JPH01272968A - Preparation of specimen in order to execute laser magnetic immunosassay - Google Patents
Preparation of specimen in order to execute laser magnetic immunosassayInfo
- Publication number
- JPH01272968A JPH01272968A JP10291288A JP10291288A JPH01272968A JP H01272968 A JPH01272968 A JP H01272968A JP 10291288 A JP10291288 A JP 10291288A JP 10291288 A JP10291288 A JP 10291288A JP H01272968 A JPH01272968 A JP H01272968A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- specimen
- antigen
- magnetic
- particles
- 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.)
- Granted
Links
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Landscapes
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Abstract
Description
（産業上の利用分野〕
本発明は極めて微量の検体から特定の抗体または抗原を
定ｈＸ的に検出可能なレーｌ′ｆ磁気免疫測定方法に用
い−Ｃ好適な検体調整力法に関するものである。
〔従来の技術〕
後天性免疫不全症候群、成人Ｔ細胞白血病等のような新
型ウィルス竹疾病、あるい(Industrial Application Field) The present invention relates to a sample adjustment force method suitable for use in a magnetic immunoassay method capable of detecting a specific antibody or antigen from an extremely small amount of sample using a constant hX method. [Prior art] New viral bamboo diseases such as acquired immunodeficiency syndrome, adult T-cell leukemia, etc.
【３１各秤ガンの早期検査法
として、抗原抗体反応を利用した免疫測定法の開発が、
睨在、世界的規模で推進されている。
従来から知られる一次反応を利用した微量免疫測定方法
としては、ラジオイムノアッセイ（以下、ＲＩ△法と記
′ＴＩ）、耐累イムノアッヒイ（ＥＩＡ）、蛍光イノ１
ノアツレイ法等が既に実用化され（いる。これらの方法
は、それぞれアイソ１〜−ブ、酵素、蛍光物質を標識と
して付加した抗原また【よ抗体を用い、これと特異的に
反応覆る抗体または抗原の有無を検出づる方法である。
ＲＩ Ａ ｖ：は、標識化されたアイソトープの放的線
紹を測定することにより抗原抗体反応に寄りした検体量
を定δ１づるｂので・あり、ビー」グラム程度の超微足
測定が可能４ｆ現右暗−の方ｄ１である。しかしながら
、この方法は放ＤＪ竹物質を利用覆るので、特殊設備を
必要とし、また、半減期客にＪ、る標識効果の減衰等を
考１煮しな（Ｊればなら４ｆいので、実施には人込゛な
制約がある。更に、散開性廃棄物処理が和会問題とな−
）でいる現状を書庫りると、その実施は自ずと制限され
る。
一方、酵素、蛍光体を標識どして用いる方法は、抗原抗
体反応に寄与した検体量を、発色や発光を観測りること
にＪ、り検出り゛る方ｒ人であり、ＲＩＡ法の如き実施
上の制約はない。しかしながら、発色あるいは発光を精
密に定がすること０９１困難であり、検出限界はプノグ
ラム程度である。
また、レーリ゛光を利用して抗原抗体反応の右フ１（（
を検出りる方法として、例えば、主に肝臓病の検出を目
的どじで開発された△ＦＰ（アルファ・ノー■１〜プ１
１−フィン）を利用した方法がある。
この方法は、Ａ Ｆ Ｐに対Ｊる抗体をプラスダック微
粒子にイτｊ加し、抗原抗体反応によってプ゛ラスデッ
ク粒子が凝集してノＩしる質耐変化から調べる方法で゛
あり、１Ｏ−１０ｙの検出感度を達成している。これは
、従来のレーリ゛−光を用いた方法の白イ８以−Ｌの感
度であるが、ＲＩＡ法に比較Ｊるど自分の−・以下に過
ぎない。史に、この方法が水溶液中にａ３ Ｌ−Ｊる抗
Ｉ京抗体複合物のブラウン運動の変化を利用しくいるた
めに、抗体を含む水溶液の舘１ａ、揺乱の影響あるいは
水溶液に混在ザる不純物粒子の影響を極めて受【Ｊ易く
、これ以上に検出感度を４′２めることは■；１即的に
望外のものＣ゛ある。Ｊ、た、このＪ、うな木質的欠点
があるため、多量の検体を必要どじてぃた。
！述のｊ、うに、従来の免疫測定手段においては、高い
検出感度を右Ｊる「り［Ａ法は、放０４刊物７゛１を使
用づるために、イの実流（ンついて（Ｊ多くの制約があ
り、一方、実施の容易な酵素イムノアラレイ法、蛍光イ
ムノアッレイｄ、等は感度が低く、精密な定ｉ７１的測
定がＣさなか・）だ。
てこで、本発明者らは、従来の方法とは原理を異に覆る
免疫測定方法のω］究を行ない、先に、ｒ」願昭６１−
２２４５６７ ；′Ｊ、特願昭６１−２５２４２７８、
特願昭６１−２５４１６４号、特願昭６２−２２０６２
号、特願昭６２−２２０６３号、特願昭６２−１５２７
９１月、特願昭６２−１５２７９２＠、特願昭６２−１
ε１９０２号、特願昭６２−２６４３１９号、特ｗ１昭
６２−２６７／１３３１ｇどしてレーザ磁気免疫測定方
法及び測定装置についての発明を特許出願している。こ
れらの新しい免疫測定方法は標ｉａ月利としてＩａ刊微
オイｌ子を用いる点に特徴があり、アイソ１〜−プを用
いないでビニ１グラムの超微量検出が可能である。検出
ｙＪ法は検体に照射したレーリ゛光の散乱光、透過光、
反ＱＪ光、］渉光重回折光の何れを検出しでもＪ、い、
。
本発明者らは１述の秘ｈ′［に２；↓づさ、磁ｆＬ微粉
了を抗原あるい【Ｊ抗体に標識し、初めてウィルスの検
出等を行なった。この新しいレーザ磁気免疫測定方法は
、従来形も検出感度が高いとされている「くＩＡ法Ｊ、
リーし検出悪疫が高いことが確認されつつある。、 ｐ
Ａえぼ、発明発布らが日本ウィルス学会第３Ｌ）回総会
（昭和６２く［１１月 講演番＋： ４０１１［新しく
開発した免疫測定装置を用いたウィルスの検出実験−ｊ
）て発表したように、不活刺止したインフルＪンリ“ウ
ィルスΔ、Ｂ型をウィルスのモデルとして用いて、ウィ
ルス検出実験を行なったところ、１ｄ中に１個程度のウ
ィルスが存在覆る場合でも検出できた。１
〔発明が解決しようとする課題〕
どこ／）で・、一般に、［でＩ△、１日Ｉ△、Ｆ■Δ並
びに本発明でのレーリ“ｖＡ気免疫測定方法のように標
識物質を用いる方法は検体調整の際に、検体と反応しな
かった未反応の標識体を分−１・除去Ｊる必要がある。
例えば、「［Ａ法の一秤である「１−Ｉｓ八へでは既知
の抗原が同相化されたマイク［−１プレートに、検体溶
液を反応させた後に洗浄を行なって、未反応の検体溶液
を除去し、次に酵素標識抗体を加えて酵素標識を行な・
）だ後、さらに未反応の酵素標識抗体を洗浄覆る方法が
とられている。この洗浄−１程は通！ｊｊ ５・へ・６
回行う必要があった。洗浄−工程を簡略化覆ればマイク
［Ｉブレー１へ十−Ｇ ＝
に標識酵累が残留し、１ｌｌｌｌ定に妨害をりえるため
である。
また、マイク１」ブレー１・に抗原を固相比重る従来の
方法は抗原抗体反応がンイクロブレー１−の表面に限ら
れることから極微量の検体の検出には、抗原ＩＩＬ体反
応口、１間を長ｕ４間にりる必要があった。。
本発明は未反応の磁性体標識抗体の分離を１回の操作Ｃ
確実に行うと共に、抗原抗体反応の表面積を増やすこと
ににって、検体ど磁性体標識抗体の遭遇の機会を増やし
、極微量の検体をも確実（ご磁性体標識抗体で捕捉・標
識づることによって、検出感度の向Ｌ１１１．びに測定
時間の短縮を図ることを課題としている。
〔課題を解決りるための手段〕
本発明は、前記課題を解決ηるためになされたもので、
本発明によれば、
磁性体標識抗体よりも充分に人きな質量を右りるＪ１１
磁性粒子に検体を固定さ−ける第１の１程と、前記検体
と前記磁性体標識抗体とを抗原抗体反応さ（！る第２の
工程と、前記第２の工程で１ｆＩられた磁性体標識検体
複合体と前記第２の−Ｌ稈（・残存りる未反応の前記磁
性体標識抗体とを遠心により分離リ−ることを特徴とす
るレーリ゛磁気免疫測定方法を実施するための検イホ調
整方法が提供される。
本発明の実施態様としＣ１非磁個体粒−ｒに検体を固定
さける前記第２のＴ稈には、非磁性体粒子の表面を活性
化して検体を非特異的に吸着さＩＪる方法あるいは昇磁
↑〕を体粒子の表面上に予め既知の抗体あるいは抗原を
固定し−ＣＪ３ざ検体を抗原抗体反応によって特異的に
結合させる方法が目的に応じて選択できる。
〔作用〕
本発明に係るレーク“磁気免疫測定方法を実施りるため
の検体調整１ノ法は、標識物質として磁性体微粒子を利
用し、この磁性体微粒子に抗体を結合して１１ノられる
磁性体標識抗体Ｊ、リム充分に人さな質量を右する昇磁
ｆ［体粒子の表面上で検体を捕捉した後、前記磁性体標
識抗体ど検体とを抗原抗体反応さける。検体を捕捉り−
るための前記非磁性体粒子は磁性体であ・）−Ｕ Ｃ：
Ｌならイｒい。何故イ、−らぽ、検体を捕捉１１２磁性
体標識抗体１″′標識づる意味が７７（（り４’Ｌるか
らである。前記非磁性体微粒子としては、平均粒？！０
．１へ・１０７１７１１程度の微粒子が好Ｊ、しい。０
．１μｍ以下ではウィルスや磁性体標識抗体の人ささと
同程度になり、次の］稈の遠心分１１１１１に不都合に
なるからである。また、１０μｍ以上Ｃは表面積が小さ
くなるためウィルスの検出窓ｆσ並びにレーク“磁気免
疫測定の際の測定６１瓜が低下１Ｊるため（゛ある。
ｊ）０記非磁性体粒子は、例えばアクリルボリン−樹脂
−）ｂボリスヂレン樹脂等のプラスチック微小球、ある
いはシリカやアルミナ等の無機］ロイド粒子などが好ま
しい３、さらに好ましく【９１、これらの−１１磁性体
粒子は前記磁性体標識体よりも密度が大きいこと゛（・
ある。何故ならば、非磁性体粒子は沈澱さけ、未反応の
磁性体標識抗体をＪ＝ ？ｉ′ｒどし−Ｃ分離りる遠心
分離操作に右利になるからである。密度の人さｈ非磁性
体粒子を得る方法どして、鉛等のＪｆ ｔｉｎ ＋４金
属を核に持つ（１機あるいは烈機の複合月利から作製り
る方法が好Ｊ、しい。
−〇 −
非磁性体粒子の表面に検体を捕捉覆る方法の一′つどし
て、Ｊ１１磁性粒子の表面を１１５慴（ヒして検体を非
特異的に吸着Ｊる方法をどることができる。
この方法は、スクリーニング検査１″）、患貨のうがい
液からインフル１−ンリーウイルスを検出づるＪ、うな
場合に有効である。うがい液にはウィルスは多くても数
百側程度しか存在しないし、Ｊ、た、Δ型、１Ｂ型舌の
複数の変早（株があるｈ日ら、まずウィルスを特定せず
に確実に前記非磁性体粒子に捕捉Ｊる目的に適している
。１
らう一つの方法として、非ｆ！ｌ竹体粒子の表面上に予
め既知の抗体あるいは坑口；ミを固定しておき検体を抗
原抗体反応にＪ：って特異的に結合させる方法がある３
、この方法１．□１、非特異反応をできる限りＣ１除し
て特定のウィルスのみを確実に検出づる精密検査に適し
ている３゜
さて、前記の検体捕捉工程の後、捕捉された検体と磁性
体標識抗体とを抗原抗体反応さける。この肋、磁性体標
識抗体は、検体と確実に抗原抗体反応ざ已るために、検
体Ｊ、リム過剰に加えることが望ましい３１例えば−例
として、ビ］グラム台の検体を検出りるためには磁性体
標識抗体１０’？／程度加えればＪ、い。
次に、前記の抗原抗体反応工程で磁性標識された抗原抗
体複合体（磁性体標識検体複合体）と前記■稈で夕＆存
づる未反応の前記Ｉ６磁性標識抗体とを分離させる。こ
の分離方法として、本発明の効果を最大に発揮させる遠
心による分離が最す望、Ｊ、しい。例えば、１μｍのア
クリルポリマー樹脂を非磁性体粒子どじて用いた場合、
非磁性体粒子に捕捉された抗原抗体複合体は１５００
ｐ ｐ ｍ、１５分間の低速遠心で沈澱づる。一方、未
反応の磁性体標識抗体は沈澱ｉ！ザ、上清として留まる
。沈澱物を採取して、例えば、ｔ」［Ｐ ＬＥ Ｓ （
１）、Ｊ、う泡・緩シ＋ｉｊ液中に抗原抗体複合体を分
散さける。
この、Ｊ、うにして、本発明のレーザ磁気免疫測定方法
を実施りるための検体調整がイＣされる。
本発明の検体調整方法を適用した後、本発明にらが先に
出願したレーＩＪ’ Ｉａ気免疫測定方方法び測定′￥
、２′７に技術開示している方法ぐ極微量のウィル−’
＋１−
スあるいは工程の検出を行うことができる。
以下に図面を参照して本発明をより具体的に詳述するが
、以下に示すもの＋、Ｉ、本発明の一実施例に過ぎず、
本発明の技術的範囲を何等制限Ｊるものではイヱい。
〔実施例１〕
第１図は本発明の検体調整方法の一実施例を説明するＩ
−稈図であ・つて、図中（ａ ） Ｔ、ＬＪｌ磁性体を
液中分散づる工程、（ｂ）は検体を捕捉Ｊる工程、（ｃ
）ＬＪｖ１１気標識−［稈、（ｄ ） ４．を遠心分１
ｉｌＩｔ丁稈、（ｅ）は測定に稈である。
本実施例においでは実験」二安金性の畠い不活＋１化し
たインフル゛［ンリ゛ウィルスを用いＣ本発明の検体調
整法の検出限界を調べる目的で実施した。
平均粒径１μｍのアクリルポリマーから４する非磁性体
粒子１の表面には、ウリギを免疫して冑られたインフル
エンザウィルスに対づ−る高度免疫抗面消が抗体２とし
て固相化され−Ｃいる。凍結乾燥保存されでいる前記非
磁性体粒子１は］７稈（ａ）において反応容器に入れら
れｌ）Ｂ Ｓ緩衝液中に分散される３゜
前記工程（ｂ）は−し′）刀し検体として既知の′ａ度
のインフル］−シｔＦウィルス（Δ／石川（＋−１３Ｎ
２ ））を前記までの工程Ｔ：′得られたＸＩ＋磁＋
＋ （ホ粒子分散液に加えて、前記インフルエンザウィ
ルスを捕捉づる工程である。本実施例ではウィルスの検
出限界を調べるために濃Ｉα１・〜１千万個／威の範囲
で１０倍階段稀釈した各淵曵のウィルス溶液を用いＩご
、。
前記工程（Ｃ）は、磁１７１体標識抗体４を添加して、
検体との間で抗原抗体反応さけ、磁気標識・ノるＩ稈て
・あって、磁性体標識抗体４はｒキス１〜ランで被覆し
た平均粒子４ Ｑ ｎ ｍのマグネタイ１へからなる磁
性微粒子４ ａにノ】レツ１〜を免疫し−Ｃｙ１られる
抗血Ｆ？’＋からＭｉ ＭｌしたＩ ＣＪ Ｇ抗体４ｂ
を共有結合したものである１、前記磁気標識は３５℃、
２゜５時間のイン：１コベー１−の条ｆ１て・行った。
。
前記−「稈（ｄ）は、前記までのＴ稈で得られた抗原抗
体複合体く磁性体標識検体複合体）５と未反応の磁ｆ［
体標識抗体４どを分１ｉＩＩＩりる工程であつ−１３＝
て、１５００ ｐ ｐ ｍ、５分間（１） ｉ、４心ニ
に −） Ｔ、前記抗原抗体複合体５〕は沈澱し、未反
応の磁性体標識抗体４は下漬どして４ＥＪらねた。
前記工程（ｅ ）は前記までの工程で得られた沈澱物を
採取してレーザ磁気免疫測定方法で測定する工程であ・
）で、］−口Ｐ ［Ｓ緩衝液１ｍｌに前記沈澱物を加え
、本発明化らが先に発明した干渉法（特曳１昭６２−１
８４９０２号）で′ウィルスの検出を行ｔ１つだ。その
結果、ウィルス１個程度を検出ケることが出来た。
第２図は比較対照例であって、現在、エイズ抗体検査等
に広く用いられている［ＬＩＳＡ法の検体調整］稈図で
ある。Ｆｌ−ＩＳＡ法の場合、（ａ）固相化工程、（ｂ
）検体捕捉−工程、（Ｃ）洗浄工程、（ｄ）酊累標識二
り稈、（Ｃ）洗浄］工程、（ｆ）気質反応−［稈、（０
）反応停止Ｔ稈、（ｈ）測定工程、等の数多くのＴ稈を
経て検体調整される。
１Ｊなわち、既知の抗原１０をマイク【］プレート１１
に同相化しくａ）、この同相化され１＝マイクロプレー
ト１１に検体３の溶液を反応さＢ （ｂ ）、＝ １４
−
その後に洗浄を行ｔｒって未反応の検体３の溶液を除去
しくＣ）、次に酵素標識抗体１２を加えて酊糸標識を行
ない（ｄ）、その後、さらに未反応の酵素標識抗体１２
を洗浄、除去しくｅ）、前記；１、での工程Ｃ得られた
標識検体段合体１３に気Ｖ−ｉ １４を反応さｔ！（ｆ
）、この反応を停止液で停止（す）さけ／、：後に、紫
外線を照（ｌ（ｌ、て測定（１１）していた１、これら
の■程のうち、洗浄丁卯（９Ｌ通１；ｉ、５・〜・６回
反覆洗浄されている。洗？１ｒが不完全／、ｉ：隻（合
、測定■稈においてバックグランドが土４ηるので、検
出感度の低手あるいは誤判断の原因になる１３本発明の
方ｖ１とＥｌ、ＩＳＡ法とを比較Ｊるど、本発明の方が
３３二Ｆ程短縮できる利点がある。
本発明の場合、表面積の大きな非！Ｈ！１体粒子の表面
（・３次元的に検体を捕］）？シ、磁性体標識抗体と抗
原抗体反応Ｊるから、従来の２次元的に反応さける方法
Ｊ、りも反応時間を短縮りることができる。また、この
特徴のため、極微量の検体の捕捉にも右利である。、捕
捉・反応過程Ｃ撹拌処Ｊ！Ｉｌを０［ＨＥ ＦＪれぽ更
に捕ｊ；Ｃ・反応処理］１゜１間の短縮ができる。
−１！’ｊ−
また、本発明の場合、遠心力によって分１ｌｌＩづるか
ら、洗浄Ｊ、りも確実２．ｚ分離が行なわれる３、遠心
条件は使用覆る非磁性体粒子とｖＡ竹鉢体標識抗体の比
重〉ｆに応じて最適り条（’ｌが決められる３□〔実施
例２〕
本実施例は患者のうがい液からインフルエンザウィルス
を検出づる実験例（−ある。
患者のうがい液を３０−採取し、まず、前処理どじで３
０００ ｒ′Ｌ） ｍ、１０分間の遠心にか【プて貸物
等を沈澱さけて除去した後、十清を２０００Ｑｒｐｍ、
３０分間超超遠心上か【づインフルエンザウィルスを沈
澱させ、沈澱物１ｄを採取して検イ木どじムコ。
次に、ウィルスが非ｑ：！ｉ ｑ的にイ」着するように
活性化したアクリルボリン−からなる非磁性体粒子を用
いて３５℃、１０ｔ１５間インー１−ユベートの条件で
検体を捕捉した。検体を捕捉処理後、前記非磁性体粒子
のウィルスがイ」盾しくいない表面をＢＳ八で被覆し、
＋’＋Ｆｒ記非磁性体粒子を不活性化した。
この後の検体調整：「稈は実施例１の（０）以降と同じ
方法で行イｆつだ。検体調整を終えた検体を前記レー１
ｆ磁気免疫測定方法で測定したところ、Ａ型のインフル
丁ンリ゛ウィルスが検出された。
従来、インフルエン量アウイルスを検出するためには、
うがい液中の機作な・ウィルスを鶏卵でＪｇ差し、１；
「万個以」−にまでウィルスを増イ１した後、血液凝固
仏で検出りる７’Ｊ沃が採られでいｌＪ０従来法で・は
検査結果が出るまて゛１カ月程度かかつていた。本発明
の方法は、ウィルスを培養ｕずに、直接検出でさる感度
を右しているから、本実施例のインノルエン１ｆウイル
スに限りヂ、あらゆるウィルスにム適用て゛さる。１例
えば、ウィルスの培養が困難である未知のウィルスの検
出にも適用でさる１゜（発明の効果〕
以上詳述のように、本発明に係るレーリ゛磁気免疫測定
方法を実／Ｉｉ！！ηるｌζめの検体調整力法ｔ３１、
検体を捕捉するだめの非磁性体粒子と標識物質として！
ｌｌ機微粒子用いて、遠心分Ｈ１にＪ、って検体を調整
づることか１４徴である。ＲＩΔ法以十の検出感度を右
しているから、従来不可能であった感染直後の抗原検査
が実施でさる。更に、Ｊ１磁何体粒子、標識体として用
いる磁性超微粒子は、放射線あるいはＡ刊の点では問題
なく、検体に対して安定なものを容易に入手できる。
本発明は、実施例に十げＩζウィルスの検出に１によら
ず、癌の早期診断、アレルギー、細菌等の検合ヤ〕従来
ＲＩＡ法が適用されＣいたベブブード小ルーしン笠の種
々のホルモンあるいは種々の酵素、ビタミン、薬剤など
の測定にも応用することが可能である。従って、従来は
限定された／１ｔ！ＢＪでＲ１へ法にＪ、らイヱ【づれ
ぼ実施て・さなか−）だ精密な測定を、−殻内な環境で
広〈実施Ｊることが可能となる。
集団検診雪−のようイア船釣的な状況Ｃ１各種のウィル
ス、癌等のスクリーニング検査等の精密な測定が広〈実
施でされば、癌あろい（１１ウイルス＋１疾患等の早期
診断が可能となり、有効な早期治療を的確に実施するこ
とが可能どなる。このＪ、うに、本発明が医学・医療の
分野で宋たり効果は計り知れない１゜[31 The development of an immunoassay method using antigen-antibody reactions as an early detection method for various types of scale cancer.
Currently, it is being promoted on a global scale. Microimmunoassay methods using conventionally known primary reactions include radioimmunoassay (hereinafter referred to as RIΔ method), cumulative immunoassay (EIA), and fluorescent immunoassay (TI).
The Nortzley method and other methods have already been put into practical use. RI A v: is a method to detect the presence or absence of a labeled isotope. It is possible to measure ultra-microscopic feet of about 4f. (If J is 4f, there are constraints on implementation.Furthermore, dispersible waste treatment is a problem of Japanese society.)
), its implementation will naturally be limited. On the other hand, methods using labeled enzymes and fluorophores detect the amount of specimen that contributed to the antigen-antibody reaction by observing color development and luminescence, and RIA methods. There are no such implementation restrictions. However, it is difficult to precisely determine color development or luminescence, and the detection limit is about a pnogram. In addition, the right side of the antigen-antibody reaction (((
As a method for detecting
There is a method using 1-fin). In this method, an antibody against AFP is added to Plasdac microparticles, and the antigen-antibody reaction causes the Plasdac particles to agglutinate, and the change in quality is investigated. A detection sensitivity of 10y has been achieved. This sensitivity is higher than that of the conventional method using Rayleigh light, but it is only lower than that of the RIA method. Historically, this method takes advantage of changes in the Brownian motion of the anti-Ikyo antibody complex in an aqueous solution, so it is difficult to prevent the aqueous solution containing the antibody from being affected by turbulence or mixing in the aqueous solution. It is extremely susceptible to the influence of impurity particles, and increasing the detection sensitivity by 4'2 is immediately undesirable. J, this J, has wood defects, so a large amount of specimen was required. ! As mentioned above, conventional immunoassay means have a high detection sensitivity. On the other hand, easy-to-implement enzyme immunoarray methods, fluorescent immunoarrays, etc. have low sensitivity, and precise and constant measurements are difficult. We investigated the immunoassay method, which differs in principle from the
224567 ;'J, patent application No. 61-2524278,
Patent Application No. 1983-254164, Patent Application No. 1983-22062
No., Patent Application No. 1982-22063, Patent Application No. 1982-1527
January 1986, Special Application 1982-152792@, Special Application 1987-1
The company has filed patent applications for inventions relating to laser magnetic immunoassay methods and measuring devices in No. ε1902, Japanese Patent Application No. 62-264319, and Japanese Patent Application No. W1 Sho 62-267/1331g. These new immunoassay methods are characterized in that they use Ia volume as a standard, and can detect an ultra-trace amount of 1 gram of vinyl without using iso-1--. The detection yJ method detects scattered light, transmitted light, and
No matter which one of the anti-QJ light and the beam diffracted light is detected,
. The present inventors labeled the magnetic fL microparticles described in 1 above with antigens or [J antibodies, and detected viruses for the first time. This new laser magnetic immunoassay method is based on the ``KIA method J'', which is said to have high detection sensitivity even in the conventional type.
It is being confirmed that the number of infections detected is high. , p
Aebo, invention and others presented the 3rd L) Annual General Meeting of the Japanese Society of Virology (November 1986, lecture number +: 4011 [Virus detection experiment using a newly developed immunoassay device-j
), we conducted a virus detection experiment using inactive influenza J virus types Δ and B as a virus model, and found that even if there was only about one virus per 1 d. 1 [Problem to be Solved by the Invention] In general, [at I△, 1 day I△, F■Δ and the Lely “vA Qi immunoassay method of the present invention] In the method using a labeled substance, it is necessary to remove unreacted labeled substances that have not reacted with the sample during sample preparation. For example, in ``1-Is8, which is one of the scales of method A, a sample solution is reacted on a microphone plate containing a known antigen in the same phase, and then the unreacted sample solution is washed. is removed, and then an enzyme-labeled antibody is added to perform enzyme labeling.
) After that, a method is used to further wash and cover the unreacted enzyme-labeled antibody. This cleaning-1 degree goes well! jj 5・to・6
I had to do it twice. This is because if the washing process is simplified, labeled enzymes will remain on the microphone [I Brake 1 to G=], which can cause constant interference. In addition, in the conventional method of solid-phase loading the antigen on the microbrake 1, the antigen-antibody reaction is limited to the surface of the microbrake 1. I had to go to U4 for a long time. . The present invention allows separation of unreacted magnetically labeled antibodies in one operation C.
By increasing the surface area for the antigen-antibody reaction, we increase the chance of encountering the sample with the magnetically labeled antibody, and reliably capture and label even minute amounts of the sample (capturing and labeling with the magnetically labeled antibody). The object of the present invention is to improve the detection sensitivity and shorten the measurement time.
According to the present invention, J11 has a much smaller mass than a magnetically labeled antibody.
A first step in which a specimen is immobilized on magnetic particles, a second step in which the specimen and the magnetically labeled antibody are subjected to an antigen-antibody reaction (!), and a second step in which the specimen is immobilized with the magnetic material labeled in the second step. A test for carrying out a Rayleigh magnetic immunoassay method, characterized in that the labeled specimen complex and the second -L culm (and the remaining unreacted magnetically labeled antibody are separated by centrifugation). In an embodiment of the present invention, the second T culm in which the specimen is immobilized on the C1 non-magnetic solid particles-r is provided with a method for non-specific adjustment of the specimen by activating the surface of the non-magnetic particles. Depending on the purpose, a method in which a known antibody or antigen is immobilized in advance on the surface of a body particle and the CJ3 sample is specifically bound by an antigen-antibody reaction can be selected depending on the purpose. [Function] The first method for preparing a sample for carrying out the RAKE magnetic immunoassay method according to the present invention uses magnetic fine particles as a labeling substance, and binds an antibody to the magnetic fine particles to generate a magnetic After capturing the analyte on the surface of the body particle, the magnetically labeled antibody J avoids an antigen-antibody reaction with the analyte. Capturing the analyte -
The non-magnetic particles are magnetic.)-UC:
L is bad. Why is it that the meaning of labeling is 77 ((ri4'L)? The meaning of labeling is 77 ((ri4'L).The non-magnetic fine particles are average particles?!0
．． Go to 1. Fine particles of about 1071711 are preferable. 0
．． This is because if it is less than 1 μm, it will be comparable to the size of a virus or magnetically labeled antibody, which will be inconvenient for the next centrifugation of the culm. In addition, since the surface area of C of 10 μm or more becomes small, the detection window fσ of the virus and the measurement value during magnetic immunoassay decrease by 1J (゛j). -Resin-)bPlastic microspheres such as borisdyrene resin, or inorganic]roid particles such as silica and alumina are preferable3, and more preferably [91.These -11 magnetic particles have a density higher than that of the magnetic label. Big thing゛（・
be. This is because non-magnetic particles should be avoided by precipitation, and unreacted magnetically labeled antibodies should be J = ? This is because it is useful for centrifugal separation operations that separate i'r and C. How to obtain non-magnetic particles with a density of Jf tin +4 metal such as lead as a core (preferably a method of making them from a single unit or a composite monthly rate of reki. -〇 - One of the methods of capturing and covering the surface of non-magnetic particles is to cover the surface of J11 magnetic particles with a method of non-specifically adsorbing the sample. Screening test 1'') is effective in detecting influenza virus from patient's gargle fluid.There are only a few hundred viruses in gargle fluid, , Δ type, and 1B type tongue (strains) are suitable for the purpose of reliably capturing the virus in the non-magnetic particles without first identifying it. One method is to immobilize a known antibody or wellhead on the surface of non-f!l bamboo particles in advance, and then specifically bind the sample to the antigen-antibody reaction3.
, this method 1. □1. Suitable for detailed testing to reliably detect only a specific virus by removing C1 from non-specific reactions as much as possible. 3.After the sample capturing step, the captured sample and magnetically labeled antibody are combined. Avoid antigen-antibody reactions. It is desirable to add this magnetically labeled antibody to an excess of the sample to ensure an antigen-antibody reaction with the sample. Is magnetically labeled antibody 10'? / If you add the degree, J, yes. Next, the antigen-antibody complex (magnetically labeled specimen complex) magnetically labeled in the antigen-antibody reaction step and the unreacted I6 magnetically labeled antibody present in the culm are separated. As this separation method, separation by centrifugation is most desirable because it maximizes the effects of the present invention. For example, when using 1 μm acrylic polymer resin with non-magnetic particles,
The antigen-antibody complex captured by non-magnetic particles is 1500
Precipitate by low speed centrifugation at p p m for 15 minutes. On the other hand, unreacted magnetically labeled antibodies are precipitated i! The supernatant remains. The precipitate is collected, for example, t' [PLE S (
1) Disperse the antigen-antibody complex in the foaming/reducing solution. In this manner, sample preparation for carrying out the laser magnetic immunoassay method of the present invention is performed. After applying the sample preparation method of the present invention, the present invention can be applied to the IJ'Ia immunoassay method and measurement, which was previously filed by Nira.
, 2'7, the method disclosed in 2'7 can reduce the amount of virus in an extremely small amount.
+1- Process or process detection can be performed. The present invention will be described in more detail below with reference to the drawings, but the following is only one embodiment of the present invention.
This is not intended to limit the technical scope of the present invention in any way. [Example 1] Figure 1 is a diagram illustrating an example of the sample preparation method of the present invention.
- This is a culm diagram, in which (a) the process of dispersing T and LJl magnetic substances in the liquid, (b) the process of capturing the specimen, (c)
) LJv11 air sign - [culm, (d) 4. Centrifuge for 1 minute
(e) is the culm used for measurement. In this example, an experiment was carried out using a diantrope-based inactivated influenza virus for the purpose of investigating the detection limit of the sample preparation method of the present invention. On the surface of non-magnetic particles 1 made of acrylic polymer with an average particle size of 1 μm, a hyperimmune antibody against the influenza virus that had been immunized against Urigi was immobilized as antibody 2. There is. The non-magnetic particles 1, which have been freeze-dried and stored, are placed in a reaction vessel at 7 culms (a) and dispersed in a BS buffer solution. The specimen was a known influenza virus with a degree of
2)) Step T up to the above: 'obtained XI+magnetic+
+ (This is the step of capturing the influenza virus in addition to the influenza virus dispersion. In this example, in order to examine the detection limit of the virus, the influenza virus was diluted stepwise by 10 times to a concentration of Iα1.about.10 million particles/Virus. Using the virus solution from each source, step (C) involves adding magnetic 171-labeled antibody 4,
To avoid an antigen-antibody reaction with the specimen, magnetically labeled antibodies 4 are magnetic fine particles consisting of average particles 4 Q nm of magnetites 1 coated with r-kiss 1 to run. 4 a] Is it anti-blood F that can be immunized with -Cy1? '+ to Mi Ml I CJ G antibody 4b
1, the magnetic label is at 35°C,
2.5 hours in: 1 column 1 - article f1 and went. . The culm (d) contains the antigen-antibody complex obtained in the T culm (magnetically labeled specimen complex) 5 and unreacted magnetic f[
The antigen-antibody complex 5] is precipitated, and the antigen-antibody complex 5] is precipitated, and the antigen-antibody complex 5] is precipitated, and the antigen-antibody complex 5] is precipitated, and the antigen-antibody complex 5 is incubated at 1500 ppm for 5 minutes (1). The reaction magnetically labeled antibody 4 was submerged and subjected to 4EJ. The step (e) is a step of collecting the precipitate obtained in the previous steps and measuring it by a laser magnetic immunoassay method.
), the precipitate was added to 1 ml of S buffer solution, and the interference method (Tokuhiki 1, 1986-1
No. 84902), there was one virus detection process. As a result, we were able to detect about one virus. FIG. 2 is a comparative example, and is a culm diagram of [sample preparation by LISA method] which is currently widely used for AIDS antibody testing and the like. In the case of the Fl-ISA method, (a) solid phase step, (b
) Specimen capture-process, (C) washing process, (d) intoxication labeled two culms, (C) washing] process, (f) temperamental reaction-[culm, (0
) The sample is prepared through a number of T-culms such as reaction termination T-culm and (h) measurement process. 1J, that is, the known antigen 10 is placed on the microphone plate 11.
(b), = 14
- After that, washing is performed to remove the unreacted sample 3 solution (C), then the enzyme-labeled antibody 12 is added to carry out the labeling (d), and then the unreacted enzyme-labeled antibody 12 is further added.
e) The labeled sample plate aggregate 13 obtained in step C above is reacted with air Vi 14. (f
), this reaction was stopped with a stop solution (11). ;i, washed repeatedly 5 to 6 times. Washing is incomplete/, i: boat (if the background is 4η in the measurement culm, the detection sensitivity is low or there is a misjudgment. 13 Comparing the method of the present invention v1 and the El, ISA method, the present invention has the advantage of being able to shorten the time by 332 F. In the case of the present invention, the non-!H! Since the surface of the particle (capturing the sample three-dimensionally) is the antigen-antibody reaction between the magnetically labeled antibody and the conventional two-dimensional reaction method, the reaction time can be shortened. In addition, because of this feature, it is also useful for capturing extremely small amounts of specimens.Capture/reaction process C stirring process J! Il 0 -1!'j- In addition, in the case of the present invention, since the centrifugal force separates the separation, the separation can be carried out reliably. The optimal length ('l) is determined according to the specific gravity of the magnetic particles and the vA bamboo pot-labeled antibody. - Yes. Take 30 samples of patient's gargle fluid and first pre-process it for 30 minutes.
000 r'L) m, centrifuge for 10 minutes.
Precipitate the influenza virus using ultra-ultracentrifugation for 30 minutes, collect 1 d of precipitate, and test. Next, the virus is non-q:! The specimen was captured using non-magnetic particles made of acrylboline activated to adhere to iQ at 35° C. under conditions of in-1-jubate for 10t15. After the sample is captured, the surfaces of the non-magnetic particles that are not resistant to viruses are coated with BS8,
+'+Fr non-magnetic particles were inactivated. Sample preparation after this: "The culm is prepared in the same manner as from (0) in Example 1.The sample after sample preparation is
When measured using f-magnetic immunoassay method, type A influenza virus was detected. Traditionally, to detect influenza viruses,
Insert the virus in the gargle with a chicken egg, 1;
After increasing the number of viruses to ``more than 10,000,'' the 7'J virus detected by blood coagulation tests could not be obtained.With the conventional method, it took about a month for test results to be obtained. Since the method of the present invention has the sensitivity of direct detection without culturing the virus, it can be applied to all viruses, not just the innoluene 1f virus of this example. 1. For example, it can be applied to the detection of unknown viruses that are difficult to culture. 1. (Effects of the Invention) As detailed above, the Rayleigh magnetic immunoassay method according to the present invention can be implemented/Ii!! Sample adjustment force method t31 for ηrlζ,
As non-magnetic particles for capturing specimens and as a labeling substance!
There are 14 ways to prepare the sample using microparticles and centrifugation H1 and J. Because it has a higher detection sensitivity than the RIΔ method, it is now possible to conduct antigen tests immediately after infection, which was previously impossible. Furthermore, the J1 magnetic particles and the magnetic ultrafine particles used as labels do not pose any problems in terms of radiation or A publication, and those that are stable to the specimen can be easily obtained. The present invention is not limited to the detection of the Izeta virus, but also to the early diagnosis of cancer, the detection of allergies, bacteria, etc.] The present invention is applicable not only to the detection of the Izeta virus, but also to the early diagnosis of cancer, allergy, bacteria, etc.; It can also be applied to the measurement of hormones, various enzymes, vitamins, drugs, etc. Therefore, conventionally the /1t! With BJ, it becomes possible to carry out precise measurements widely in an in-shell environment. A situation similar to that of a snowy mass screening C1 If accurate measurements such as screening tests for various viruses and cancers are widely implemented, early diagnosis of cancer (11 viruses + 1 disease, etc.) will become possible. , it becomes possible to accurately carry out effective early treatment.The effects of this invention in the medical and medical fields are immeasurable.
第１図は本発明の検体調整方法の一実施例を説明づるＴ
程図であって、（Ｅ））は：１１−磁ｆ」体粒子を液中
分散覆る一１程図、（ｂ）は検体を捕捉１Ｊるｌ稈図、
（Ｃ）（よ磁気標識］二程図、（ｄ）は遠心分１ｌＩＩ
Ｉ］−稈図、（Ｃ） Ｔｉｔ測定−「稈図であろ１゜イ
）２図は比較λ１照例で゛あって、現在、］イズ抗体検
査等に広く用いられている、ＦｉｌＳＡ法の検体調整］
程図である。ＦＬｆＳＡ７／、の場合、（ａ）は固相化
Ｔ程図、（ｂ ）は検体捕捉］程図、（Ｃ） ｉ；↓洗
浄−１−稈図、（ｄ）は酵素４Ｓ：識］稈図、（ｅ）は
洗浄■「稈図、（ｆ ） Ｂ、１． ｊ；！質反応］程
図、（す）は反応ＰＦＩドＩ−稈図、（ｈ ） １．Ｌ
測定［稈図である。
１・・・Ｊ１磁ｆ１体粒子、２・・抗体、３・・・検体
、４・・・磁＋ｔ−（Ａ標識抗体、５・・・＋７１：
Ｂａ抗体複合体（磁性体Ｆ識検体複合体）。FIG. 1 illustrates an embodiment of the sample preparation method of the present invention.
(E) is a diagram showing the process of dispersing the 11-magnetic particles in the liquid; (b) is the diagram of the process of capturing the specimen;
(C) (magnetic labeling) two-stage diagram, (d) centrifugation 1lII
I] - Culm diagram, (C) Tit measurement - "Culm diagram 1゜A) Figure 2 is an example of comparison λ1, and is a sample of the FilSA method, which is currently widely used for ]is antibody tests, etc. Adjustment]
This is a diagram. In the case of FLfSA7/, (a) is the solid-phase T process diagram, (b) is the sample capture] process diagram, (C) i;↓Washing-1-culm diagram, (d) is the enzyme 4S: identification] culm diagram. Figure, (e) is the cleaning ■ culm diagram, (f) B, 1.j;
Measurement [culm diagram] 1...J1 magnetic f1 particle, 2...antibody, 3...analyte, 4...magnetic+t-(A-labeled antibody, 5...+71:
Ba antibody complex (magnetic material F identification analyte complex).
Claims (2)
非磁性体粒子に検体を固定させる第１の工程と、前記検
体と前記磁性体標識抗体とを抗原抗体反応させる第２の
工程と、前記第２の工程で得られた磁性体標識検体複合
体と前記第２の工程で残存する未反応の前記磁性体標識
抗体とを遠心により分離することを特徴とするレーザ磁
気免疫測定方法を実施するための検体調整方法。(1) a first step of immobilizing the specimen on non-magnetic particles having a sufficiently larger mass than the magnetically labeled antibody; a second step of causing an antigen-antibody reaction between the specimen and the magnetically labeled antibody; Carrying out a laser magnetic immunoassay method characterized in that the magnetically labeled specimen complex obtained in the second step and the unreacted magnetically labeled antibody remaining in the second step are separated by centrifugation. Sample preparation method for
れた既知の抗体と検体とを抗原抗体反応によって特異的
に結合させることを特徴とする請求項１記載のレーザ磁
気免疫測定方法を実施するための検体調整方法。(2) The laser magnetic immunoassay according to claim 1, wherein the first step involves specifically binding a known antibody immobilized on the surface of a non-magnetic particle to the specimen through an antigen-antibody reaction. Sample preparation method for carrying out the method.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63102912A JPH0750112B2 (en) | 1988-04-26 | 1988-04-26 | Sample preparation method for carrying out laser magnetic immunoassay method |
| US07/915,022 US5238810A (en) | 1986-09-22 | 1992-07-15 | Laser magnetic immunoassay method and apparatus thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63102912A JPH0750112B2 (en) | 1988-04-26 | 1988-04-26 | Sample preparation method for carrying out laser magnetic immunoassay method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01272968A true JPH01272968A (en) | 1989-10-31 |
| JPH0750112B2 JPH0750112B2 (en) | 1995-05-31 |
Family
ID=14340068
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63102912A Expired - Lifetime JPH0750112B2 (en) | 1986-09-22 | 1988-04-26 | Sample preparation method for carrying out laser magnetic immunoassay method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0750112B2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5236824A (en) * | 1988-04-26 | 1993-08-17 | Nippon Telegraph And Telephone Corporation | Laser magnetic immunoassay method and method by a magnetophoresis apparatus therefor |
| US5238811A (en) * | 1988-04-26 | 1993-08-24 | Nippon Telegraph And Telephone Corporation | Laser magnetic immunoassay method and apparatus therefor and superparamagnetic material-labeled body and method for the manufacture of same |
| JP2004512497A (en) * | 2000-06-23 | 2004-04-22 | ミナーヴァ・バイオテクノロジーズ・コーポレーション | Interaction of colloid-fixed species with species on non-colloidal structures |
| JP2004526124A (en) * | 2000-06-23 | 2004-08-26 | ミナーヴァ・バイオテクノロジーズ・コーポレーション | Rapid and sensitive detection of protein aggregation |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4115535A (en) * | 1977-06-22 | 1978-09-19 | General Electric Company | Diagnostic method employing a mixture of normally separable protein-coated particles |
| JPS57132060A (en) * | 1980-12-23 | 1982-08-16 | Boehringer Mannheim Gmbh | Peculiarly bonding protein and method of measuring one component in reaction of material able to be bonded thereto |
| JPS60152954A (en) * | 1984-01-20 | 1985-08-12 | Hitachi Ltd | Measurement of biological sample |
| JPS6379070A (en) * | 1986-09-22 | 1988-04-09 | Nippon Telegr & Teleph Corp <Ntt> | Laser magnetic immunoassay |
-
1988
- 1988-04-26 JP JP63102912A patent/JPH0750112B2/en not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4115535A (en) * | 1977-06-22 | 1978-09-19 | General Electric Company | Diagnostic method employing a mixture of normally separable protein-coated particles |
| JPS57132060A (en) * | 1980-12-23 | 1982-08-16 | Boehringer Mannheim Gmbh | Peculiarly bonding protein and method of measuring one component in reaction of material able to be bonded thereto |
| JPS60152954A (en) * | 1984-01-20 | 1985-08-12 | Hitachi Ltd | Measurement of biological sample |
| JPS6379070A (en) * | 1986-09-22 | 1988-04-09 | Nippon Telegr & Teleph Corp <Ntt> | Laser magnetic immunoassay |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5236824A (en) * | 1988-04-26 | 1993-08-17 | Nippon Telegraph And Telephone Corporation | Laser magnetic immunoassay method and method by a magnetophoresis apparatus therefor |
| US5238811A (en) * | 1988-04-26 | 1993-08-24 | Nippon Telegraph And Telephone Corporation | Laser magnetic immunoassay method and apparatus therefor and superparamagnetic material-labeled body and method for the manufacture of same |
| JP2004512497A (en) * | 2000-06-23 | 2004-04-22 | ミナーヴァ・バイオテクノロジーズ・コーポレーション | Interaction of colloid-fixed species with species on non-colloidal structures |
| JP2004526124A (en) * | 2000-06-23 | 2004-08-26 | ミナーヴァ・バイオテクノロジーズ・コーポレーション | Rapid and sensitive detection of protein aggregation |
| JP2015096855A (en) * | 2000-06-23 | 2015-05-21 | ミナーヴァ・バイオテクノロジーズ・コーポレーション | Rapid and sensitive detection of protein aggregation |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0750112B2 (en) | 1995-05-31 |
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