WO2003060519A1 - Method of assaying agglutination reaction - Google Patents

Method of assaying agglutination reaction Download PDF

Info

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
French (fr)
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/en
Priority to JP2003560562A priority patent/JPWO2003060519A1/en
Publication of WO2003060519A1 publication Critical patent/WO2003060519A1/en

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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

A method of assaying an immune agglutination reaction widely applicable to, in particular, clinical examinations, etc. whereby an agglutination reaction can be accurately assayed simply by observing under a fluorescent microscope without resort to any expensive autoanalyzer or electron microscope. Namely, a method of assaying an agglutination reaction characterized by comprising fluorescent-labeling a virus (or an antibody) against an antibody (or a virus) to be assayed and observing the agglutination thereof in a solution.

Description

明細 凝集反応の測定方法 発明の属する技術分野：  Description Method for measuring agglutination reaction Technical field to which the invention pertains:
本発明は、 抗体とウィルスとの免疫凝集反応などの凝集反応を測定する方法に 関する。  The present invention relates to a method for measuring an agglutination reaction such as an immunoagglutination reaction between an antibody and a virus.
従来の技術： Conventional technology:
臨床検査の分野では、 生体試料 （血液、 尿など） を用いて種々の疾患の診断を 行っているが、 これらを診断する方法として、 種々の測定法が開発され利用され ている。 これらの測定法の代表的方法として、 酵素反応を利用する生化学測定法 や抗原抗体反応を利用する免疫測定法が挙げられる。 近年においては、 生体試料 中の成分を精度よく測定することが望まれ、 特異性の高い抗原抗体反応を利用し · た免疫測定法が盛んに用いられている。  In the field of clinical testing, various diseases are diagnosed using biological samples (blood, urine, etc.), and various measuring methods have been developed and used as methods for diagnosing them. Representative methods of these assays include a biochemical assay using an enzyme reaction and an immunoassay using an antigen-antibody reaction. In recent years, it has been desired to accurately measure components in a biological sample, and immunoassays utilizing highly specific antigen-antibody reactions have been actively used.
免疫測定法としては、免疫比濁法（T I A法）、 ラテックス比濁法（L I A法）、 酵素免疫測定法 （E I A法） 、 放射免疫測定法 （R I A法） などが挙げられ、 目 的に応じて使い分けされている。  Examples of immunoassays include immunoturbidimetry (TIA), latex nephelometry (LIA), enzyme immunoassay (EIA), and radioimmunoassay (RIA). It is used properly.
近年、 生体試料中の微量成分の測定においては、 癌などの早期発見やエイズゥ ィルスなどの感染初期を診断するため、 超微量でも測定できる方法が要望されて いる。超微量測定が可能な手法としては、特に E I A法が重用されている。 また、 その測定法自体の精度を上げるため、 あるいは測定の簡素化を図るための各種応 用法が研究されており、 例えば、 E I A法の抗原または抗体を標識する物質とし て酵素の代わりに発光物質を利用する方法 （特開平 5— 3 4 3 4 6号公報） や強 磁性粒子に抗体を固定化し蛍光標識を行い、 凝集を生じさせたのち、 蛍光濃度測 定を行う方法 （特開平 5— 9 9 9 2 6号公報） 、 測定対象物質をキャリアータン パク質に複合ィ匕させた後、 該キャリア一タンパク質を不溶性担体に担持させるこ とにより、 BZF分離と呼ばれる操作 （Bは免疫反応等により結合した成分、 F は未反応の成分） 'を不要とした方法 （特開 2 0 0 0— 3 3 8 1 0 8号公報） が提 案されている。 In recent years, in the measurement of trace components in a biological sample, there has been a demand for a method capable of measuring even a very small amount in order to early detect cancer or the like and diagnose early stages of infection such as AIDS virus. The EIA method is especially heavily used as a method capable of ultra-trace measurement. In addition, various application methods have been studied to improve the accuracy of the measurement method itself or to simplify the measurement.For example, a luminescent substance instead of an enzyme is used as a substance for labeling an antigen or antibody in the EIA method. (Japanese Unexamined Patent Publication No. Hei 5-334436) or by immobilizing an antibody on ferromagnetic particles and performing fluorescent labeling to cause aggregation and then measuring the fluorescence concentration (Japanese Unexamined Patent Publication No. 5-99926) discloses a method of separating BZF by combining a substance to be measured with a carrier protein and then supporting the carrier-protein on an insoluble carrier. (B is a component bound by an immune reaction, F is an unreacted component) ”(Japanese Patent Application Laid-Open No. 2000-338018) has been proposed. .
しかしながら、 上記方法は、 それ以前の従来手法に比べれば測定時間等の点で 簡素化されているものの、 汎用自動分析装置や専用自動分析装置、 或いは電子顕 微鏡による観察を必要とし、 簡易検査もしくは予備的検査の手法としては十分満 足できるものではなかった。 発明の開示  However, although the above method is simplified in terms of measurement time, etc., as compared with the previous conventional method, it requires observation using a general-purpose automatic analyzer, a dedicated automatic analyzer, or an electron microscope, and thus requires a simple inspection. Or, it was not enough as a preliminary inspection method. Disclosure of the invention
本発明は、 上記従来技術の課題に鑑みなされたものであり、 高価な自動分析装 置や電子顕微鏡を必要とせず、 蛍光顕微鏡による観察のみで、 免疫凝集反応など を正確に測定することができ、 臨床検査等への応用の広い免疫凝集反応の測定方 法の提供を目的のひとつとする。  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.
即ち、 本発明は、 測定対象物質である凝集反応しうる物質に蛍光標識を施し、 溶液中において、 それらの凝集を観察することを特徴とする凝集反応の測定方法 である。  That is, 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.
例えば、 測定対象物質である抗体 （またはウィルス） に対応したウィルス （ま たは抗体） に蛍光標識を施し、 溶液中において、 それらの凝集を観察する免疫凝 集反応を測定する方法である。  For example, there is a method in which 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.
凝集反応が一次または二次以上の多次であり、 免疫反応、 多価結合分子反応、 またはその組み合わせであることが好ましい。  Preferably, the agglutination reaction is primary or secondary or higher order, and is an immune reaction, a polyvalent binding molecule reaction, or a combination thereof.
測定対象物質が多価結合能を有する分子である場合も含む。 測定対象物質は凝集反応を惹起または誘発しうる物質または凝集反応を起こす 物資を含む。 This includes the case where 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.
また、 本発明は、 測定対象物質である凝集反応しうる物質に蛍光標識を施して なる凝集反応する物質の検出試薬を提供する。 測定対象物質である抗体 （または ウィルス） に対応したウィルス （または抗体） に蛍光標識を施レてなる、 抗体ま たはゥィルスを検出する試薬も含む。  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.
これらの凝集を蛍光顕微鏡により観察することで免疫凝集反応などの凝集反応 を正確に測定できる。  By observing these agglutinations with a fluorescence microscope, agglutination such as immunoagglutination can be accurately measured.
発明の詳細な説明： DETAILED DESCRIPTION OF THE INVENTION:
以下に本発明を詳しく説明する。 ウィルスに対応する抗ウィルス抗体を反応さ せるとウィルスの凝集体が形成されることは公知であるが、 その観察には電子顕 微鏡が必要になる。本発明では、 測定対象物質である抗体 （またはウィルス） と、 蛍光標識を施したウィルス （または抗体） を含む溶夜とを混ぜ合わせ、 該蛍光標 識が取り込まれたウイルスの凝集体を形成させ、 それを蛍光顕微鏡により観察す るという簡単な操作により免疫凝集反応を測定可能としたため、 簡易検査もしく は予備的検査として非常に有用である。  Hereinafter, 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. In the present invention, 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. However, since 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.
即ち、 本発明は、 通常では光学顕微鏡レベルでは目視しえないウィルスという 微生物を（目視しえない）蛍光標識した抗体で凝集させることにより非常に鮮明な 凝集体として目視しうるものとした点に特徴を有する。 即ち、 2つ以上の固相化 していない物質で、本来、溶液中で均一に分布するものが、 ある特性（ここでは抗 原抗体反応）を通じて極性を持つ分布（=凝集）に変化すること、かつ、蛍光色素は 均一分布である場合区別できませんが、 分布に極性をもたらすことによって蛍光 強度の勾配をもたらし、 光学レベルで目視しうるものなりうるということ （名付 けるなら 「非固相系蛍光体再分布可視化特性」） を利用した方法である。 本発明の免疫凝集反応の測定方法は、 測定対象物質である抗体 （またはウィル ス） に対応したウィルス （または抗体） に蛍光標識を施しておき、 抗体またはゥ ィルスの検出試薬、 即ち従来法にはない迅速かつ簡便な検査キットとして応用す ることができる。 ここで、 蛍光標識は、 ウィルスに対してでも抗体に対してでも、 どちらでもよく、 必要に応じ選択すればよい。 また、 蛍光標識を施したウィルス (または抗体） は、 適当な緩衝液 （リン酸緩衝液、 トリス緩衝液） などにより希 釈して用いられる。 That is, 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. Has features. In other words, two or more non-immobilized substances, which are originally distributed uniformly in a solution, change to a distribution (= aggregation) that has polarity through a certain property (here, an antigen-antibody reaction). 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. This is a method that utilizes the phosphor redistribution visualization characteristics ”). In the method for measuring an immunoagglutination reaction of the present invention, 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. Here, 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.
ここで、 本発明に係る免疫凝集反応より測定される測定対象物質としては、 生 体試料中のウィルスまたは抗体が挙げられ、 例えば、 肝炎 （B型もしくは C型な ど） 由来ウィルスまたは抗体、 H I Vウィルスまたは抗体、 インフルエンザ由来 ウィルスまたは抗体、 大腸菌その他に感染するウィルスであるバクリオファージ 等を例示することができるが、 特にこれらに限定されるものではない。 即ち、 上 記の如き、 天然由来のウィルスや抗体に限らず、 アブタマ一、 遺伝子組み換えに よるリコンビナント抗体等、 人工的な物質であっても、 凝集反応を惹起するもの であれば、 本発明の対象となり得る。  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.
上記免疫反応の他に、 一分子あるいは一複合体で多価結合能を持つ物質、 例え ばアビジンなどの凝集反応を同様に検出できる。 '  In addition to the above immune reaction, an agglutination reaction of a substance having a multivalent binding ability in one molecule or one complex, for example, avidin can be similarly detected. '
また、本発明では、反応が一つの抗体により起こる一段階の凝集反応に限らず、 一つの抗体 （一次抗体） で凝集が不充分であれば、 1次抗体に結合する抗体 . (二 次抗体） を用いたものなど多段階の反応による凝集も対象となりうる。 即ち、 本 発明は一次または二次以上の多次である免疫凝集反応に適用できる。  In the present invention, 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.
また、 本発明で用いられる蛍光標識、 並びに蛍光標識の付与手法には特に制限 はなく、 従来技術が適用でき、 必要量の蛍光色素を対象物質に付与すればよい。 蛍光顕微鏡観察の場合、 その色素に応じたフィル夕一が必要となるため、 この点 から蛍光色素として最も一般的なフルォロセィンィソチオシァネ一ト（F I T C ) の使用が便利である。 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. In the case of fluorescence microscopy observation, it is necessary to set a filter according to the dye. Therefore, it is convenient to use fluorinated thiosocyanate (FITC), which is the most common fluorescent dye.
ほかに、 蛋白性の蛍光色素など、 他の蛍光色素も使用できる。 例えば、 green f luorescent proteinなどの蛍光蛋白である。 また、 蛍光色素含有シリカ球のよ うな大きな蛍光体の表面に抗原をつけ抗体を反応させ凝集体を観察できる。  In addition, other fluorescent dyes such as protein fluorescent dyes can be used. For example, 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.
図面の簡単な説明： BRIEF DESCRIPTION OF THE DRAWINGS:
図 1は本発明の実施例における凝集反応の観察を示す顕微鏡写真である。 実施例  FIG. 1 is a micrograph showing the observation of the agglutination reaction in Examples of the present invention. Example
実施例 1 Example 1
大腸菌に感染するウィルスであるバクテリオファージに対して、 蛍光標識を行 つた抗 M13抗体を反応させて観察を行つた。  Bacteriophage, a virus that infects Escherichia coli, was observed by reacting it with a fluorescently labeled anti-M13 antibody.
く材料〉 Material)
'線維状ファージ M13K07 helper phage (Gibco BRL社）  '' M13K07 helper phage (Gibco BRL)
-抗 M13抗体 （抗 M13K07 helper phageに結合するモノクローナル抗体） （アマシ ャムバイオテク社製） '  -Anti-M13 antibody (monoclonal antibody that binds to anti-M13K07 helper phage) (Amersham Biotech) ''
• 虽光標識 ( Fluorescence protein label ing ki t (Roche 社） (5 (6) - carboxyfluorescein-N-hydroxysuccinimide ester, Sephadex G - 25カラムなどを 含む)。  • Fluorescence protein labeling kit (Roche) (including 5 (6) -carboxyfluorescein-N-hydroxysuccinimide ester, Sephadex G-25 column, etc.).
く抗体の標識〉 Antibody label>
抗 M13抗体（lmg/ml) 200 1 にリン酸緩衝食塩水を 300 /2 1加え、 500 z l とし Dimethylsul foxideに溶解した 5 (6) -carboxyf luorescein-N- ydroxysuccinimide es ter (20mg/inl)を 1 1加え、 緩やかな攪拌を行いながら 2時間、 室温にて反応 させた。 反応後、 Sephadex G-25カラムを用いて標識した抗体と未反応の 5 (6) - carboxyf luorescein-N- ydroxysuccinimide esterを分離した。 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.
くファージの検出〉 Detection of phage>
ファージ溶液（ 1〜^7。^ ; colony forming uni ts/ 1) 1 n 1 に標識抗体 (1. 5mg/ml)を 1 1加え攪拌後、すぐに 2 1の反応液をスライドガラス上にのせ、 カバーガラスをかけて蛍光顕微鏡にて観察した。 図 1はその観察写真である。 反応直後から明瞭な蛍光を発する凝集体を 104〜107/ 1の? のファージ反応 溶液で検出することができた。 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.
一方、 対照実験として、 抗 M13抗体の代わりに蛍光標識した抗マウス抗体、 抗 ゥサギ抗体を使用したところ、 凝集体は検出できなかった。  On the other hand, when a fluorescently-labeled anti-mouse antibody or anti-Egret antibody was used instead of the anti-M13 antibody, no aggregates could be detected.

Claims

請求の範囲 The scope of the claims
1 . 測定対象物質である凝集反応しうる物質に蛍光標識を施し、 溶液中におい て、 それらの凝集を観察することを特徴とする凝集反応の測定方法。 1. A method for measuring an agglutination reaction, which comprises applying a fluorescent label to an agglutinable substance to be measured and observing the agglutination thereof in a solution.
2 . 測定対象物質である抗体 （またはウィルス） に対応したウィルス （または 抗体） に蛍光標識を施し、 溶液中において、 それらの凝集を観察する免疫凝集反 応を測定する請求項 1に記載した方法。  2. The method according to claim 1, wherein a virus (or antibody) corresponding to the antibody (or virus) to be measured is fluorescently labeled, and an immunoagglutination reaction is measured in a solution for observing their aggregation. .
3 . 凝集反応が一次または二次以上の多次であり、 免疫反応、 多価結合分子反 応またはその組み合わせである請求項 1に記載した方法。  3. The method according to claim 1, wherein the agglutination reaction is primary or secondary or higher order, and is an immune reaction, a multivalent binding molecule reaction or a combination thereof.
4 . 測定対象物質が多価結合能を有する分子である請求項 1に記載した方法。 4. The method according to claim 1, wherein the substance to be measured is a molecule having multivalent binding ability.
5 . 測定対象物質は凝集反応を惹起または誘発しうる物質または凝集反応を起 こす物資である請求項 1または 3に記載した方法。 5. The method according to claim 1, wherein the substance to be measured is a substance capable of inducing or inducing an agglutination reaction or a substance causing an agglutination reaction.
6 . 測定対象物質である凝集反応しうる物質に蛍光標識を施してなる凝集反応 する物質の検出試薬。  6. A reagent for detecting agglutinating substances obtained by applying a fluorescent label to an agglutinable substance that is a measurement target substance.
7 . 測定対象物質である抗体 （またはウィルス） に対応したウィルス （または 抗体） に蛍光標識を施してなる、 抗体またはウィルスを検出する請求項 4に記載 した試薬。  7. The reagent according to claim 4, wherein a virus (or antibody) corresponding to the antibody (or virus) to be measured is fluorescently labeled to detect the antibody or virus.
PCT/JP2001/011573 2001-12-27 2001-12-27 Method of assaying agglutination reaction WO2003060519A1 (en)

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 (en) 2001-12-27 2001-12-27 Method of assaying agglutination reaction
JP2003560562A JPWO2003060519A1 (en) 2001-12-27 2001-12-27 Method for measuring agglutination

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2001/011573 WO2003060519A1 (en) 2001-12-27 2001-12-27 Method of assaying agglutination reaction

Publications (1)

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

Family

ID=11738085

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2001/011573 WO2003060519A1 (en) 2001-12-27 2001-12-27 Method of assaying agglutination reaction

Country Status (3)

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

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010261791A (en) * 2009-05-01 2010-11-18 Fujikura Kasei Co Ltd Method for detecting target substance
CN103760346A (en) * 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 Dot fluorescence immunoassay method for quantitatively detecting plant virus

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 (en) * 1997-04-11 1998-10-23 Matsushita Electric Ind Co Ltd Fluoroimmonoassay method

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 (en) * 1997-04-11 1998-10-23 Matsushita Electric Ind Co Ltd Fluoroimmonoassay method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010261791A (en) * 2009-05-01 2010-11-18 Fujikura Kasei Co Ltd Method for detecting target substance
CN103760346A (en) * 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 Dot fluorescence immunoassay method for quantitatively detecting plant virus

Also Published As

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

Similar Documents

Publication Publication Date Title
US10732111B2 (en) Automated immunoanalyzer system for performing diagnostic assays for allergies and autoimmune diseases
JP6695280B2 (en) Improved assay method
Andreotti et al. Immunoassay of infectious agents
CN100420947C (en) Method for quantitative determination of specific analyte with single trapping agent and reagent kit therefor
FI76888B (en) NYA MEDEL OCH FOERPACKNINGAR FOER IMMUNOLOGISKA ANALYSER.
CN103443626B (en) Streptavidin-bonded magnetic particles and manufacturing method for same
US9274056B2 (en) Use of non-chelated fluorochromes in rapid test systems
KR20000071894A (en) Multipurpose diagnostic systems using protein chips
JP2007519933A (en) Systems, methods, and reagents for detection of biological and chemical materials using dynamic surface generation and imaging
JP2000019172A (en) Multi-wavelength fluorescence polarimetry
EP2639584A1 (en) Real time diagnostic assays using an evanescence biosensor
JPH07508102A (en) assay
CN112362867A (en) Detection method using aggregation-induced emission combined immunomagnetic beads and kit thereof
CN113156119A (en) Method for detecting coronavirus by adopting angiotensin converting enzyme II (ACE2)
US20030119030A1 (en) Immunoassay diagnostic probe and a method for use thereof
FI111194B (en) Dual-position immunoassay for an antibody using chemiluminescent chips and biotin-bound ligand
Daneshvar et al. Detection of biomolecules in the near-infrared spectral region via a fiber-optic immunosensor
Parry et al. Detection of rubella antibody using an optical immunosensor
WO2003060519A1 (en) Method of assaying agglutination reaction
WO2008068554A1 (en) Complement-based analyte assay
Li et al. Preparation and characterization of a homogeneous immunoassay for point-of-care testing (POCT) of procalcitonin (PCT)
Zhang et al. Ball-lens assisted sensitivity improvement of fluorescence immunoassay in microchannels
US20230221309A1 (en) Method, use of the method and kit for detecting bioindicators in a sample
WO1993025905A1 (en) Immunoassay method
WO2024102748A1 (en) Methods, diagnostic instruments, and kits for detecting diseases

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2003560562

Country of ref document: JP

122 Ep: pct application non-entry in european phase