WO2003060519A1 - Procede permettant de tester une reaction d'agglutination - Google Patents

Procede permettant de tester une reaction d'agglutination Download PDF

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Publication number
WO2003060519A1
WO2003060519A1 PCT/JP2001/011573 JP0111573W WO03060519A1 WO 2003060519 A1 WO2003060519 A1 WO 2003060519A1 JP 0111573 W JP0111573 W JP 0111573W WO 03060519 A1 WO03060519 A1 WO 03060519A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
virus
reaction
substance
measured
Prior art date
Application number
PCT/JP2001/011573
Other languages
English (en)
Japanese (ja)
Inventor
Michihiro Nakamura
Original Assignee
Techno Network Shikoku Co., Ltd.
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 Techno Network Shikoku Co., Ltd. filed Critical Techno Network Shikoku Co., Ltd.
Priority to AU2002216423A priority Critical patent/AU2002216423A1/en
Priority to PCT/JP2001/011573 priority patent/WO2003060519A1/fr
Priority to JP2003560562A priority patent/JPWO2003060519A1/ja
Publication of WO2003060519A1 publication Critical patent/WO2003060519A1/fr

Links

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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to a method for measuring an agglutination reaction such as an immunoagglutination reaction between an antibody and a virus.
  • immunoassays include immunoturbidimetry (TIA), latex nephelometry (LIA), enzyme immunoassay (EIA), and radioimmunoassay (RIA). It is used properly.
  • TIA immunoturbidimetry
  • LIA latex nephelometry
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • the present invention has been made in view of the above-mentioned problems of the related art, and does not require an expensive automatic analyzer or an electron microscope, and can accurately measure an immunoagglutination reaction and the like only by observation with a fluorescence microscope.
  • One of the objectives is to provide a method for measuring immunoagglutination that is widely applied to clinical tests and the like.
  • the present invention is a method for measuring an agglutination reaction, which comprises applying a fluorescent label to a substance to be measured, which can undergo an agglutination reaction, and observing the agglutination thereof in a solution.
  • a virus (or antibody) corresponding to an antibody (or virus) to be measured is fluorescently labeled, and an immunoaggregation reaction is measured in a solution by observing their aggregation.
  • the agglutination reaction is primary or secondary or higher order, and is an immune reaction, a polyvalent binding molecule reaction, or a combination thereof.
  • the substance to be measured is a molecule having multivalent binding ability.
  • the substance to be measured includes a substance capable of inducing or inducing an agglutination reaction or a substance causing an agglutination reaction.
  • the present invention also provides a reagent for detecting an agglutinating substance obtained by applying a fluorescent label to an agglutinable substance that is a measurement target substance. Also includes reagents for detecting antibodies or viruses, which are obtained by applying a fluorescent label to a virus (or antibody) corresponding to the antibody (or virus) to be measured.
  • agglutination such as immunoagglutination can be accurately measured.
  • the present invention will be described in detail. It is known that when an anti-virus antibody corresponding to a virus is reacted, an aggregate of the virus is formed, but observation thereof requires an electron microscope.
  • the antibody (or virus) to be measured is mixed with a lysate containing a fluorescently labeled virus (or antibody) to form an aggregate of the virus into which the fluorescent label has been incorporated.
  • the immunoagglutination reaction can be measured by a simple operation of observing it with a fluorescence microscope, it is very useful as a simple test or a preliminary test.
  • the present invention is characterized in that microorganisms, which are normally invisible at the level of an optical microscope, can be visually observed as very clear aggregates by agglutinating with a (non-visible) fluorescently labeled antibody.
  • agglutinating with a (non-visible) fluorescently labeled antibody has features.
  • Fluorescent dyes are indistinguishable if they are homogeneously distributed, but by giving a polarity to the distribution, a gradient of fluorescence intensity can be created, which can be visible at the optical level.
  • a virus (or an antibody) corresponding to an antibody (or a virus) to be measured is labeled with a fluorescent label, and a detection reagent for the antibody or the virus, ie, a conventional method, is used. It can be applied as a quick and simple test kit.
  • the fluorescent label may be either a virus or an antibody, and may be selected as necessary.
  • the virus (or antibody) labeled with a fluorescent dye is used after being diluted with an appropriate buffer (phosphate buffer, Tris buffer) or the like.
  • the substance to be measured by the immunoagglutination reaction according to the present invention includes a virus or an antibody in a biological sample, for example, a virus or an antibody derived from hepatitis (such as B or C), an HIV, A virus or antibody, a virus or antibody derived from influenza, a bacilli phage which is a virus that infects Escherichia coli, and the like can be exemplified, but not particularly limited thereto. That is, as described above, the present invention is not limited to naturally occurring viruses and antibodies, but may also be an artificial substance such as porcine or recombinant antibody obtained by genetic recombination as long as it induces an agglutination reaction. Can be targeted.
  • a virus or an antibody in a biological sample for example, a virus or an antibody derived from hepatitis (such as B or C), an HIV, A virus or antibody, a virus or antibody derived from influenza, a bacilli phage which is a virus that in
  • an agglutination reaction of a substance having a multivalent binding ability in one molecule or one complex, for example, avidin can be similarly detected.
  • the reaction is not limited to a one-step agglutination reaction caused by one antibody. If one antibody (primary antibody) is insufficiently aggregated, an antibody that binds to the primary antibody. ) Can also be targeted for aggregation by multi-step reactions. That is, the present invention can be applied to primary or secondary or higher order immunoagglutination.
  • the fluorescent label used in the present invention and the method of applying the fluorescent label are not particularly limited, and conventional techniques can be applied, and a necessary amount of a fluorescent dye may be applied to the target substance.
  • a fluorescent dye may be applied to the target substance.
  • fluorinated thiosocyanate FITC
  • fluorescent dyes such as protein fluorescent dyes can be used.
  • it is a fluorescent protein such as green fluorescent protein. Aggregates can be observed by attaching an antigen to the surface of a large fluorescent substance such as a fluorescent dye-containing silica sphere and allowing the antibody to react.
  • FIG. 1 is a micrograph showing the observation of the agglutination reaction in Examples of the present invention.
  • Bacteriophage a virus that infects Escherichia coli, was observed by reacting it with a fluorescently labeled anti-M13 antibody.
  • Fluorescence protein labeling kit (Roche) (including 5 (6) -carboxyfluorescein-N-hydroxysuccinimide ester, Sephadex G-25 column, etc.).
  • Anti-M13 antibody (lmg / ml) 200 1 was added with 300/2 of phosphate buffered saline, adjusted to 500 zl, and dissolved in dimethylsulfoxide. 1 1 was added and reacted at room temperature for 2 hours with gentle stirring. After the reaction, unreacted 5 (6)-with the antibody labeled using Sephadex G-25 column The carboxyf luorescein-N-ydroxysuccinimide ester was separated.
  • Phage solution ( 1 to ⁇ 7 ; ⁇ ; colony forming units / 1) Add 1 1 of labeled antibody (1.5 mg / ml) to 1 n 1 and stir. Immediately after that, place 21 reaction solution on a slide glass. The sample was covered with a cover glass and observed with a fluorescence microscope. Fig. 1 is a photograph of the observation. Aggregates that emit distinct fluorescent immediately after the reaction could be detected at 10 4 to 10 7/1? Phage reaction solution.

Abstract

L'invention concerne un procédé permettant de tester une réaction d'agglutination immune généralement applicable, en particulier, pour des examens cliniques, etc., procédé selon lequel une réaction d'agglutination peut être testée avec précision uniquement par observation au microscope fluorescent, sans recourir à des moyens onéreux tels qu'un auto-analyseur ou un microscope électronique. Le procédé permettant de tester une réaction d'agglutination est caractérisé en ce qu'il consiste à effectuer le marquage fluorescent d'un virus (ou d'un anticorps) contre un anticorps (ou un virus) à tester, et à observer l'agglutination de celui-ci dans une solution.
PCT/JP2001/011573 2001-12-27 2001-12-27 Procede permettant de tester une reaction d'agglutination WO2003060519A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2002216423A AU2002216423A1 (en) 2001-12-27 2001-12-27 Method of assaying agglutination reaction
PCT/JP2001/011573 WO2003060519A1 (fr) 2001-12-27 2001-12-27 Procede permettant de tester une reaction d'agglutination
JP2003560562A JPWO2003060519A1 (ja) 2001-12-27 2001-12-27 凝集反応の測定方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2001/011573 WO2003060519A1 (fr) 2001-12-27 2001-12-27 Procede permettant de tester une reaction d'agglutination

Publications (1)

Publication Number Publication Date
WO2003060519A1 true WO2003060519A1 (fr) 2003-07-24

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Application Number Title Priority Date Filing Date
PCT/JP2001/011573 WO2003060519A1 (fr) 2001-12-27 2001-12-27 Procede permettant de tester une reaction d'agglutination

Country Status (3)

Country Link
JP (1) JPWO2003060519A1 (fr)
AU (1) AU2002216423A1 (fr)
WO (1) WO2003060519A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010261791A (ja) * 2009-05-01 2010-11-18 Fujikura Kasei Co Ltd 標的物質の検出方法
CN103760346A (zh) * 2014-01-25 2014-04-30 云南省农业科学院生物技术与种质资源研究所 一种定量检测植物病毒的荧光免疫斑点法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6435373A (en) * 1987-07-31 1989-02-06 Fujirebio Kk Method and device for high-sensitivity immunoassay
JPH10282098A (ja) * 1997-04-11 1998-10-23 Matsushita Electric Ind Co Ltd 蛍光免疫測定法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6435373A (en) * 1987-07-31 1989-02-06 Fujirebio Kk Method and device for high-sensitivity immunoassay
JPH10282098A (ja) * 1997-04-11 1998-10-23 Matsushita Electric Ind Co Ltd 蛍光免疫測定法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010261791A (ja) * 2009-05-01 2010-11-18 Fujikura Kasei Co Ltd 標的物質の検出方法
CN103760346A (zh) * 2014-01-25 2014-04-30 云南省农业科学院生物技术与种质资源研究所 一种定量检测植物病毒的荧光免疫斑点法

Also Published As

Publication number Publication date
AU2002216423A1 (en) 2003-07-30
JPWO2003060519A1 (ja) 2005-05-19

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