on a solid support such as a plastic adhesive sheet.
  • the solid support is composed of a material that does not interfere with the capillary flow of the sample and conjugate.
  • the immunochromatographic test strip may be fixed on a solid support with an adhesive or the like.
  • the adhesive component and the like are also composed of a substance that does not interfere with the capillary flow of the sample and the conjugate.
  • the immunochromatographic test strip is stored in an appropriate container (housing) taking into account the size of the immunochromatographic test strip, the method and position of sample addition, the position where the insoluble membrane detector is formed, and the signal detection method. -It can be mounted and used, and the state stored and mounted in this way is called "device".
  • the antibody or antigen that immunologically reacts with the substance to be detected used in the present invention is an antibody or antigen that can bind to the substance to be detected.
  • the substance to be detected is a virus or an antigen
  • the antibody or the substance to be detected When is an antibody, an antigen is preferred.
  • An antibody or antigen that reacts immunologically with a substance to be detected is immobilized on a label and a detection part described later.
  • the antibody or antigen immobilized on the label and the detection unit may be the same, but it is preferable that the label and the detection unit are different.
  • Marker As the label used in the present invention, a known label conventionally used for a test strip for immunochromatography can be used.
  • colloidal metal particles such as gold colloid particles and platinum colloid particles, color latex particles, magnetic particles, and fluorescent particles are preferable, and gold colloid particles and color latex particles are particularly preferable.
  • the conjugate used in the present invention is obtained by immobilizing an antibody or antigen that immunologically reacts with a substance to be detected on the label as described above.
  • the conjugate is preferably one in which an anti-rotavirus monoclonal antibody and an anti-adenovirus monoclonal antibody are immobilized on colloidal gold particles.
  • Examples of a method for immobilizing an antibody or antigen that immunologically reacts with a substance to be detected to a label include physical adsorption and chemical bonding, and are generally immobilized by physical adsorption.
  • the immunochromatographic test strip of the present invention may further contain other reagents and components depending on measurement conditions and samples.
  • examples of other reagents include blocking agents that prevent non-specific reactions.
  • Other configurations include, for example, an absorption pad that controls the development of the sample by absorbing the sample that has moved and passed through the insoluble membrane.
  • the production of the immunochromatographic test strip of the present invention can be carried out by appropriately modifying or altering the method described in the examples.
  • the detection kit using the immunochromatography of the present invention only needs to include the immunochromatographic test strip.
  • the detection kit may further include a reagent necessary for detection, a sample diluent, a test tube, a swab for collecting stool, an instruction manual, a housing for storing a test strip, and the like.
  • upstream or downstream is used to mean the upstream side or the downstream side in the direction of sample flow.
  • the test pad of the present invention is laminated so that the sample pad, conjugate pad, third pad, and insoluble membrane partially overlap from the top, the sample pad is the most upstream and the insoluble membrane is the downstream.
  • the end pad may be stacked on the downstream side of the insoluble membrane so as to overlap with each other. In this case, the end pad is the most downstream.
  • FIG. 1 is a schematic configuration diagram of the immunochromatographic test strip of the present invention. Place the insoluble membrane (b) on the plastic adhesive sheet (a), then place and install the third pad (g), conjugate pad (d), sample pad (e) in this order, and the absorbent pad on the opposite end. (f) was placed and mounted.
  • the conjugate pad is impregnated with a conjugate of gold colloid sensitized with an anti-adenovirus monoclonal antibody, and the insoluble membrane contains anti-adenovirus monoclonal antibody (c1) and control reagent (c2) in the flow direction. It is fixed on the line vertically.
  • Each pad is laminated so that the upper and lower pads are in contact with a part thereof.
  • a line made of the anti-adenovirus monoclonal antibody (c1) is called a test line, and a line made of the control reagent (c2) is called a control line.
  • Test results are shown in Table 1. From this result, when a third pad made of polysulfone and cellulose acetate was used as the third pad, the nonspecific reaction could be suppressed, and in particular, the case of polysulfone could suppress the nonspecific reaction better. In this case, the average pore diameter of the porous holes of the third pad was 20 to 50 ⁇ m. On the other hand, when a third pad made of polyamide, polycarbonate, polyethersulfone, or glass fiber was used, the nonspecific reaction could not be suppressed. In addition, even when the third pad itself was not used, coloring was observed on the test line, and the nonspecific reaction could not be suppressed (not shown in the table).
  • the immunochromatographic test strip including a third pad made of a specific material according to the present invention can detect a substance to be detected in a stool-derived sample without causing a non-specific reaction peculiar to stool. Therefore, the substance to be detected in stool can be accurately detected using immunochromatography.

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Abstract

La présente invention aborde le problème consistant à fournir : un procédé de détection utilisant l'immunochromatographie pour détecter une substance détectable dans un échantillon dérivé de selles par développement dans une membrane insoluble, le procédé consistant également à inhiber une réaction non spécifique unique aux fèces ; et une bande de test d'immunochromatographie destinée à être utilisée dans ledit procédé. L'invention concerne : un procédé pour inhiber une réaction non spécifique même dans des échantillons dérivés de fèces en utilisant une bande de test dans laquelle un troisième tampon poreux comprenant une polysulfone ou de l'acétate de cellulose est positionné en amont de la membrane insoluble ; et une bande de test d'immunochromatographie destinée à être utilisée dans ledit procédé.
PCT/JP2015/074138 2014-08-29 2015-08-27 Bande de test d'immunochromatographie WO2016031892A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018043687A3 (fr) * 2016-08-31 2018-04-26 積水化学工業株式会社 Particules fluorescentes pour agent de diagnostic et réactif de dosage immunologique l'employant
JP2018205273A (ja) * 2017-06-09 2018-12-27 東洋紡株式会社 イムノクロマト試験片およびキットおよび測定方法
CN109900898A (zh) * 2019-03-18 2019-06-18 上海艾瑞德生物科技有限公司 一种免疫侧流层析试剂条及其制备方法和应用
WO2022265065A1 (fr) 2021-06-16 2022-12-22 積水メディカル株式会社 Procédé de dosage immunologique du sars-cov-2 et kit de dosage immunologique, et anticorps monoclonal ou fragment d'anticorps associé
WO2022265066A1 (fr) 2021-06-16 2022-12-22 積水メディカル株式会社 Procédé de dosage immunologique du sars-cov-2 et kit de dosage immunologique

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JP2002148266A (ja) * 2000-11-10 2002-05-22 Rohto Pharmaceut Co Ltd 検出装置及びその製造方法
JP2003344406A (ja) * 2002-05-24 2003-12-03 Towns:Kk イムノクロマトグラフィー用展開溶媒、測定法およびキット
JP2007259995A (ja) * 2006-03-28 2007-10-11 Kawasumi Lab Inc 血漿採集キット、血漿採集キット付き体外循環回路及び血液から血漿成分を分離する方法
JP2007531882A (ja) * 2004-03-30 2007-11-08 ワットマン インコーポレイテッド 側方流動方式、材料、及び方法
WO2010001598A1 (fr) * 2008-06-30 2010-01-07 積水メディカル株式会社 Phase solide poreuse pour essai de liaison, et procédé d'essai de liaison l'utilisant

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002148266A (ja) * 2000-11-10 2002-05-22 Rohto Pharmaceut Co Ltd 検出装置及びその製造方法
JP2003344406A (ja) * 2002-05-24 2003-12-03 Towns:Kk イムノクロマトグラフィー用展開溶媒、測定法およびキット
JP2007531882A (ja) * 2004-03-30 2007-11-08 ワットマン インコーポレイテッド 側方流動方式、材料、及び方法
JP2007259995A (ja) * 2006-03-28 2007-10-11 Kawasumi Lab Inc 血漿採集キット、血漿採集キット付き体外循環回路及び血液から血漿成分を分離する方法
WO2010001598A1 (fr) * 2008-06-30 2010-01-07 積水メディカル株式会社 Phase solide poreuse pour essai de liaison, et procédé d'essai de liaison l'utilisant

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018043687A3 (fr) * 2016-08-31 2018-04-26 積水化学工業株式会社 Particules fluorescentes pour agent de diagnostic et réactif de dosage immunologique l'employant
JPWO2018043687A1 (ja) * 2016-08-31 2019-08-15 積水化学工業株式会社 診断薬用蛍光粒子及びそれを用いた免疫測定試薬
JP2018205273A (ja) * 2017-06-09 2018-12-27 東洋紡株式会社 イムノクロマト試験片およびキットおよび測定方法
CN109900898A (zh) * 2019-03-18 2019-06-18 上海艾瑞德生物科技有限公司 一种免疫侧流层析试剂条及其制备方法和应用
WO2022265065A1 (fr) 2021-06-16 2022-12-22 積水メディカル株式会社 Procédé de dosage immunologique du sars-cov-2 et kit de dosage immunologique, et anticorps monoclonal ou fragment d'anticorps associé
WO2022265066A1 (fr) 2021-06-16 2022-12-22 積水メディカル株式会社 Procédé de dosage immunologique du sars-cov-2 et kit de dosage immunologique

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