JPS5990056A - 免疫化学的定量方法 - Google Patents

免疫化学的定量方法

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JPS5990056A
JPS5990056A JP58165330A JP16533083A JPS5990056A JP S5990056 A JPS5990056 A JP S5990056A JP 58165330 A JP58165330 A JP 58165330A JP 16533083 A JP16533083 A JP 16533083A JP S5990056 A JPS5990056 A JP S5990056A
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antibody
capillary
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JPS6134097B2 (ja
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アンデルス・オロフ・グルツブ
ウ−ラ・クリスチナ・グラツド
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Syva Co
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    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/81—Tube, bottle, or dipstick

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Abstract

(57)【要約】本公報は電子出願前の出願データであるた
め要約のデータは記録されません。

Description

【発明の詳細な説明】 技術分野 本発明は免疫化学的定量用検査キットに関し。
更に詳しくは毛細管作用をなす多孔質担体物質を含む免
疫化学的定量用検査キットに関する。
発明の目的及び（芦成 本発明は臨床検査等において目的とする検査を免疫什学
的定量により実施するのに際し、所望の免疫イヒ学的定
ｔ’を迅速、簡便かつ安定にしかも高精度で行なうこと
ができる免疫化学的定量用検査キットを提供することを
目的とする。
本発明に従った免疫化学的定量用検査キットは。
抗体全結合せしめた多孔質担体物質から成る条片と、呈
色指示薬に結合させた抗体とを含んで成る。
本発明に従った免疫化学的定量用検査キットの抗体を結
合せしめた多孔質担体物質は、好ますくは、ポリ塩化ビ
ニル、趨イヒビエループロピレン共重合体又は塩化ビニ
ル−酢酸ビニル共重合体のマトリックス中に実質上均一
に埋封されたシリカ微粉から成る。
本発明の臨床検査材は、抗体を結合させた毛細管の作用
をなす多孔質担体から成り、例えば吸着または臭化シア
ンあるいはダルタールアルデヒドのような物質により公
知の方法に、Ｊ：す共有結合で抗体に結合させた多孔η
の毛細管の作用を利用することを特徴とするものである
。
ｒ！ｌ！ら、本発明１１で従、えば、定量し、ようとす
る抗原を含む試別を、毛細管の作用をなシしめる吊片に
浸透せしめたのち、吊片の抗原を含有する部分は、螢光
化合物又は発色反応を触媒する酵素のような適肖な呈色
指示薬に結合され、た抗体を添加１−ることによりＹａ
色させて検出するものである。
従来の免疫化学的定量法１ｄ、例えばマンジー二法やオ
ーテヤロニー法のような免疫拡散法あるいはローレルの
免疫電気泳動法によるものであるが、これらの方法（て
おいてζは、その変法も含めて拡散や電気泳動過程によ
って抗原と抗体との相互作用全必要とするが１本発明の
場合は、抗体を結合させた多孔質担体物質の毛細管の作
用によって抗原と杭体の相互作用をなさしめるものであ
る。
本発明の実施のための毛細管作用盆する物質としては、
濾紙、クロマトグラフ用濾紙、イオン交換組、セルロー
スアセテート膜、セルロースアセテートディスク、セル
ロース薄層クロマトグラフ用ディスク等のようなセルロ
ース繊維を含む物質や、デンプン、セファデックス（三
次元構造の網状組織又はエピクロルヒドリンと交さ結合
したデキストラン句のマトリ、クス、ファルマシアファ
インケミカルス社、ウプサラ、スウェーデン１１Ｍ）の
ような物質や、ポリ塩化ビニル、塩化ビニル−プロピレ
ン共重合体％塩化ビニルー酢酸ビニル共重合体のような
プラスチック物質、セラミック物ａおよびポリ塩化ビニ
ル・ソリ力を結合させたものが利用できるａ′ｉｉ：た
本発明において用いられる抗体は、公知の免疫法に用い
られるものである。
実施例 以下に本発明の実施例を示す。
臨床検査材の製法 例１　毛糾１管＃Ａ質における臭什シアンによる抗体の
不溶化 セルロース含有物Ｎ１０７を、蒸留水に５分間浸し、こ
れに３００　ｍｌの蒸留水に臭什７アン８２を溶かした
溶液を加え、１規定苛性ンーダでｐＨ’ｔ１０．５に調
整する。このｐＨ値に保つことにょシ反応は２０分間で
完結するから、溶招（を祷で、更にセルロース物質を２
ｔの０．００５モル炭酸水素す）　ＩＪウムで洗浄し、
洗浄液を除去したのち、そのセルロース物ａを、０．１
モル炭酸水素ナトリウム１１１ｍ／ＩＣ５００■の抗ヒ
トガンマグロブリン（ＩｒＧ、　Ｆｃ　　分画）家兎ガ
ンマグロブリン含有浴Ｗｌ中４℃で１夜据とうし、四に
０．　ＯＣＪ　５モルエタノールアミン溶液１００　ｍ
ｔ金用いて３時ｍ１振とうし、しかるＰＫｏ、５モル炭
酸水素ナトリウム５００ｍ１．次にｐ）Ｊ　４．０の０
．１モル酢酸緩衝ｆ（ｆ、　２００　ｍｅで洗浄して４
０．３％アルブミンを加えた０、０７５モルバルビッー
ル酸ナトリウムＨ％　Ｎ　５００　ｍｅ　中に保存すλ
）。この保存湛度は４℃とする。
例２　毛却１管物質におけるゲルタールアルデヒドによ
る抗体の不溶化 七ルロースアセテート膜０．６　ｆｉｔ　ｆ、　ｐＨ６
，８（７）０．１モルリン酸緩衝ｆｊ　１０　ｍｌ中に
抗ヒトガンマグｏ　７’　ＩＪ　ｙ　（ＩｆＧ、　Ｆｃ
分画〕家兎ガンマグロブリン１００■を溶解した溶液中
で３０分間振とうし、その溶ｆｆ’（ｒ捨てたのち、膜
を１％ゲルタールアルデヒド溶ｎ１０　ｍｅ中で更に３
０分分間上うし、ついで１．０モルメチルアミン１０ｒ
ｎＡ中で１５分間振とうする。しかる後に膜を１００ｍ
／のバルビッール酸ナトリウムで洗浄し、これを同じ緩
衝液に、０．３πアルブばンを加えて、その溶液中にお
いて４℃で保存→・る。
例３　毛細管担体における吸着による抗体の不溶化 ポリ塩化ビニル・シリカ（ミクロボーラスプラスチック
シート−アメリカエスナコーボレーンＢン社１１）１０
ｆｉ、ｐｌ−１６，８（７）　０．１　モルリン酸緩＠
ｇ１００−に、抗ヒトガンマグロブリン（ＩｙＧ。
Ｆｃ分画）家兎ガンマグロブリン５００■を含む溶液中
において４℃で１夜振とうし、完全に反応さぜたのち、
目的物を　ｐＨＺＯにリン酸で緩衝化された生理食塩液
１ｔを用いて洗浄し、これを濾紙に挾んで乾燥させる。
例４　螢光標識抗体の判決 （ａ）抗ヒトガンマグロブリン（、ＩＪＧ、　　Ｆｃ分
画）家兎ガンマグロブリン７２０　ｍｙ　、　ｔｇ化ナ
トリウムＱ、５７　Ｆ　、炭酸水素ナトリウム０．２５
９Ｆおよび炭酸ナトリウ・ム・１０水和物０．０４５　
ｆを蒸留水７２ｍｅＦＣ（ＩＩ解し、反応溶液を水浴中
で冷力１し、攪拌シながらフロ１／ツ七インインチオシ
アネート３６■を力（１乏−１さらｆ溶液を攪拌しなが
ら１８時時間割１−する。その混合物を透析溶液が螢光
を失う壕で、リン酸で緩衝什されたｐＨＺＯの生理食塩
液に対して透析し、イ尋らねた標識抗体を凍結して保存
する。
（１））抗ヒトガンマグロブリン（ＩｒＧ、　　Ｉｉ’
ｃ分画）家兎ガンマグロブリン７２０ｑ、塩化ナトリウ
ムＱ、　５７　Ｆ　、炭酸水素ナトリウムｃ＋、　２５
９　ｆおＪ：び炭酸ナトリウム・１０水利物０．０４９
　ｆを７２−の蒸留水（（溶解し、反応溶液を水浴で冷
却し、こり、全攪拌しながら、ロダミン９巧′ｆ：溶解
したアセトン２　ｍＩ！全これに加え、その溶液を１８
時間冷却する。こねを透析溶液が螢光を失うまで、リン
酸でｐＨＺＯに緩？Ｒｊ什された生理食塩液に対して透
析する。このようにして得られた標識抗体を凍結して保
存する。
（Ｃ）抗ヒトガンマグロブリン（ＩＩＦ（］、　　Ｆｃ
分画〕家兎ガンマグロブリン７２０■、塩化ナトリウム
０、５７　ｆ　、炭酸水素ナトリウムＱ、　２５９　Ｆ
および炭酸ナトリウム・１０水和物０．０４９Ｆ牙、７
２ｍｅの蒸留水に溶解し、その反応液を水浴で冷却し、
これを攪拌しつ＼１８時間冷加し１、［２かる後に。
その混合物音、リン酸で緩衝什されたｐＩＪ７．０の生
理食塩液に対して透析液の螢光がなくなる迄、透析する
。斯し１得られた標識抗体を凍結保存する。
例５　　ＬＤＩ：Ｉアイン酵累Ｈ４欄識抗体の製法硫酸
アンモニウムで沈澱さぜたＬ　Ｄ　ＩＡアイン酵素１−
１４４．５　ｍｙ　（７）　懸濁液ヲ、ｐＨ６，８ノｏ
、　１　モルリン酸緩衝液で透析し、攪拌しつ＼内液に
抗ヒトガンマグロブリン（１ｆｃ１．　　Ｆｃ分画〕家
兎ガンマグロブリン２．２５■を加え１．リン酸緩衝液
で最終迩ｆ　１．５５　ｍｅとし、全ての家兎ガンマグ
ロブリンが溶解したのち、これに０．１９にゲルタール
アルデヒドｎ　Ｒ４５メｔ　１を流加し、その反応ｉを
室温中で２時間放置し、更にその混合物をバルビッール
酸ナトリウム緩衝液に対して透析し、得られた標識体を
４℃において保存する。而してこｈ’、−使用中ろ場合
には、２腎アルプばンを加えたバルビッール酸緩衝液で
希釈するのである。
例６　不溶化の点検 前述の例に従って抗体と処理した物質の２つの条片と、
未処理の２つの条片とを試験に用いる。
即ち、抗体処理条片１つおよび未処理条片１つを、バル
ビッール酸緩Ｆｉ＃１　ｍｅ当りヒトガンマグロブリン
（Ｉｆｆ）ｉｍｇの溶液に浸漬する。つぎに４つの条片
ｋ　０．１５モル塩化ナトリウムを含むバルビッール酸
１．′ｅ衝７ｉ！２０ゴにより１０分間洗浄したのチ、
戻にそれら全フロレツセインインテオゾアネート柄識抗
体中７１１０分間振とりする。洗浄をくり返シ２７大の
ち、ぐ１片を乾燥し、ろ４０　ｎｍの波長の紫外貌で検
査し、ＩｙＧ溶液で撮とうした不溶化抗体を１澄し、た
条片にのみ螢光が現われることｔ確館する。（（ＩＪ　
３の方法により槽造した小片？検査する場合には、ウジ
アルブミンで処理した条片を対照と１−２て用いる。
免疫化学的定端法 Ｖ；１１７ 本発明によって製造した臨宋悼査材を、免疫化学約定゛
陛として診断の目的のために用いる場合には次の方法に
よる。
即ち、１〜３ｔＩｔの既知容隋の炒検物質と、既知附の
抗原を含む相応最の標準溶液およびそｆ′Ｌ？：腫々希
釈したものを、同じく担体物質上に付着する。この場合
、移動相が毛細管内を移行し易いように、例えば０．３
％アルブミンを添加した０、０７５モルバルビッール酌
ナトリウム緩衝ｆｊヲ用い１毛細管による滲透作用は、
開放状態または密閉試験容器中で行わせる。その滲透は
上向オたけ下向で差支えないが、特に密閉容器と上向浸
透の組み合わせが望ましい。毛細管の浸透が開始した後
の適当な時期に、湿った担体物質金、フロレツセ１ンイ
ンチオシアネート寸たは乳酸デヒドロゲナーゼ（ＬＬ”
ｌＨ）標識抗体ケ含む溶液に移すのであるが、この屑籠
中での振とり時間は１０分間稈背が望まし７い。振とり
後、斜体物ＪＲを３７℃の流水で５分間程［Ｗ洗浄し、
更ＫＯ９２％アルブεンと０．１５モル塩塩化ナトリウ
ム含む’２０ｍ１のバルビッール酸すトリウｌ−プ備４
ダマ”５分１１）ｉずつ２回１１４とう−する。
ｌｒ’＋ゴ木の移小１１］？巨斧（を泪１巧ドするメと
めに、り（少ｒわ′［−イ末が用いら）１イ）／バ、そ
の観察全ジ４０　ｎｍの紫外線で行う。？’「Ｌ７、Ｊ
、　ｌ）　ＩＩ　Ｊブ１″体〃：飛が用いられる場合６
τ＆；ｌ：、４１コ休物質ｔｔ、＋−１−；ｉ＋　：の
組成の染色浴で処理−する、。
即ち、ニトロブルーテトラゾリウム３０　＋、ｙ　、ｌ
：　ＮＡ１）４０１ｌｖｔ　　ｐＨ７，４の［］、０５
モルトリス緩留１液留６１ｙｅ　ｉτ汁γｙ１了し、こ
のｉｌｌ　＆看ろ、６モル乳序ジリチウム１．６０ｍ１
　、　Ｑ、　０６モルシアン什カルンウム５　ｒｅおよ
びｖ才）ンＬ７）７７チルフアナソ゛リウムメトサルフ
エート１．２１ｎ／の９　ｉ：　７ｉぐｋｎ　Ｐ　ＩＷ
しｔｒ−Ｆ　ｙｉ　ｔ　ｙＪｎ　、ｔ−１ろ７て：の染
色浴中て１毛細管物ノＰｊ　Ｋ明１１Ｑ−Ｇ色彩ノ５：
律５察さノ１．るプ°で担と分子＜’−＞　、色彩の顕
色１．Ｊ、乳酸−トＮΔ■）ニビルビン１い’　十ＮＡ
Ｉ）＋１２　　の反応に、１１１月１７〉：触〃１シと
し１作月Ｊ　ｊ、　、　　ＮＡ　Ｉ）ＩＪ２　　が定゛
田：　ｌ′１４１　ＫニトロブルーテトラゾリウムをＳ
Ｘｖ元ｔ、−Ｃ，、不溶イｔ１′：の淡紫［′（−々素
ケ作るためである。
臨庄梓査利の＋ｒ原で抜色さシ１だ部分の呈色にし、ネ
ルわｔ物質のシフ１原の含何隣が増す０でつれて強ＩＷ
金増寸。（名子に結合さｊｉ−に抗体分子は１毛細ｖＫ
′よる琶透中に交換反応によって松原の移動を遅延させ
る。抗原の温間が高くなるに従って、抗体に結合されて
いる抗原分子の平均移動時ｒ！４１が減少ゴるために、
遅延効果ガ少なくなるのである。
例８ 例えば本発明によって製造≦］また毛細管作用をなす抗
体結合相体物質の榮片を％被検物質の少城の溶液Ｋ　３
　ｍｍの深さに浸し１毛細管の浸透を開始させて３〜２
０分間続けた後、φ片ｒ溶腋から引上げて、抗原の移動
距咄を例７において述べた要領で測定する。
こ＼して１ｒＪ一部の実柳例について説明したが、この
実施例は抑々の４ｑ価を変更や＠俟が可能である。
木登１４ｎＶＣおいて使用する臨床検査ゼは多孔性（，
１２＋１ち１毛細？ｇの作用をな−ｆ　）　４Ｆ３休ｌ
吻質の条片よりなる、「糸片１という用語１ｊ：　Ｊ、
？味のあるろ細や、膜状又Ｇ・１円板状、しＷにｒｄ薄
い５Ｆたい棹状のもの等を広ノア１／１′含むものであ
る。
「担体」という用１悟は抗体に対し例えば吸着或いは共
有結合で結合しうる物価を意味するもので、らｌ　：、
）ｌ’、　／７−１物？コＸｙ限るもので妙ない。
イ列６のり孔＋′（゛ポリマーマトリックスに西２置さ
）１ブ軌均−しこ８１）１０什式ヲまたシリカを有→°
るＩＪ＜’ｆｌビニールのミクロポーラスプラスグー、
ツクシート４；ｔ：他の適当な浸透性又（１名（けｊ、
σ）、同様にシリカ微粉が埋１、ｔ−ｇねた（ＩＡ什ヒ
ビニルプロピレン共重合体或いは塩什ビニルー酊酔ビニ
ル共重合体等の連続する重合マトリックスに代えてもよ
い、ユ又そのミクロポーラスプラスチ、クソートにおき
代オ、られる。ミクロポーラスシートの孔の直径はり、
　１〜１ミクロンである。又全体の孔のηｒ］嘗クロり
−ラスフ゛ラスグックゾートの１グラムあたり１．１〜
１．８ミ１）リットルである〇 ／ｉ？凶出願出 願人　カンノ二二一 ’ｌ’ｌ’ｉｉ’ｌ’　１．１．ｌ偵代４理人弁理士　
　宵　　木　　　　　朗 弁理士　　西　　舘　　１１１　　　之−弁理士　　石
　　　１）　　　　　　敬弁理士　　山　　口　　昭　
　之

Claims (1)

  1. 【特許請求の範囲】 １、抗体を結合せしめた多孔質担体物質から成る条片と
    、呈色指示薬に結合させた抗体とを含んで成る免疫化学
    的定量用検査キット。 ２、前記抗体を結合せしめた多孔質担体物質がポリ塩化
    ビニル、塩化ビニループロピレン共重合体又は塩化ビニ
    ル−酢酸ビニル共重合体の連続マトリックス中に埋封さ
    れたシリカ微粉から成る毛細管含有担体物質である特許
    請求の範囲第１項記載のキット。
JP58165330A 1975-01-27 1983-09-09 免疫化学的定量方法 Granted JPS5990056A (ja)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE7500841A SE388694B (sv) 1975-01-27 1975-01-27 Sett att pavisa ett antigen exv i prov av kroppvetskor, med utnyttjande av till porost berarmaterial bundna eller adsorberande antikroppar
SE75008417 1975-01-27

Publications (2)

Publication Number Publication Date
JPS5990056A true JPS5990056A (ja) 1984-05-24
JPS6134097B2 JPS6134097B2 (ja) 1986-08-06

Family

ID=20323501

Family Applications (2)

Application Number Title Priority Date Filing Date
JP51007875A Pending JPS51101122A (ja) 1975-01-27 1976-01-27
JP58165330A Granted JPS5990056A (ja) 1975-01-27 1983-09-09 免疫化学的定量方法

Family Applications Before (1)

Application Number Title Priority Date Filing Date
JP51007875A Pending JPS51101122A (ja) 1975-01-27 1976-01-27

Country Status (18)

Country Link
US (1) US4168146A (ja)
JP (2) JPS51101122A (ja)
AT (1) AT347601B (ja)
AU (1) AU508642B2 (ja)
BE (1) BE837896A (ja)
CA (1) CA1074228A (ja)
DE (1) DE2603004C2 (ja)
DK (1) DK149968C (ja)
FI (1) FI760074A7 (ja)
FR (1) FR2298798A1 (ja)
GB (1) GB1502563A (ja)
IL (1) IL48722A (ja)
IT (1) IT1123602B (ja)
NL (1) NL7600128A (ja)
NO (1) NO760233L (ja)
NZ (1) NZ179830A (ja)
SE (1) SE388694B (ja)
ZA (1) ZA76421B (ja)

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US4168146A (en) 1979-09-18
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AT347601B (de) 1979-01-10
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IL48722A (en) 1978-07-31
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DK29176A (da) 1976-07-28
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FR2298798A1 (fr) 1976-08-20
IT1123602B (it) 1986-04-30

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