WO2022259990A1 - 糞便検体の検査方法及びそのためのイムノクロマト試験片 - Google Patents

糞便検体の検査方法及びそのためのイムノクロマト試験片 Download PDF

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Publication number
WO2022259990A1
WO2022259990A1 PCT/JP2022/022712 JP2022022712W WO2022259990A1 WO 2022259990 A1 WO2022259990 A1 WO 2022259990A1 JP 2022022712 W JP2022022712 W JP 2022022712W WO 2022259990 A1 WO2022259990 A1 WO 2022259990A1
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Prior art keywords
antibody
specimen
immunochromatographic
sample
norovirus
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PCT/JP2022/022712
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English (en)
French (fr)
Japanese (ja)
Inventor
巧士 平岡
祐司 齋藤
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Denka Co Ltd
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Denka Co Ltd
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Priority to US18/566,740 priority Critical patent/US20240272158A1/en
Priority to EP22820171.1A priority patent/EP4350353A4/en
Priority to JP2023527842A priority patent/JPWO2022259990A1/ja
Priority to KR1020237038259A priority patent/KR20240019074A/ko
Publication of WO2022259990A1 publication Critical patent/WO2022259990A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present invention relates to a fecal specimen testing method and an immunochromatographic test strip therefor.
  • immunoassays using antigen-antibody reactions are generally known as simple test methods.
  • a complex of a capture substance (capture substance) that specifically binds to the analyte and a label that specifically binds to the analyte is formed on the membrane, and the label is detected/quantified. , to detect (measure or quantify) the object to be detected.
  • Immunochromatography is widely used for the detection of a wide variety of substances to be detected because the measurement apparatus is simple and the method is excellent in terms of cost.
  • a detection unit in which an antibody that specifically binds to the substance to be detected is immobilized as a capture substance on a membrane strip such as nitrocellulose, and a label that specifically binds to the substance to be detected.
  • a test sample containing the analyte is added dropwise to a test device having a labeled body portion containing to detect or quantify the label (for example, Patent Documents 1 and 2).
  • JP 2008-268043 A Japanese Patent Application Laid-Open No. 2008-203135
  • An object of the present invention is to suppress the influence of interfering substances in immunochromatography contained in stool samples, and to enable accurate and specific measurement of substances to be detected in test samples. It is to provide a method and an immunochromatography test piece therefor.
  • the inventors of the present application have found that by performing immunochromatography in the presence of taurodeoxycholic acid, the effects of interfering substances can be suppressed, and the substances to be detected in stool specimens can be tested.
  • the inventors have found that it is possible to measure accurately and specifically regardless of the amount to be measured, and completed the present invention.
  • the present invention provides the following.
  • a method for testing a stool sample including testing a stool sample by immunochromatography in the presence of taurodeoxycholic acid or a salt thereof.
  • the influence of interfering substances in a stool sample is suppressed, and the substance to be detected in the stool sample is accurately and specifically measured regardless of the amount of the stool sample subjected to the test. It is possible.
  • FIG. 1 shows a preferred form of a typical immunochromatographic test strip.
  • the present invention relates to an inspection method that prevents false negatives occurring in fecal specimens and enables accurate measurement of a substance to be detected, and an immunochromatographic test strip therefor.
  • false negative means that no positive signal is generated in the assay even though the sample contains the substance to be detected.
  • the immunochromatographic method immunochromatographic method
  • signal generation is inhibited due to reasons such as the above complexes not being formed.
  • the immunochromatographic test strip consists of a support having a detection region on which an antibody (antibody 1) that captures an object to be measured (antigen, etc.) is immobilized, a labeled region having a movable labeled antibody (antibody 2), and a sample drop. It comprises a sample pad to be applied, an absorbent band for absorbing the developed specimen liquid, and a backing sheet for bonding these members together.
  • the number of detection regions and the type of labeled antibody contained in the labeled region are not limited to one. It can be measured in strips.
  • FIG. 1 is a diagram showing a preferred form of a typical immunochromatographic test strip. Note that the immunochromatographic test piece is not limited to that shown in FIG. In FIG. 1, a indicates a support, b indicates a label region, c indicates a detection region, d indicates a sample pad, e indicates an absorption band, and f indicates a backing sheet.
  • FIG. 1 The upper part of FIG. 1 is a top view, and the lower part is a cross-sectional view.
  • a support having two detection regions formed on a backing sheet, an absorption band, a marker region, a sample pad, and the like are laminated. Then, as shown, one end of the absorption band and one end of the support, the other end of the support and one end of the label area, the other end of the label area and the sample pad. are overlapped to form a continuous lateral flow channel.
  • the support is a material that has the ability to immobilize the antibody that captures the test substance (antigen), and has the ability to prevent the horizontal passage of liquids.
  • it is a porous thin film with capillary action and a material capable of transporting liquids 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. Among them, a thin film made of nitrocellulose is more preferable.
  • the labeled body region consists of a porous substrate containing a labeled antibody, and the material of the substrate can be commonly used glass fiber, non-woven fabric, or the like.
  • the substrate is preferably in the form of a pad with a thickness of about 0.3 mm to 0.6 mm in order to be impregnated with a large amount of labeled antibody.
  • the detection area refers to a partial area of the support on which the antibody that captures the test substance (antigen) is immobilized.
  • the detection region provides at least one antibody-immobilized region for capturing an antigen.
  • the sample pad is a part where the specimen or a sample prepared using the specimen is added dropwise, and is a porous material with water absorption.
  • the material commonly used cellulose, glass fiber, non-woven fabric, and the like can be used.
  • it is preferably in the form of a pad with a thickness of about 0.3 mm to 1 mm. Note that the sample pad and the marker region described above are purely functional distinctions, and do not necessarily need to be separate materials. That is, it is also possible for a partial area of the material placed as the sample pad to have the function of the marker area.
  • the absorption band is a member for absorbing components supplied to the support and not involved in the reaction in the detection area.
  • As the material filter papers, sponges, and the like having high water retention properties made of general natural polymer compounds, synthetic polymer compounds, etc. can be used. .
  • the backing sheet is a member for attaching and fixing all the above-mentioned materials, ie, the support, sample pad, marker area, absorption band, etc., with partial overlap.
  • a backing sheet is not absolutely necessary as long as these materials are arranged and fixed at optimum intervals, but it is generally preferable to use it from the standpoint of convenience in terms of manufacturing or use.
  • the specimen passes through a porous channel formed by a series of connections of the sample pad, label region, support, detection region, absorption band, and others. Therefore, in the present embodiment, all of these are sample moving regions.
  • a porous channel formed by a series of connections of the sample pad, label region, support, detection region, absorption band, and others. Therefore, in the present embodiment, all of these are sample moving regions.
  • there may be a form in which the specimen passes through the interface without penetrating the interior of the material. is also included in the scope of the present specification.
  • the present invention includes taurodeoxycholic acid or a salt thereof on a specimen suspension or specimen extract, or on an immunochromatographic test strip.
  • Preferred salts of taurodeoxycholic acid are sodium and potassium salts.
  • taurodeoxycholic acid or a salt thereof may be simply referred to as “taurodeoxycholic acid” or “TDOC” unless otherwise clearly indicated by the context.
  • the concentration of taurodeoxycholic acid in the sample suspension buffer or extraction buffer is usually 0.005% to 10% by mass, preferably 0.05% to 5% by mass.
  • a solution of taurodeoxycholic acid preferably an aqueous solution using water or an aqueous buffer as a solvent, is preferably added by impregnation or dropwise addition.
  • the amount of taurodeoxycholic acid added to the immunochromatographic test piece is usually about 0.01 mg to 10 mg, preferably about 0.05 mg to 5 mg.
  • the fecal specimen can be diluted with a buffer solution or used as is without dilution.
  • the substance to be detected is not limited at all, and may be any substance to be detected. Specific examples include virus antigens such as influenza virus, adenovirus, respiratory syncytial virus, HAV, HBs, HIV, and norovirus; bacterial antigens such as MRSA, group A streptococcus, group B streptococcus, and Legionella spp.; toxins produced by bacteria; Antigens such as mycoplasma, Chlamydia trachomatis, hormones such as human chorionic gonadotropin, C-reactive protein, myoglobin, cardiac troponin, various tumor markers, pesticides, and endocrine disruptors. Antibodies can be mentioned.
  • the present invention also provides an immunochromatographic test strip formed as described above.
  • the configuration of the test device itself may be a known one, except that the above surfactant is added to the specimen suspension/extraction buffer.
  • a schematic diagram of a specific example of the immunochromatographic test strip of the present invention is shown in FIG. 1, but the test strip of the present invention is not limited thereto.
  • a specimen sample is prepared by suspending or extracting a fecal specimen in a buffer for specimen suspension or extraction.
  • a nitrocellulose membrane (a) laminated on a plastic plate (f) includes a stabilized dry label pad formed by the method described above, in which an antibody that reacts with an antigen-antibody reaction with a substance to be detected is labeled with colored latex particles.
  • the test device is equipped with a label portion (b) and a detection portion (c) in which an antibody that reacts with an antigen-antibody reaction with the substance to be detected is immobilized in a linear form as a capture substance.
  • the detection part (c) is an antibody or an antigen-binding antibody thereof capable of antigen-antibody reaction with the substance to be detected and simultaneously binding to the substance to be detected or the antibody or antigen-binding fragment thereof on the colored latex particles. This is a region in which the sex fragments are solid-phased in a line.
  • the absorbent pad (e) there are two detectors (c), which are intended to capture two types of detection targets, such as influenza A virus and influenza B virus. is. By providing a plurality of such detectors (c), it is possible to simultaneously perform immunoassays for a plurality of types of analytes.
  • the immunochromatographic test strip of the present invention which uses, as a stabilized dry label, latex particles bound with an antibody that reacts with an antigen-antibody reaction with a substance to be detected or an antigen-binding fragment thereof, the substance to be detected can be measured quickly and easily. .
  • Example 1 Addition to TDOC specimen suspension 1 . Immobilization of anti-norovirus antibody to nitrocellulose membrane
  • Anti-norovirus antibody diluted with purified water to 1.0 mg/mL and anti-mouse IgG antibody were prepared, and a nitrocellulose membrane sample pad lined with PET film was prepared.
  • An anti-norovirus antibody was applied linearly on one side, and an anti-mouse IgG antibody was linearly applied on the absorber side. Thereafter, the nitrocellulose membrane was dried at 45°C for 30 minutes to obtain an anti-norovirus antibody-immobilized membrane.
  • This membrane is called an "antibody-immobilized membrane" in this example.
  • Immobilization of anti-norovirus antibody to colored polystyrene particles Dilute anti-norovirus antibody with purified water to 0.5 mg/mL, add colored polystyrene particles to 0.1%, stir, and add 1% carbodiimide. and stir further. The supernatant was removed by centrifugation and resuspended in 50 mM Tris (pH 9.0) and 3.0% BSA to obtain anti-norovirus antibody-bound colored polystyrene particles. These particles are referred to as "antibody-immobilized particles" in this example.
  • test piece The antibody-immobilized membrane prepared in 1 and the dry pad prepared in 3 were adhered to other members (sample pad, backing sheet, absorbent band) and cut to a width of 5 mm to obtain a norovirus test piece.
  • the test piece has, from upstream along the sample flow, a sample pad, a dry pad (label region), an antibody-immobilized membrane (detection region), and an absorption band.
  • Specimen suspension TDOC sodium taurodeoxycholate in the examples and comparative examples
  • Examples 2 and 3 Comparative Example 2 Tests using fecal specimens 1. Immobilization of anti-norovirus antibody to nitrocellulose membrane Anti-norovirus antibody diluted with purified water to 1.0 mg/mL and anti-mouse IgG antibody were prepared, and a nitrocellulose membrane sample pad lined with PET film was prepared. An anti-norovirus antibody was applied linearly on one side, and an anti-mouse IgG antibody was linearly applied on the absorber side. Thereafter, the nitrocellulose membrane was dried at 45°C for 30 minutes to obtain an anti-norovirus antibody-immobilized membrane. This membrane is called an "antibody-immobilized membrane" in this example.
  • Immobilization of anti-norovirus antibody to colored polystyrene particles Dilute anti-norovirus antibody with purified water to 0.5 mg/mL, add colored polystyrene particles to 0.1%, stir, and add 1% carbodiimide. and stir further. The supernatant was removed by centrifugation and resuspended in 50 mM Tris (pH 9.0) and 3.0% BSA to obtain anti-norovirus antibody-bound colored polystyrene particles. These particles are referred to as "antibody-immobilized particles" in this example.
  • norovirus test piece The antibody-immobilized membrane prepared in 1, the dry pad prepared in 3, and the impregnated nonwoven fabric prepared in 4 were laminated with other members (backing sheet, absorbent band), cut into 5 mm width, and cut into a norovirus test piece. and A test piece using the impregnated nonwoven fabric as a sample pad is referred to as an "improved test piece" in this example.
  • the test piece has, from upstream along the sample flow, a sample pad, a dry pad (label region), an antibody-immobilized membrane (detection region), and an absorption band.
  • TDOC suppresses the effects of interfering substances in stool specimens, and accurately and specifically measures detectable substances in stool specimens regardless of the amount of the stool specimen used in the test. was possible.

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PCT/JP2022/022712 2021-06-07 2022-06-06 糞便検体の検査方法及びそのためのイムノクロマト試験片 Ceased WO2022259990A1 (ja)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US18/566,740 US20240272158A1 (en) 2021-06-07 2022-06-06 Fecal sample test method and immunochromatographic test piece therefor
EP22820171.1A EP4350353A4 (en) 2021-06-07 2022-06-06 FECAL SAMPLE TEST METHOD AND ASSOCIATED IMMUNOCHROMATOGRAPHIC TEST PIECE
JP2023527842A JPWO2022259990A1 (https=) 2021-06-07 2022-06-06
KR1020237038259A KR20240019074A (ko) 2021-06-07 2022-06-06 분변 검체의 검사 방법 및 그것을 위한 이뮤노크로마토그래피 시험편

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JP2021-095332 2021-06-07
JP2021095332 2021-06-07

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WO2022259990A1 true WO2022259990A1 (ja) 2022-12-15

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US (1) US20240272158A1 (https=)
EP (1) EP4350353A4 (https=)
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KR (1) KR20240019074A (https=)
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JP2008268043A (ja) 2007-04-23 2008-11-06 Denka Seiken Co Ltd 検査デバイスの検出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
JP2012050391A (ja) * 2010-09-02 2012-03-15 Life:Kk 便検体から細菌類を検出する方法
JP2012211849A (ja) * 2011-03-31 2012-11-01 Fujifilm Corp 高感度なイムノクロマトグラフ方法
WO2017163341A1 (ja) * 2016-03-23 2017-09-28 デンカ生研株式会社 イムノクロマト法検査デバイスの試料添加部の形成方法及びイムノクロマト法検査デバイス
WO2018066588A1 (ja) * 2016-10-05 2018-04-12 デンカ生研株式会社 赤血球の凝集方法及び分離方法並びに赤血球凝集用試薬
WO2019138899A1 (ja) * 2018-01-11 2019-07-18 東洋紡株式会社 測定試料希釈液およびキットおよび測定方法

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WO2018012517A1 (ja) * 2016-07-13 2018-01-18 積水メディカル株式会社 イムノクロマトグラフィーを利用した検出方法

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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
JP2008268043A (ja) 2007-04-23 2008-11-06 Denka Seiken Co Ltd 検査デバイスの検出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
JP2012050391A (ja) * 2010-09-02 2012-03-15 Life:Kk 便検体から細菌類を検出する方法
JP2012211849A (ja) * 2011-03-31 2012-11-01 Fujifilm Corp 高感度なイムノクロマトグラフ方法
WO2017163341A1 (ja) * 2016-03-23 2017-09-28 デンカ生研株式会社 イムノクロマト法検査デバイスの試料添加部の形成方法及びイムノクロマト法検査デバイス
WO2018066588A1 (ja) * 2016-10-05 2018-04-12 デンカ生研株式会社 赤血球の凝集方法及び分離方法並びに赤血球凝集用試薬
WO2019138899A1 (ja) * 2018-01-11 2019-07-18 東洋紡株式会社 測定試料希釈液およびキットおよび測定方法

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Title
See also references of EP4350353A4

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