JP2510551B2 - Method for measuring antigen-antibody reaction - Google Patents
Method for measuring antigen-antibody reactionInfo
- Publication number
- JP2510551B2 JP2510551B2 JP62019966A JP1996687A JP2510551B2 JP 2510551 B2 JP2510551 B2 JP 2510551B2 JP 62019966 A JP62019966 A JP 62019966A JP 1996687 A JP1996687 A JP 1996687A JP 2510551 B2 JP2510551 B2 JP 2510551B2
- Authority
- JP
- Japan
- Prior art keywords
- antigen
- antibody
- reaction
- latex
- magnetic latex
- Prior art date
- Legal status (The legal status 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 status listed.)
- Expired - Lifetime
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Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は抗原抗体反応の測定方法に関する。TECHNICAL FIELD The present invention relates to a method for measuring an antigen-antibody reaction.
（従来の技術と発明が解決しようとする問題点） 従来、ラテックス等の不溶性担体粒子に担持させた抗
体または抗原を抗原または抗体と液体媒体中で反応さ
せ、その反応の進行に伴う反応混合物の透過率の減少、
すなわち吸光度の増加からその抗原抗体反応の速度を測
定し、その速度から検体中の抗原または抗体の濃度を定
量する方法が知られている（特公昭58-11575号公報記
載）。この方法により抗原または抗体の濃度を高い精度
で迅速に定量できる。(Problems to be Solved by Conventional Techniques and Inventions) Conventionally, an antibody or an antigen supported on insoluble carrier particles such as latex is reacted with an antigen or an antibody in a liquid medium, and a reaction mixture accompanying the reaction progresses. Decrease in transmittance,
That is, a method is known in which the rate of the antigen-antibody reaction is measured from the increase in absorbance, and the concentration of the antigen or antibody in the sample is quantified from the rate (described in Japanese Patent Publication No. 58-11575). By this method, the concentration of the antigen or antibody can be rapidly quantified with high accuracy.
しかしながら、この方法では感度の点で数ng/ml程度
の濃度までしか信頼しうる測定値を得ることができず、
生長ホルモン、インスリン、ジゴキシン等のさらに高感
度の測定を必要とする項目については必ずしも満足でき
るものではなかった。また従来のラテックス等の不溶性
担体粒子を利用した測定方法においては血清中等の抗原
または抗体を測定する際に反応系中に存在する共存物質
の影響を受けやすいという欠点を有していた。However, in this method, it is possible to obtain a reliable measurement value only at a concentration of about several ng / ml in terms of sensitivity,
Items such as growth hormone, insulin, and digoxin that require more sensitive measurement were not always satisfactory. Further, the conventional measuring method using insoluble carrier particles such as latex has a drawback that it is easily affected by a coexisting substance existing in the reaction system when measuring an antigen or an antibody in serum or the like.
（問題点を解決するための手段） そこで本発明者らはより低濃度の抗原又は抗体の測定
を可能にし、反応系の共存物質の影響を受けない測定方
法を見い出すべく鋭意研究を行った結果、本発明に到達
したものである。(Means for Solving Problems) Therefore, the present inventors have conducted diligent research to find a measurement method that enables measurement of a lower concentration of antigen or antibody and is not affected by coexisting substances in the reaction system. The present invention has been reached.
すなわち、本発明の要旨は、磁性ラテックスに担持さ
せた抗体または抗原と液体溶媒中に存在する抗原または
抗体とを反応させた後に、未反応の磁性ラテックス及び
磁性ラテックスに担持している抗原抗体反応物を、磁場
を付与することにより集めて液体溶媒を除去し、つい
で、溶出液を添加し磁性ラテックスに担持している抗体
または抗原に反応している抗原または抗体あるいはその
混合物を溶出させ、再び磁場を付与することにより磁性
ラテックスを集めて溶出した抗原または抗体あるいはそ
の混合物を含有している溶出液と磁性ラテックスとを分
離し、該溶出液を抗原抗体反応に適したpH条件に調整
後、抗体または抗原を担持した不溶性担体粒子を分注し
て反応させ、その反応混合物の凝集の度合を光学的に透
過光または散乱光の強度変化、あるいは目視でとらえる
ことを特徴とする抗原抗体反応の測定方法に存する。That is, the gist of the present invention is to react unreacted magnetic latex and antigen-antibody reaction carried on the magnetic latex after reacting the antibody or antigen carried on the magnetic latex with the antigen or antibody present in the liquid solvent. The substances are collected by applying a magnetic field to remove the liquid solvent, and then an eluent is added to elute the antibody carried on the magnetic latex or the antigen or the antibody or the mixture thereof which has reacted with the antigen, and then again. The magnetic latex is collected by applying a magnetic field to separate the eluate containing the eluted antigen or antibody or a mixture thereof from the magnetic latex, and the eluate is adjusted to a pH condition suitable for the antigen-antibody reaction, The insoluble carrier particles carrying the antibody or antigen are dispensed and reacted, and the degree of aggregation of the reaction mixture is optically determined by the intensity of transmitted light or scattered light. Reduction, or consists in the measurement method of the antigen-antibody reaction, wherein the capture visually.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明方法においては磁性ラテックスに担持させた抗
体又は抗原を液体媒体中で対応する抗原または抗体と反
応させた後に磁性ラテックスと液体溶媒とを磁場を付与
して分離し、液体溶媒を除去する。In the method of the present invention, an antibody or antigen supported on a magnetic latex is reacted with a corresponding antigen or antibody in a liquid medium, and then the magnetic latex and the liquid solvent are separated by applying a magnetic field to remove the liquid solvent.
次いで、溶出液を加え、磁性ラテックスと反応した抗
原または抗体あるいはその混合物を溶出させる。その後
に溶出液に磁場を付与して磁性ラテックスを集めて、溶
出液と磁性ラテックスを分離、除去する。次にこの溶出
液を抗原抗体反応に適したpHに調整後、抗体または抗原
を担持させた（感作させた）ラテックス等の不溶性担体
粒子と反応させ、反応混合物の凝集の度合を光学的にま
たは目視によって測定する。Then, an eluate is added to elute the antigen or antibody or the mixture thereof which has reacted with the magnetic latex. Then, a magnetic field is applied to the eluate to collect the magnetic latex, and the eluate and the magnetic latex are separated and removed. Next, this eluate is adjusted to a pH suitable for the antigen-antibody reaction, and then reacted with insoluble carrier particles such as latex carrying (sensitized) antibody or antigen to optically determine the degree of aggregation of the reaction mixture. Alternatively, measure visually.
本発明に用いられる磁性ラテックスとしては、粒状、
板状等のものが通常採用される。The magnetic latex used in the present invention is granular,
Plates and the like are usually adopted.
本発明において測定物および反応物となる抗原として
は、例えば蛋白質、ポリペプチド、ステロイド、多糖
類、脂質、花粉等種々のものが挙げられる。また、抗体
としては、例えば上記した抗原に対する抗体である蛋白
質が挙げられる。磁性ラテックスへの抗体または抗原を
担持させる方法は常法によることができる。Examples of the antigen to be measured and reacted in the present invention include various substances such as proteins, polypeptides, steroids, polysaccharides, lipids and pollens. Examples of the antibody include proteins that are antibodies against the above-mentioned antigens. A method for supporting an antibody or an antigen on the magnetic latex can be a conventional method.
すなわち、被検体中の抗原または抗体と反応しうる抗
体または抗原を物理的に吸着させるか、もしくはカップ
リング剤等を用いて化学的に結合させてもよい。抗体ま
たは抗原を担持した（感作させた）磁性ラテックスと液
体媒体中の抗原または抗体との反応は、その種類によっ
ても異なるがよく攪拌させた後、通常約５分〜30分で行
われる。反応の温度は室温で反応を行うのが通常である
が、時間を短縮するために30℃〜40℃程度で反応させて
もよい。That is, an antibody or an antigen capable of reacting with an antigen or an antibody in a subject may be physically adsorbed, or chemically bound using a coupling agent or the like. The reaction between the magnetic latex carrying (sensitized) the antibody or the antigen and the antigen or the antibody in the liquid medium is usually about 5 to 30 minutes after thoroughly stirring, although it varies depending on the kind. The reaction temperature is usually room temperature, but in order to shorten the time, the reaction may be performed at about 30 ° C to 40 ° C.
液体媒体中の抗原または抗体と反応した磁性ラテック
スと液体媒体との分離、抗原及び抗体を溶出した後に溶
出液中からの磁性ラテックスの分離・除去は、磁石を用
いて磁場を付与することにより磁性ラテックスを沈降さ
せ分離する方法により行うことができる。Separation of the magnetic latex that has reacted with the antigen or antibody in the liquid medium from the liquid medium, and separation / removal of the magnetic latex from the eluate after elution of the antigen and antibody are performed by applying a magnetic field using a magnet. It can be carried out by a method of settling and separating the latex.
磁性ラテックスに担持された抗体または抗原と反応し
ている抗原または抗体（抗原抗体反応物）を溶出させる
溶出液としては最も一般的には0.2Mグリシン/HC1緩衝液
（pH2.3〜3.0）が使用される。その他、リン酸−クエン
酸（pH2.8）、1Mプロピオン酸、3M NaSCN（pH7.5）等が
利用される。溶出液量は、特に制限されないが、濃縮効
果をもたせるためには、測定する液体媒体の液量より少
なくするのが好ましい。この液量は希望する濃縮倍率に
よって決められる。溶出させる反応時間は通常約５分〜
20分で平衡に達する。The most commonly used eluent for eluting the antigen or antibody (antigen-antibody reaction product) that has reacted with the antibody or antigen carried on the magnetic latex is 0.2M glycine / HC1 buffer (pH 2.3 to 3.0). used. In addition, phosphoric acid-citric acid (pH 2.8), 1M propionic acid, 3M NaSCN (pH 7.5) and the like are used. The amount of the eluate is not particularly limited, but it is preferably smaller than the amount of the liquid medium to be measured in order to have the concentration effect. This volume is determined by the desired concentration ratio. The reaction time for elution is usually about 5 minutes
Equilibrium is reached in 20 minutes.
抗原または抗体を溶出した後、磁性ラテックスを前述
の分離手段で溶出液中から除去した後、抗原または抗体
を含む溶出液をたとえばトリスアミノメタン緩衝液によ
りpHを通常６〜９に調整する。After elution of the antigen or antibody, the magnetic latex is removed from the eluate by the above-mentioned separation means, and then the eluate containing the antigen or antibody is adjusted to a pH of usually 6 to 9 with, for example, a trisaminomethane buffer.
本発明に用いられる不溶性担体粒子としては、粒径0.
01μ〜１μのラテックス粒子が最適であり、通常0.001
重量％以上、好ましくは0.01〜１重量％程度の不溶性担
体粒子の懸濁液として用いる。この不溶性担体粒子に抗
体または抗原を感作し、これを上記のpH6〜９程度に調
整した溶出液中の抗原または抗体と凝集反応させる。凝
集の度合は光学的に透過光または散乱光の強度変化とし
て定量的にとらえることができる。また、スライド上で
溶出液中の抗原または抗体と不溶性担体粒子に担持され
た抗体または抗原を反応させ、その凝集の度合を視覚的
にとらえることもできる。The insoluble carrier particles used in the present invention have a particle size of 0.
Latex particles from 01μ to 1μ are optimal, usually 0.001
It is used as a suspension of insoluble carrier particles in an amount of not less than wt%, preferably about 0.01 to 1 wt%. The insoluble carrier particles are sensitized with an antibody or an antigen, and this is allowed to undergo an agglutination reaction with the antigen or the antibody in the eluate adjusted to about pH 6 to 9 described above. The degree of agglutination can be optically quantitatively captured as a change in intensity of transmitted light or scattered light. Alternatively, the degree of aggregation can be visually recognized by reacting the antigen or antibody in the eluate with the antibody or antigen carried by the insoluble carrier particles on the slide.
上記の方法により、測定液体媒体より目的とする抗原
または抗体を濃縮するとともに測定液体媒体と目的とす
る抗原または抗体を分離することにより、測定液体媒体
中に含まれる共存物質の影響を受けることなく、予め濃
度既知の試料より作製した抗原または抗体の検量線を用
いて、測定液体媒体中の抗原または抗体の濃度を精度よ
く測定することができる。By the method described above, by concentrating the target antigen or antibody from the measurement liquid medium and separating the measurement liquid medium and the target antigen or antibody, without being affected by coexisting substances contained in the measurement liquid medium. The concentration of the antigen or the antibody in the measurement liquid medium can be accurately measured by using the calibration curve of the antigen or the antibody prepared from the sample of which the concentration is known in advance.
（実施例） 以下、実施例により本発明を更に詳細に説明するが、
本発明はその要旨を超えない限り以下の実施例に限定さ
れるものではない。(Examples) Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to the following examples unless it exceeds the gist.
実施例１ 血清中の微量AFP（アルファ フエト プロテイン）の
測定 固相として平均粒径0.7μｍの磁性ラテックス（ロー
ヌプーラン社製）を用いた。Example 1 Measurement of trace amount of AFP (alpha-fetoprotein) in serum As a solid phase, a magnetic latex having an average particle size of 0.7 μm (manufactured by Rhone Poulenc) was used.
まず１％V/V磁性ラテックスにAFPを感作する。試験
管内にAFP陽性検体260μｌと１％V/VAFP磁性ラテック
ス40μｌを分注し、約１分間攪拌する。攪拌の約10分後
試験管の壁面に磁石を置き、AFPと凝集反応したラテッ
クスを集める。溶媒とラテックスが完全に分離したのを
確認した後、上清を除去する。次に、水300μｌを試験
管に加え、攪拌し、再び磁石によりラテックスを集め、
上清を除去し洗浄を行なう。試験管の壁に残っている凝
集したラテックスをpH2.3グリシン−HCl緩衝液100μｌ
を加え、よく攪拌、分散させ、AFP抗原を溶出させる。
溶出し終った磁性ラテックスを磁石により集め、上清10
0μｌを反応セルに分注し、緩衝液50μｌ、安定化液110
μｌ〔三菱化成工業株式会社（現三菱化学株式会社）、
免疫測定装置「LPIA 100」用AFP測定用〕を混合し、そ
の後免疫測定装置「LPIA 100」〔三菱化成（現三菱化
学）製〕AFPラテックス40μｌを分注し、攪拌して、
反応速度を測定する。図１に反応速度ＶとAFP濃度との
検量線を示す。First, sensitize AFP to 1% V / V magnetic latex. Dispense 260 μl of AFP positive sample and 40 μl of 1% V / VAFP magnetic latex into a test tube, and stir for about 1 minute. Approximately 10 minutes after stirring, place a magnet on the wall of the test tube and collect the latex that has undergone agglutination reaction with AFP. After confirming that the solvent and the latex are completely separated, the supernatant is removed. Next, add 300 μl of water to the test tube, stir, collect the latex again with a magnet,
Remove the supernatant and wash. The aggregated latex remaining on the wall of the test tube was washed with 100 μl of pH 2.3 glycine-HCl buffer.
And stir well to disperse to elute the AFP antigen.
The magnetic latex that has been eluted is collected by a magnet and the supernatant 10
Dispense 0 μl into the reaction cell, buffer 50 μl, stabilizing solution 110
μl [Mitsubishi Chemical Industries (currently Mitsubishi Chemical Corporation),
Immunoassay device “LPIA 100” for AFP measurement] is mixed, and thereafter, immunoassay device “LPIA 100” [manufactured by Mitsubishi Kasei (now Mitsubishi Kagaku)] AFP latex 40 μl is dispensed and stirred,
The reaction rate is measured. FIG. 1 shows a calibration curve of reaction rate V and AFP concentration.
（発明の効果） 本発明方法によれば現在、ラテックス等の不溶性担体
粒子による免疫測定方法で測定することが不可能であっ
た検体中の微量の抗原または抗体を測定可能な濃度に濃
縮することにより、これを測定可能にし、また、検体中
の共存物質に影響されない高精度の分析を行うことがで
きる。(Effect of the Invention) According to the method of the present invention, it is possible to concentrate a trace amount of an antigen or antibody in a sample, which could not be measured by an immunoassay method using insoluble carrier particles such as latex, to a measurable concentration. As a result, this can be measured, and highly accurate analysis that is not affected by coexisting substances in the sample can be performed.
図１は、実施例１で得られた検量線（反応速度ＶとAFP
濃度の関係）を示す図である。FIG. 1 shows the calibration curve (reaction rate V and AFP) obtained in Example 1.
It is a figure which shows the relationship of density.
Claims (2)
原と液体溶媒中に存在する抗原または抗体とを反応させ
た後に、未反応の磁性ラテックス及び磁性ラテックスに
担持している抗原抗体反応物を、磁場を付与することに
より集めて液体溶媒を除去し、ついで、溶出液を添加し
磁性ラテックスに担持している抗体または抗原に反応し
ている抗原または抗体あるいはその混合物を溶出させ、
再び磁場を付与することにより磁性ラテックスを集め
て、溶出した抗原または抗体あるいはその混合物を含有
している溶出液と磁性ラテックスとを分離し、該溶出液
を抗原抗体反応に適したpH条件に調整後、抗体または抗
原を担持した不溶性担体粒子を分注して反応させ、その
反応混合物の凝集の度合を光学的に透過光または散乱光
の強度変化、あるいは目視でとらえることを特徴とする
抗原抗体反応の測定方法。1. An unreacted magnetic latex and an antigen-antibody reaction product carried on the magnetic latex after reacting an antibody or an antigen supported on a magnetic latex with an antigen or an antibody present in a liquid solvent. The liquid solvent is collected by applying a magnetic field to remove the liquid solvent, and then an eluate is added to elute the antibody carried on the magnetic latex or the antigen or the antibody or the mixture thereof reacting with the antigen,
The magnetic latex is collected by applying a magnetic field again, the eluate containing the eluted antigen or antibody or a mixture thereof is separated from the magnetic latex, and the eluate is adjusted to a pH condition suitable for the antigen-antibody reaction. After that, insoluble carrier particles carrying an antibody or an antigen are dispensed and reacted, and the degree of aggregation of the reaction mixture is optically detected by intensity change of transmitted light or scattered light, or by visual observation. How to measure reaction.
ド上で抗原抗体反応に適したpH条件に調整後、抗体また
は抗原を担持した不溶性担体粒子を加えて反応させ、そ
の反応混合物の凝集の度合を視覚的にとらえることを特
徴とする特許請求の範囲第１項記載の方法。2. The eluate from which the magnetic latex has been separated is adjusted to a pH condition suitable for an antigen-antibody reaction on a slide, and then insoluble carrier particles carrying an antibody or an antigen are added and reacted, and the degree of aggregation of the reaction mixture. The method according to claim 1, characterized by visually recognizing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62019966A JP2510551B2 (en) | 1987-01-30 | 1987-01-30 | Method for measuring antigen-antibody reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62019966A JP2510551B2 (en) | 1987-01-30 | 1987-01-30 | Method for measuring antigen-antibody reaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63187157A JPS63187157A (en) | 1988-08-02 |
| JP2510551B2 true JP2510551B2 (en) | 1996-06-26 |
Family
ID=12013931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62019966A Expired - Lifetime JP2510551B2 (en) | 1987-01-30 | 1987-01-30 | Method for measuring antigen-antibody reaction |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2510551B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5053089B2 (en) * | 2004-08-03 | 2012-10-17 | ベクトン・ディキンソン・アンド・カンパニー | Use of magnetic materials for direct isolation of compounds and fractionation of multicomponent samples |
| CN103765214B (en) | 2012-08-31 | 2015-10-21 | 株式会社东芝 | Detection bodies testing fixture |
| DK2881740T3 (en) * | 2013-12-03 | 2019-05-13 | Immunodiagnostic Systems Ltd | Method for quantifying an analyte and automatic analyzer configured to perform the method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5896251A (en) * | 1981-12-04 | 1983-06-08 | Asahi Medical Kk | Measuring method for antigen or antibody and reagent for measurement |
| JPS61217765A (en) * | 1985-03-23 | 1986-09-27 | Tadashi Hara | Method and apparatus for assaying immunizing protein |
-
1987
- 1987-01-30 JP JP62019966A patent/JP2510551B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63187157A (en) | 1988-08-02 |
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