JPH0251151B2 - - Google Patents
Info
- Publication number
- JPH0251151B2 JPH0251151B2 JP57058612A JP5861282A JPH0251151B2 JP H0251151 B2 JPH0251151 B2 JP H0251151B2 JP 57058612 A JP57058612 A JP 57058612A JP 5861282 A JP5861282 A JP 5861282A JP H0251151 B2 JPH0251151 B2 JP H0251151B2
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- component
- solid phase
- measured
- receptor
- 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
Links
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- 239000004395 L-leucine Substances 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
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- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
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- 239000011574 phosphorus Substances 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
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- 229940034208 thyroxine Drugs 0.000 description 1
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】
本発明は、反応媒体の表面積を拡大せしめる媒
体用エレメント担体を用いてなる微量の生体成分
の多成分同時測定法に関する。従来より、抗原
(パプテンを含む)と抗体とに基く免疫反応や薬
物やホルモン等の物質とレセプターとに基くレセ
プター反応など(以下、免疫反応と略す)により
定量的に測定する手段は数多く知られている。
このような方法としては、主として血清、尿、
唾液などの体液中に微量に存在する成分の定量、
定性などに応用されてきた。
微量の成分としては、生体内成分としての種々
のホルモン類、それらに対するレセプター、生体
内酵素、病態における特異的成分、投与された薬
物やそれらに対するレセプター、さらにこれらの
代謝物、その他、細菌やウイルスなどの微生物、
これらに対する抗体など種々の生体内に存在する
微量成分が挙げられる。
そのような微量成分は、主として抗原抗体反応
などの免疫反応、例えばホルモンとそれに対する
抗体またはレセプターを応用して測定されてい
る。
免疫反応の結果、それらの結合物が不溶性にな
つたり、多数の抗原−抗体分子からなる大きな集
合体が形成されることを利用する方法は、すでに
古典的な方法として知られている。
これらは反応管内に沈澱する量を直接測定する
方法や、ゲル内で沈降される免疫拡散法、あるい
は補体を用いる方法などの種々の方法により測定
されている。また、抗原、抗体やレセプターを何
んらかの方法で標識しておき、免疫反応せしめた
後その標識活性を測定する種々の方法もよく知ら
れている。これらの内でも高感度測定法として広
く用いられる方法に放射性物質を標識物質とする
ラジオイミユノアツセイ(RIA)がよく知られて
おり、また、RIAは放射性物質を利用するもので
あるがより安全な代りうる方法としてこの放射性
物質標識の代りに酵素で標識したものを用いる測
定法、即ち酵素免疫測定法(EIA)が近年広く利
用されている。
EIAは、抗原または、抗体やレセプターなどの
受容体に酵素を結合させておき、その酵素活性を
標識として免疫反応の程度を測定し、その結果か
ら抗原または受容体の量を測定しようとするもの
である。このような場合、測定すべき成分に対し
て特異的結合能を有する受容体または抗原を用
い、これに測定すべき成分としての抗原または受
容体と酵素標識した抗原または受容体を競争反応
せしめ、次いで免疫反応によつて生じた結合物と
非結合物とを分離し、または分離することなくそ
の酵素活性を測定する場合や、測定すべき物質に
対して特異的結合能を有する受容体または抗原を
用い、これに測定すべき抗原または受容体を反応
せしめ、次いでその結合物に酵素標識した抗原に
対して結合能を有する化合物または受容体に対し
て結合能を有する化合物を反応せしめてサンドイ
ツチせしめその後、その酵素活性を測定する場合
などがある。
このような種々の免疫反応を利用するに当つ
て、従来では、測定すべき成分の1つに対して1
つの反応を行なつているにすぎず、多種の成分を
測定するには非常の多くの欠点があつた。即ち、
多種成分測定に当つては、目的とする成分の個数
相当分の血清等の多量の試料を必要とし、かつ各
測定を各々行なうために用いる試薬類も多種多様
のものとなり、さらに長時間を要するものであつ
た。これに対し、担体に2種の抗体を固定化した
固相体を用い、これに測定すべき2種類の抗原を
含む試料を加えて反応せしめ、まずその間の1種
の抗原の標識化合物を反応せしめ、その標識活性
を測定し、次いでその後、他の1種の抗原の標識
化合物を反応せしめ、この標識活性を測定してな
る方法(特開昭56−78598号同52−15815号、同56
−96248号参照)が知られている。これらの測定
法では、従来から固相体として使用されていたビ
ーズと試験管を抗体として利用するか、区別し得
る種々の粒子径の微粒子を担体として利用する
か、またはビーズや微粒子の代わりに複数のフイ
ンを有する担体を用いて、この担体上に2種また
はそれ以上の抗体を固定化せしめた固相体を用い
るために、微量成分を極めて少量の試料を対象と
して反応せしめることが困難であり、かつこの固
定化された抗体の力価の算出が極めて困難であ
り、また測定に当つても、各々の成分を順次測定
するための反応を要することから、従来に比べ、
より長時間の測定時間を必要とする欠点を生じる
ものであつた。
本発明者らは、生体内の種々の微量成分を、簡
便かつ短時間に正確に多成分にわたつて同時測定
し得る方法について研究開発したものであつて、
その目的とするところは免疫反応のための反応管
を1つの担体とし、かつ免疫反応の反応媒体に加
えられる担体として、その担体が反応管の免疫反
応部に挿入可能な形状であつて、外径が反応管の
内径と僅かな間〓を保つように形成され、反応管
とエレメント担体と間〓が同一となるように形成
されたもので、反応媒体の表面を拡大せしめる媒
体用エレメント担体であつて、この担体を1以上
の担体として用いることにより、少量の試料を対
象として反応せしめるに当たり、媒体用エレメン
ト担体により微少量の試料液を含む反応媒体の表
面積を拡大せしめながら反応せしめる得ることが
できるもので、微少量の試料液にて測定でき、簡
便で、良好な多成分同時測定法を提供するもので
ある。
本発明は、EIAおよびRIAにも応用でき、また
競争反応やサンドイツチ免疫反応にも応用できる
ものである。まず本発明に使用される担体の1つ
の反応管としては、従来より種々のEIAやRIAに
おいて用いられる免疫反応用の反応試験管等のも
のであればよく、例えば、ガラス製やプラスチツ
ク製のものが任意の形にて用いられているもので
ある。また、エレメント担体としては、その担体
が反応管の免疫反応部に挿入可能な形状であつ
て、外径が反応管の内径と僅かな間〓を保つよう
に形成され、反応管とエレメント担体と間〓が同
一となるように形成されたもので、反応媒体の表
面積を拡大せしめる媒体用エレメント担体であつ
て、適宜1以上の分割可能な形状であればよく、
また免疫反応の際、測定すべき成分と特異的結合
能を有してなる受容体または抗原を固定化し得る
もので、前記反応管内に加える形状のものであれ
ばよく、通常、ガラス製、不溶性多糖類、不溶性
蛋白質など種々のものが、粒状、板状、弾丸状、
槍形状などの形状にて用いられる。
本発明で使用されるこのような担体としては、
一般のEIAやRIAに用いられる不溶性担体が使用
される。特に抗体や抗体のフラグメントである
Fab′などを不溶化するに当つては、一般的に云
つて不溶性担体に結合させる対象が抗体蛋白質で
あるから、酵素蛋白質を固定化する固定化酵素の
製造の場合に用いられる担体の様なものが使用さ
れる。この担体としては、例えばグルタルアルデ
ヒドなどにて処理してなるアルブミンやゼラチン
などの不溶性蛋白質担体、アガロース、セルロー
スやデキストリンなどのエピクロルヒドリン処理
または臭化シアン処理、さらにそれらのアミノ化
試薬処理してなる不溶性半合成高分子担体(多糖
類)、アクリロニトリル、アクリル酸、アクリル
酸エステル、メタアクリル酸、メチルメタアクリ
ル酸、ビニルアルコール、酢酸ビニル、スチレ
ン、アミノスチレン、ジビニルベンゼン、アクリ
ルアミド、エチレン、無水マレイン酸、クロトン
酸などのポリマーまたはコポリマー、さらにそれ
らのアミノ化試薬処理してなる不溶性合成高分子
担体(高分子樹脂)、その他アミノ化試薬処理し
てなるシラン化合物などの不溶性無機担体などの
官能基に基いて結合せしめて使用してもよい。ま
たこの不溶性担体にアミノ基などを導入する手段
としては公知の種々の手段を用いればよく、例え
ばニトリル基の還元によるアミノ化、水酸基のγ
−アミノプロピルトリエトキシシランの反応によ
るアミノアルキル基の導入、またはヒドロキシメ
チル基の過ヨウ素酸酸化によるアルデヒド基への
変換、さらにそのアルデヒド基にジアミンを反応
せしめてなるアミノ基の導入、多糖類水酸基の臭
化シアン反応によるイミドカルボナート基への変
換、アミド基のハロゲン化リン化合物によるイミ
ノハロゲニド基への変換、さらにこの官能基を官
能基導入試薬にて処理して新たに官能基を適宜導
入してもよい。これらの不溶性担体において、ナ
イロン、ポリスチレン、ポリアクリルアミド、塩
化ビニル等の高分子樹脂、セルロース、デキスト
ランなどの不溶化多糖体、ガラス等の無機材質等
が加工するのに好適なものとして例示される。こ
のような担体におけるエレメント担体は、弾丸状
あるいは槍形状その他これに類する形状のもので
もよいが、他の固体成形物例えば金属、プラスチ
ツクの表面に本発明のエレメントの形状が保持さ
れるように付着形成されたもので、用いる免疫反
応管と同形状の反応媒体の表面積を拡大せしめる
ための媒体用エレメントが好ましい。
次に本発明のエレメント担体について図面を参
照しながら説明する。
第1図はエレメント担体としての媒体用エレメ
ント担体を符号1として総括的に示してある。エ
レメント担体1は、例えばナイロン6,6の高分
子樹脂材料から成り、エレメント本体2と該本体
2の上面部2aの中央部に突出して設けられた取
付脚3とから構成されている。取付脚3には、他
の操作棒(図示せず)が取付けられるが、取付脚
3自体を操作棒として使用するために延長させた
形状であつてもよい。エレメント本体2は弾丸状
に成型されていて、その外径は反応管4の内径と
僅かな隙間を保つ様な径に形成してある。エレメ
ント本体2の下周面の頂部にはクサビ状の突起5
が設けてあつて、該突起5により、エレメント本
体2の弧状外周面2bと反応管4との弧状内周面
4aとの間に一定の間隙部6が保持されているよ
うになつている。また、このクサビ状の突起5の
代わりに凸状の突起としてもよい。要は突起5に
より、エレメント本体2が反応管4の弧状内周面
4aと密着状態となることを防止し得る形状であ
ればよい。この突起部の高さとしては、エレメン
ト本体2と反応管4の管内壁面との幅とが同一と
なればよい。エレメント本体2の上面部2aに
は、第2図に示すように、取付脚3を中心にして
四角形状に切り込み溝8が付設してあつてもよ
い。切り込み溝8の終端はエレメント本体2の周
縁部に開口させてあつて、上面部2aの延長線上
での反応液の表面張力を弱めるように考慮してあ
る。またこの切り込み溝8の溝形状としては、断
面形状が半円状、楕円状、台形状、三角形状、四
角形状などのいずれの形状であつてもよい。
本発明のエレメント担体は、上記の様な構造で
あるため、反応管に入れた反応液中にエレメント
本体を浸漬させれば、エレメント本体の突起が反
応管の内底面に接触してエレメント本体の外周面
とそれに対応する反応管の内周面との間に僅かな
隙間が形成される(第1図参照)。したがつて、
反応液量が少量であつてもエレメント本体の浸漬
によつて、その液が隙間部に案内されるとともに
エレメント本体の上面部にまで上昇された反応液
は切り込み溝内を流れてそれ自体の表面張力作用
を失う。それ故、反応液はエレメント本体の外周
面に均一な条件(一定厚の層の形態)で接触する
ことになる。
上記の様な切り込み溝8の変形例としては、第
3図イに示すように、エレメント本体2の上面部
2aの半径方向に放射状に配設してもよい。ま
た、他の変形例としては、第3図ロに示すよう
に、エレメント本体2の上面部2aの直径方向に
直線状に配設してもよい。更に他の変形例として
は、第3図ハに示すように、エレメント本体2の
上面部2aの円周方向に左右対称の形に配設して
もよい。このように、切り込み溝8は、その終端
が上面部2aの周縁部に開口されている形状であ
ればよく、また如何なる形状に付設することもで
きる。
第4図はエレメント1の他の変形例を示したも
のである。この例におけるエレメント本体2は槍
形状に成形してある。したがつて、反応管4もエ
レメント本体2に沿うテーパー形状のものが使用
されるもので、種々市販の反応管の形状に従つ
て、そのエレメントを変形、加工すればよい。な
お、エレメント本体2のその他の構造について
は、前記第1図で示した例のものと同じであるた
め、それと構造を同じくする部分には同じ番号を
付し、説明は省略する。
さらに上述の如く、少量の反応液の表面積の増
大を兼用することなく複数の免疫反応を行なわせ
しめるには、複数のエレメントを板状または棒状
の形状になした担体を固相体として用いればよ
い。
この様な反応管やエレメント等の担体に通常公
知の手段を適用して化学的または物理的に抗体ま
たはそのフラグメントもしくは抗原、その他の薬
物の受容体を結合して不溶性抗体等(抗原、受容
体)となし、固相体を調製する。
固相体の調製法としては、前記担体にあらかじ
め抗体等を結合せしめるための官能基を導入して
特定の形状に加工してもよく、または加工した後
に官能基を導入してもよいもので、例えば反応管
やエレメントの担体がナイロン6,6の場合は、
あらかじめ前述の形状に加工し、次いでジメチル
硫酸あるいはイミクロ法による活性化をおこな
い、へキサメチレンジアミン処理、グルタルアル
デヒド処理をおこなつて、抗体等例えば第2抗体
を結合させ、更に第1抗体を免疫反応により固定
化して固相体を得ればよい。また、抗体がデキス
トランゲル、例えばセフアデツクスの場合も、あ
らかじめ前述の形状に加工し、次いでブブロムシ
アン(BrCN)による活性化をおこない、これに
抗体を結合させて固定化する。更にまた、ポリス
チレン等の場合加工後、硫酸で化学的処理した後
固定化するか、または物理的吸着による固定化を
おこなえばよい。またガラス等の場合はγ−アミ
ノアルキルトリエトキシシラン化合物などの有機
アミノ基導入シラン試薬によりアミノアルキルシ
リル化せしめ、このアミノ基に基いて公知の固定
化手段により固定化せしめればよい。さらに一般
に化学的に固定化するに当つては、担体の分子内
の官能基または導入された官能基と、抗体等の分
子内に有する官能基、例えばアミノ基、水酸基、
カルボキシル基、チオール基、アルデヒド基等の
官能基に基いて両者を結合し得る多官能性化合物
が用いられる。この公知の種々の多官能性化合物
としては、例えばヘキサメチレンジイソシアナー
ト、2,4−トルエンジイソシアナートなどのジ
イソシアナート化合物、ヘキサメチレンジイソチ
オシアナートなどのジイソチオシアナート、スク
シンアルデヒド、グルタルアルデヒド、アジポア
ルデヒドなどのジアルデヒド、N,N′−エチレ
ンビスマレイミド、N,N′−0−フエニレンジ
マレイミド、ビビスジアゾベンジン、N,N′−
ポリメチレンビスヨードアセトアミド、ジエチレ
ンマロンイミデート、ジメチルアジピンイミデー
ト、3−(2′−ベンゾチアゾリルージチオ)−プロ
ピオン酸、3−(2′−ピリジル−N−オキサイド
−ジチオ)プロピオン酸、6−N〔3−(2′−ベン
ゾチアゾリル−ジチオ)プロピオニル〕カプロン
酸などのスルフイドカルボン酸類またはそのスク
シンイミドエステル、p−ニトロフエニルエステ
ル、酸クロライド、イミデートなどの反応性誘導
体(特願昭53−85900号参照)、マレイミド安息香
酸、マレイミドフエニル酢酸、マレイミドフエニ
ルプロピオン酸などのマレイミドカルボン酸類ま
たはその反応性誘導体などが挙られる。
またこの多官能性化合物は、担体に官能基を導
入するための官能基導入試薬としても使用できる
ものである。
さらにまた、一般的に固相体を得るに当つて
は、例えばPH6〜8の緩衝液やアセトン、メタノ
ール、エタノール、ジオキサン、ジメチルスルホ
キサイド、ジメチルアセトアミド、テトラヒドロ
フランまたはそれらの混合媒体などの不活性媒体
を用いて、冷却または室温にて、担体と多官能性
化合物を反応せしめる。その後必要に応じて洗浄
し、さらに同様の不活性媒体を用いて、さらに抗
体等を加え反応せしめて不溶性担体と抗体等を反
応せしめる。
さらに第2抗体を用いて第1抗体を結合せしめ
てなる抗体−第2抗体−第1抗体結合物を得るに
当つては、例えば目的の形状に加工した抗体を用
いて、上述の如くの固相体を得る手段に基いて、
まず第2抗体を結合せしめる。次いでこれに、第
1抗体を加えて免疫反応に基いて結合せしめれば
よく、このようにして得られた第1抗体の免疫活
性はほとんど劣化されないために特に好ましい固
相体である。
このようにして得られた固相体において、固相
体の2以上を用いるに当つては、例えば反応管固
相体と、1つのエレメント固相体または2以上に
分別可能なエレメント固相体との組合せ、また
は、2以上に分別可能なエレメント固相体の複数
組合せである。特に2以上に分別可能なエレメン
ト固相体としては、例えば第1図に示すエレメン
ト担体の複数を用い、その1つのエレメントに目
的とする抗体等を固定化し、さらに別のエレメン
トには異なる目的の抗体等を固定化して、複数の
異なる抗体等を固定化した固相体を得る。次いで
これらの固相体は、各々の大きさに切断する。例
えば2種のエレメント固相体とする際は、組合せ
た結果において1つのエレメントを構成するもの
であれば適当な大きさにて切断してよく簡単には
各々対称的な半分の大きさに切断したフラグメン
トとすればよく、また面者の濃度が異なる場合に
は、その濃度に準じた比率によつて切断した各フ
ラグメントを用いてエレメントを構成せしめても
よく、例えば1/4の大きさのエレメントフラグメ
ントと3/4の大きさのエレメントフラグメントと
を用いて低濃度部は3/4のフラグメントとし、高
濃度部は1/4のフラグメントとなして接着せしめ
てエレメントとなせばよい。また3種以上のエレ
メント固相体とする際は、各々その数に応じて切
断したフラグメントとすればよく、次いでこれら
のフラグメントを合せて元の形状のエレメントと
して用いればよい。またエレメント固相体のフラ
グメントを組合せるに当つては、単にエレメント
本体の切断面に従つて分別可能な形状として一体
化してもよく、また、各々のエレメントのフラグ
メントの切断面の接合部位にエレメント本体と反
応管との間隙距離を保持せしめるための接合部位
をもつて各フラグメントにより一体化せしめても
よい。
特に、後者の際には、あらかじめエレメント本
体のフラグメントを担体として用い、これに抗体
等を固定化せしめて固相体となすことが好まし
い。さらに前述の複数の板状または棒状のエレメ
ントによる分別可能な担体からなる固相体を用い
るに当つても、各担体の表面全体を固相体として
利用可能なようにその全面に抗体等を固定化せし
めることが好ましく、また各々の固相体において
も接触近辺部位に、間隙距離を保持せしめるため
の接合部位を形成せしめればよい。
さらに測定に当つて用いられる酵素を結合させ
た成分、例えば抗体、抗原、ハプテンなどや放射
性同位元素を結合させた成分は、公知のEIAや
RIAに用いられる標識のための手段が利用され
る。また標識物質としての酵素としては、公知の
種々の酵素、例えば酸化還元酵素、加水分解酵
素、転位酵素、リアーゼ、イソメラーゼ、リガー
ゼに属する酵素が利用できる。
この様にして担体に固定化した不溶性抗体等の
2以上を用いて、この抗原等に対して特異的結合
性を有する測定すべき成分、例えば抗原、抗体の
2以上を測定するためには、公知のEIAまたは
RIAによる測定法が適用される。
例えば使用される小試験管が15×105mm(内径
12.8mm±0.1mm)の場合、エレメントは第5図に
符号AないしIで示す各部寸法のものが使用され
る。すなわち、A:半径6mmの半球部、B:円柱
部の高さ10mm、C:円錐状突起部の高さ0.4mm、
D:円錐状突起の円錐の半径0.5mm、E:半径
0.75mmの半円状溝、F:対向する溝間の中心部距
離7.5mm、G:操作棒を挿着するための凸部の直
径4mm、H:操作棒挿着孔の直径2mm、I:操作
棒挿着のための凸部の高さ3mmの寸法にて示され
る円柱部、(高さ10mm、直径12mm)と半径6mmの
半球部から成るエレメント本体で、また分別もし
くは分割可能なものとしたものでもよく、またこ
の条件下では、免疫反応用緩衝液の量は100μ
程度、標識物質を結合させた成分を含有する液は
100μ程度および被検液(血清、尿等)は100μ
程度を用いればよい。また、この際反応液の幅
(厚み)は0.4mmと均一に形成されるものである。
この前述のエレメント本体においては、一つのエ
レメント本体にはインシユリン抗体を固定化せし
め、また他の同一形状のエレメント本体にはグル
カゴン抗体を固定化せしめ、次いで各々のエレメ
ントを同一の形状、即ち第1図の切断形状に切断
した上面形状をして半円形状を有するエレメント
フラグメントとなし、得られた半円形状のインシ
ユリン抗体の固相体のフラグメントと半円形状の
グルカゴン抗体の固相体のフラグメントとを一体
化せしめて分別可能なエレメント本体の固相体と
して用いる。その他の同時測定としての例示とし
ては、例えばα−フエトプロテイン、CEA、フ
エリチンとの同時測定、T3、T4の同時測定、そ
の他種々のステロイド系ホルモンやペプチド系ホ
ルモンなどの適宜な組み合せや種々の特異的抗体
における一体化せしめた分割可能な固相体を用い
ることによりなし得る。また反応管壁を1つの分
割可能な固相体として用いてもよいことも、前述
の記載から明らかである。また反応温度時間は対
象物によつて変るが、例えばインシユリンとグル
カゴンの同時測定の場合は5℃、8−48時間であ
る。一般に測定すべき成分に応じて適当に時間、
温度および測定すべき成分の濃度を選択すればよ
い。さらに、使用される小試験管およびそれに用
いるエレメント大きさに基いて、使用する各液量
を調整すればよい。
血清中のインシユリンおよびグルカゴン含有測
定法を例に挙げると以下のごとくである。
グルカゴン抗体の固相体である反応管用小試験
管(15×105mm、内径12.8mm±0.1mm)に緩衝液
100μ、酵素標識インシユリン液50μおよび酵
素標識グルカゴン液50μおよび一定濃度のイン
シユリンおよびグルカゴン含有液100μを加え、
次いでこれに前記の大きさ、形状のインシユリン
抗体の固相体であるエレメントを加え、5℃で15
時間インキユベーシヨンをおこなう。反応終了後
蒸留水または生理食塩水5mlを加え、このエレメ
ントを洗滌した後、あらかじめ酵素基質液500〜
1000μを分注してある別の小試験管にエレメン
トを移し換え、37℃で30−120分間酵素反応をお
こなう。反応停止液1.5〜2.0mlを加え、反応を停
止させて、エレメントを除き、次いで酵素反応に
基いて消費される成分や生成される成分を測定す
る。また反応管用試験管は、洗浄液をアスピレー
ターまたはデカンテイシヨンにて除去後、上記と
同様に酵素基質液約2〜3mlを加えて酵素反応を
行なわせしめ、次いでその液の吸光度を測定す
る。既知濃度の標識液の吸光度より求めた標識曲
線より被検体試料のインシユリンおよびグルカゴ
ンの量を求める。
以上のようにして測定されるのであるが、更に
詳しく説明を加える。
6,6−ナイロンを本発明のエレメントの形状
(第5図参照)のもの(以下6,6ナイロン−測
定用エレメントという)を5塩化リンを用いるイ
ミクロ法により活性化し、ヘキサメチレンジアミ
ン、グルタルアルデヒド処理してスペーサー導入
6,6ナイロン−測定用エレメントを得る。この
ものにウサギ抗モルモツトIgG血清のIgG画分を
反応させIgGを固定化する。次いで、これにモル
モツトインシユリン抗体血清を反応させて、6,
6−ナイロン製エレメント−抗モルモツトIgG−
インシユリン抗体を調製する。同様に、ウサギ抗
モルモツトIgG血清のIgG分画を反応せしめて
IgGを固定化せしめた抗体にモルモツトグルカゴ
ン抗体血清を反応せしめて6,6−ナイロン製エ
レメント−抗モルモツトIgG−グルカゴン抗体を
調製する。
次いでこの6,6−ナイロン製エレメント−抗
モルモツトIgG−インシユリン抗体、および6,
6−ナイロン製エレメント−抗モルモツトIgG−
グルカゴン抗体の各固相体をステンレス製カツタ
ーにて半分に切断して半円形状となす。次いで
各々のエレメントフラグメントを合体せしめてエ
レメント本体となす。次いでこのエレメント本体
をβ−ガラクトシダーゼ−インシユリン結合物お
よびβ−ガラクトシダーゼ−グルカゴン結合体お
よび種々の濃度の標識インシユリンおよびグルカ
ゴンまたは両者を含有する血清などと反応させ、
洗滌後このエレメント本体を取り出し、次いで各
フラグメントを分離し、β−ガラクトシダーゼ活
性を測定して標準曲線を作成する。標準インシユ
リンおよびグルカゴンを用いる標準曲線より試料
中のインシユリンおよびグルカゴン含量を同時に
測定する。
このように本発明の分別可能な標識化合物を使
用することにより、極めて少量の試料反応液量で
2以上の免疫反応を短時間で行うことができる結
果、微量の成分を少量の試料量で正確かつ短時間
に測定することができる。
次に測定法の実施例を挙げて説明するが本発明
はこれに限定されるものでないことはいうまでも
ない。
実施例 1
インシユリンとグルカゴンのEIAによる同時測
定
〔固相体[試験管]−Aの調製法〕
ポリスチレン試験管(15×105mm、内径12.8±
0.1mm)に硫酸2mlを加え、5℃で6時間反応さ
せ、SO3C-基を導入しこれに抗モルモツトIgGウ
サギ血清のIgG画分20μgを含有する0.1Mリン酸
緩衝液(PH8.0)2mlを加え、5℃で1夜反応せ
しめ、1本当り8μg固定せしめた試験管を得た。
次いでこの試験管に抗 16-29グルカゴンフラグメ
ント血清(モルモツト)の30000倍希釈液(0.25
%BSA、5mM EDTA、3mM MgCl2、0.15
mM NaOl、0.1%NaN3含有0.01Mリン酸緩衝
液(PH7.4)よりなる希釈液)2mlを加えて、5
℃で1夜反応して[試験管−抗モルモツトIgG−
16-29グルカゴンフラグメント抗体]結合物を得
た。
〔固相体[エレメント]−Bの調製法〕
第5図で示される大きさ、および形状のポリス
チレン製エレメントを硫酸で活性化し、SO3 -基
を導入し、これを抗モルモツトIgGウサギ血清の
IgG画分20μgを含有する0.1Mリン酸緩衝液(PH
8.0)1mlに浸し、5℃で1夜反応せしめ、1本
当り10μgのIgGを固定せしめたポリスチレン製
エレメント固相体を得た。
次いでこのエレメント固相体をモルモツトのイ
ンシユリンの抗体を含有する血清の23200倍希釈
液(前項記載)1mlに浸し、5℃で1夜反応せし
めて〔エレメント−抗モルモツトIgG−インシユ
リン抗体〕結合物の固相体を得た。
〔β−ガラクトシダーゼ−インシユリンの調製
法〕
インシユリン60mgを0.1Mリン酸緩衝液(PH
8.0)17mlに溶解し、3−(2′−ベンゾチアゾリル
−ジチオ)プロピオン酸スクシンシイミドエステ
ル6.5mg含有ジメチルホルムアミド1.7mlを加え
て、冷却下1時間反応せしめ、反応後PH5.0とな
して沈澱物を回収し、この沈澱物を0.1Mリン酸
緩衝液(PH7.0)4.0mlに溶解し(インシユリンと
して8.9mg/ml)、このうち10.0μを分取し、こ
れにβ−ガラクトシダーゼ6mgを加えて1時間反
応せしめ、その後この反応液をセフアデツクスG
−100を充填したカラム(1.5×120cm)にて
0.15M NaCl含有0.01Mリン酸緩衝液(PH7.4)で
溶出せしめ、その65〜73mlの画分を集めて〔(イ
ンシユリン)−CO−(CH2)2−S−(β−ガラクト
シダーゼ)〕で略示されるインシユリンのアミノ
基とβ−ガラクトシダーゼのチオール基とによる
インシユリン−β−ガラクトシダーゼ結合物(β
−Gal標識インシユリン)を含有する溶出区分
(インシユリン2.4μg/ml、かつβ−ガラクトシ
ダーゼ1分子当り1分子のインシユリンの結合物
であり、かつインシユリン抗体に対し、インシユ
リン結合物の95%の活性を有しているもの)を得
た。
〔β−ガラクトシダーゼ− 19-29グルカゴンフラ
グメントの調製法〕
グルカゴンフラグメント(19−29)0.4mgを50
mMリン酸緩衝液(PH8.0)0.4mlに溶解し、これ
に100mM EDTA水溶液10μおよび0.1%3−
(2′−ベンゾチアゾリル−ジチオ)プロピオン酸
スクシンシイミドエステル含有ジメチルホルムア
ミド溶液625μを加えて5℃1時間反応せしめ
た。反応後これをセフアデツクスG−15(1.5×40
cm)のカラムでゲル過して、その素通り区分を
得た〔グルカゴンフラグメント(19−29)のN末
端に3−(2′−ベンゾチアゾリル−ジチオ)プロ
ピオニル基を導入した誘導体を含む〕。次いで、
この3−(2′−ベンゾチアゾリル−ジチオ)プロ
ピオニル基を導入した誘導体20μgを含有する50
mMリン酸緩衝液(PH7.0)2mlに、β−ガラク
トシダーゼ6.19mg含有50mMリン酸緩衝液(PH
7.0)を加え、室温で1時間反応した。反応液を
セフアデツクスG−150(1.5×90cm)のカラムで
ゲル過してその素通り区分を回収してグルカゴ
ンフラグメント(19−29)のN末端とβ−ガラク
トシダーゼのチオール基とをもつて結合せしめた
β−ガラクトシダーゼ−グルカゴンフラグメント
(19−29)結合体(純度95%)(β−Gal標識グル
カゴン)を得た。
インシユリンとグルカゴンのEIAによる同時測
定法
固相体−A(試験管)に免疫反応用緩衝液
(0.25%BSA、5mM EDTA、3mM MgCl2、
0.15M NaCl、0.1%NaN3含有0.01Mリン酸緩衝
液(PH7.4))100μ、β−Gal標識インシユリン
(インシユリンとして20.0pg)50μ、β−Gal標
識グルカゴン(グルカゴンフラグメントとして
2.0pg)50μ、および標準液(インシユリンとし
て5〜640μ/ml、及びグルカゴンとして25〜
3200pg/mlを有する液)100μを加え、これに
固相体−B(エレメント)を1個加え、5℃で1
夜反応を行なつた。終了後、蒸留水5mlを加えま
ず固相体Bを洗滌しながら引き抜き、あらかじめ
β−ガラクトシダーゼ活性測定液[O−ニトリフ
エニール−β−D−ガラクトピラノシド5mg/ml
を有する0.1%BSA、0.15M NaCl、1mM
MgCl2、0.1%NaN3含有0.01Mリン酸緩衝液(PH
6.7)85部および200mMメルカプトエタノール含
有メタノール溶液15部よりなる溶液]500μを
加えた小試験管に固相体−Bを加え、37℃で1時
間反応させた。反応終了後、反応停止液[100m
Mグリシン−NaOH緩衝液(PH11.0)]2mlを加
え、反応を停止させ、固相体−Bを除いた後、そ
の液の420nmの吸光度を測定した。次に、固相
体−Aは、洗滌液をデカンテーシヨンで取り除い
た後、β−ガラクトシダーゼ活性測定液(前記)
2mlを加え、37℃で2.5時間反応させ、反応終了
後、反応停止液[400mMグリシン−NaOH緩衝
液(PH11.0)]500μを加え、反応を停止させ反
応液の420nmの吸光度を測定しグルカゴンの定
量曲線を作成した。
その結果は、第7図に示す通りで、また第7図
中、〇−〇はインシユリンの定量曲線を示し、△
−△はグルカゴンの定量曲線を示すものであり、
本発明のインシユリン、グルカゴンの同時測定系
はきわめて良好な定量曲線を示すものであつた。
なお、被検試料(血清、血漿等)中のインシユリ
ン、グルカゴンを測定する場合は、標準液100μ
の代わりに試料100μを加え、前記と同様の
操作を行ない、定量曲線と対照することにより試
料中のインシユリン、グルカゴン量を求めた。
実施例 2
インシユリンとグルカゴンのRIAによる同時測
定
〔固相体[試験管]−Aの調製法〕
前記と同様に調整した。
〔固相体[エレメント]−Bの調製法〕
第5図に示される大きさおよび形状の6,6ナ
イロン製エレメント50個を5塩化リン4gとピリ
ジン4gを含むベンゼン60ml中で2日間撹拌した
後、4回ベンゼンにて洗滌し、イミクロライド化
した6,6ナイロン製エレメントを得、これに
100mMのヘキサメチレンジアミンを含有する水
溶液(PH11.5)50mlを加えて室温下、1時間反応
せしめ、0.1Mリン酸緩衝液(PH8.0)で充分洗滌
し、さらにこれに4%グルタルアルデヒド含有
0.1Mリン酸緩衝液(PH8.0)50mlを加え、室温下
で1時間反応せしめ、0.1Mリン酸緩衝液(PH
8.0)で洗滌し、スペーサーを6,6ナイロン製
エレメント(50個)を抗モルモツトIgG血清(ウ
サギ)のIgG画分5mgを含有する0.1Mリン酸緩衝
液(PH8.0)50ml中に加えて、5℃1夜反応せし
めて1個当りIgG50μgを固定せしめたナイロン
製エレメント固相体を得た。
次いで、このエレメント固相体にモルモツトの
インシユリンの抗体を含有する血清の23200倍希
釈液〔0.25%BSA、5mM EDTA、3mM
MgCl2、0.15M NaCl、0.1%NaN3含有0.01Mリ
ン酸緩衝液(PH7.4)よりなる希釈液〕50ml(250
mgのインシユリン抗体を含む)を加えて、5℃で
一夜反応して〔ナイロン製エレメント−抗モルモ
ツトIgG−インシユリン抗体〕結合物の固相体−
Bを得た。
〔 125I−インシユリンの調製法〕
125I−インシユリンの調製は、W.M.Hunter、
F.C.Greenwoodらの方法〔Nature (London)
194、495(1962)〕に順じておこない、セフアデツ
クスG−25のカラム(0.9×20cm)を用いてゲル
過して、 125I−インシユリンを得た。
〔 125I−〔Tyr18〕−グルカゴン(18−29)の調製
法〕
(A) [Tyr18]−グルカゴン(18−29)の製造
H−Tyr−Ala−Gln−Asp−Phe−Val−
Gln−Trp−Leu−Met−Asn−Thr−OHの製
造
Boc−Tyr−(Bzl−Cl2)−Ala−Gln−Asp
(OBzl)−Phe−Val−Gln−Trp−Leu−Met−
Asn−Thr(Bzl)−OBzl2.04g(1mM)にア
ニソール6ml、エタンジチオール0.94mm(10m
M)、スカトール131mg(1mM)、ジメチルス
ルフイド0.62ml(10mM)を加えた。これに、
無水弗化水素(HF)70mlを導入し、0℃で1
時間撹拌後、減圧下でHFを速やかに留去し
た。残査にEt2O100mlを加え生じた沈澱を集め
た。これを、50%酢酸100mlに溶解し、遠心分
離し、上清液を凍結乾燥する。これを8M尿素
溶液20mlと酢酸20mlにて溶解しチオグリコール
酸2mlを加え、45℃で20時間、還元した後、セ
フアデツクスLH−20のカラム(4.2×137cm)
にチヤージし、50%酢酸で溶出した。溶出液を
7mlづつ分画し、74−80本目を集め、凍結乾燥
して[Tyr18]−グルカゴン(18−29)を得た。
収量120mg、Rf20.73(セルロース)、アミノ酸分
析値:Asp1.97(2)、Thr0.95(1)、Gln2.07(2)、
Ala0.98(1)、Val0.94(1)、Met0.98(1)、Leu1(1)、
Phe0.97(1)、Tyr0.94(1)、Trp1.05(1)、上記の原
料は次のようにして製造された。
(1) P(19−29)Boc−Ala−Gln−Asp(OBzl)
−Phe−Val−Gln−Trp−Leu−Met−Asn
−Thr(Bzl)−OBzl〔1〕
P(20−29)(特開昭56−81540号公報 実
施例1 中で合成報告済)12.4g(7.5mM)
をTFA40ml、ジメチルスルフイド20ml、エ
タンジチオール20ml、およびスカトール983
mgの混液に加え、室温で50分撹拌した後、減
圧下TFAを留去する。残査にエーテルを加
え、生じた沈澱を乾燥DMF150mlとN−メチ
ルピロリドン50mlの混液に溶解した。これに
Boc−Ala−OH2.0g(1.4倍当量)HOBt1.4
g(1.4倍当量)およびWSCD1.92ml(1.4倍
当量)を加え、NMMでPH7に調整後、一夜
かきまぜ、さらにDMF50ml、HOBt0.51g、
Boc−Ala−OH0.71g、WSCD0.69mlを加
え、一夜撹拌した。DMFを減圧留去後、氷
水中に投入し、生じた沈澱を熱エタノールで
2回洗浄して目的物11.35g(収率87.8%)
を得た。
TLC;Rf10.75(シリカゲル)
アミノ酸分析;Asp1.92(2)、Thr0.88(1)、
Glu1.94(2)、Ala0.88(1)、Val0.91(1)、Met0.95
(1)、Leu1、Phe0.93(1)、Trp0.71(1)、
(2) [Tyr18]P(18−29)Boc−Tyr(Bzl−
Cl2)−Ala−Gln−Asp(OBzl)−Phe−Val−
Gln−Trp−Leu−Met−Asn−Thr(Bzl)−
OBzl〔2〕
P(19−29)〔1〕8.61g(5mM)を
TFA40ml、ジメチルスルフイド20ml、エタ
ンジチオール20ml、およびスカトール983mg
の混液に加え、室温で50分撹拌した後減圧下
TFAを留去した。残査にエーテルを加え生
じた沈澱をDMF150mlとN−メチルピロリド
ン50mlの混液に溶解した。これにBoc−Tyr
(Bzl−Cl2)−OH3.08g(1.4倍当量)
HOBt0.95g(1.4倍当量)、WSCD1.28ml
(1.4倍当量)を加え、NMMにてPH7に調整
後、室温にて48時間撹拌した。DMFを減圧
留去後、氷水を投入し、生じた沈殿を熱エタ
ノールで3回洗浄して目的物9.00g(収率
88.0%)を得た。
TLC;Rf10.76(シリカゲル)
アミノ酸分析値;Asp1.90(2)、Thr0.91(1)、
Glu1.96(2)、Ala0.89(1)、Val0.90(1)、Met0.93
(1)、Leu(1)、Phe0.95(1)、Tyr0.92(1)、
Tvp0.68.(1)、
なお、本明細書中に記載の略記号は次の意味
を有する。
Ala;L−アラニン
Gln;L−グルタミン
Asp;L−アスパラギン酸
Asn;L−アスパラギン
Phe;L−フエニルアラニン
Val;L−バリン
Trp;L−トリプトフアン
Leu;L−ロイシン
Met;L−メチオニン
Thr;L−スレオニン
Boc;t−ブトキシカルボニン
Bzl;ベンジル
OBzl;ベンジルエステル
TFA;トリフルオロ酢酸
NMM;N−メチルモルホリン
DMF;ジメチルホルムアミド
HOBt;1−ヒドロキシベンゾトリアゾール
WSCD;N−エチル、N′−3−ジメチルアミ
ノプロピル−カルボジイミド
Bzl−Cl2;2,6−ジクロロベンジル
また薄層クロマトグラフイー(TLC)の担
体および展開溶媒は、以下のものを用いた。
1;クロロホルム−メタノール−酢酸
(80:25:2)メルク社製シリカゲル
Avt5721、
2;1−ブタノール−ピリジン−酢酸−
水(15:10:3:12)メルク社製セルロース
Avt5716
(B) 125I−〔Tyr18〕−グルカゴン(18−29)の調
製
前記(A)で得られた[Tyr18]−グルカゴン
(18−29)を用いて、W.M.Hunter、F.C.
Greenwoodらの方法〔Nature(London)194、
495(1962)〕に順じて行ない、セフアデツクス
G−25のカラム(0.9×20cm)にてゲル過し、
125I−[Tyr18]−グルカゴン(18−29)を得
た。
〔RIAによるインシユリン、グルカゴン同時測
定〕
固相体[試験管]−Aに免疫反応用緩衝液(前
記)100μ、 125I−インシユリン(インシユリ
ンとして10.0pg)50μ 125I−[Tyr18]−グルカ
ゴン(18−29)(グルカゴンとして4.5pg)50μ、
および標準液(インシユリンとして5〜640μU/
ml)、グルカゴンとして25〜3200pg/ml含有)
100μを加え、これに固相体(エレメント)−B
を1個加え、5℃で1夜反応をおこなつた。終了
後、生理食塩水5mlを加え、まず固相体−Bを洗
滌しながら引き抜き、新しい試験管に移し、その
放射能を測定した。また別に、固相体−Aは洗滌
液をデカンテーシヨンで取り除いた後、その放射
能を測定した。その結果は第8図に示す通りで、
また第8図中、〇−〇はインシユリンの定量曲線
を示し、△−△はグルカゴンの定量曲線を示すも
ので、本発明のインシユリン、グルカゴン同時測
定系は、きわめて良好な定量曲線を示した。な
お、第8図において横軸は、インシユリン、グル
カゴン濃度、縦軸は、B/Bo(%)で示した。
実施例 3
CEAとフエリチンのサンドイツチ法による
EIAでの同時測定
〔固相体[試験管]−Aの調製法〕
ポリスチレン試験管(15×105mm、内径12.8±
0.1mm)に濃硫酸2mlを加え、5℃で6時間反応
させ、−SO3 -基を導入し、これに抗CEAウサギ血
清のIgG画分50μgを含有する0.1Mリン酸緩衝液
(PH8.0)2mlを加え、5℃で1夜反応せしめ、
[試験管−EIA抗体]結合物の固相体−Aを得た。
〔固相体[エレメント]−Bの調製法〕
6,6ナイロン製エレメント50個を30%(V/
V)ジメチル硫酸含有メタノール溶液50mlに加
え、37℃で50分間反応し、メタノールで充分洗滌
後、これに100mMヘキサメチレンジアミン含有
水溶液(PH11.5)50mlを加え、室温下1時間反応
せしめ、0.1Mリン酸緩衝液(PH8.0)で充分洗滌
し、さらにこれに4%(V/V)グルタルアルデ
ヒド含有0.1Mリン酸緩衝液(PH8.0)50mlを加
え、室温下で1時時反応せしめ0.1Mリン酸緩衝
液(PH8.0)で洗滌し、スペーサーを6,6ナイ
ロン製エレメントに導入せしめた。このエレメン
ト(50個)を抗フエリチンウサギ血清のIgG画分
5mgを含有する0.1Mリン酸緩衝液(PH8.0)50ml
に加えて、5℃1夜反応せしめて[エレメント−
フエリチン抗体]結合物の固相体−Bを得た。
〔β−Gal標識フエリチン抗体の調製法〕
フエリチン抗血清より硫安分画でIgG画分を
得、さらにフエリチンを固定化せしめたセフアロ
ース4B抗体を用い、アフイニテイークロマトグ
ラフイーで抗体を精製した。精製抗体4mgを
0.1Mリン酸緩衝液(PH8.0)1.8mlに溶解し、3−
(2′−ベンゾチアゾリル−ジチオ)プロピオン酸
スクシンシイミドエステル14.88μg含有ジメチル
ホルムアミド液200μ、及び100mM EDTA水
溶液20μを加えて、5℃で1時間反応せしめ
た。反応後、これをセフアデツクスG−15(1.5×
40cm)のカラムでゲル過して、その素通り画分
を得た[フエリチン抗体のアミノ基に3−(2′−
ベンゾチアゾリル−ジチオ)プロピオニル基を導
入した誘導体を含む]。次いで、この3−(2′−ベ
ンゾチアゾリル−ジチオ)プロピオニル基を導入
した誘導体1.5mgを含有する50mMリン酸緩衝液
(PH7.0)2mlにβ−ガラクトシダーゼ3.33mg含有
50mMリン酸緩衝液(PH7.0)2mlを加え、室温
で2時間反応した。反応液をセフアデツクスG−
150(1.5×90cm)のカラムでゲル過してその素
通り画分を回収して、β−ガラクトシダーゼ標識
フエリチン抗体結合体(純度98%)(β−Gal標
識フエリチン抗体)を得た。
〔β−Gal標識CEA抗体の調製法〕
CEA抗血清より硫安分画でIgG画分を得て、さ
らにCEAを固定化せしめたセフアロース4B抗体
を用い、アフイニテイークロマトグラフイーで抗
体を精製した。
精製抗体4mgを0.1Mリン酸緩衝液(PH8.0)1.8
mlに溶解し、3−(2′−ベンゾチアゾリル−ジチ
オ)ピロピオン酸・スクシンシイミドエステル
14.88μg含有ジメチルホルムアミド200μ、及び
100mM EDTA水溶液20μを加えて、5℃で
1時間反応せしめた。反応後、これをセフアデツ
クスG−15(1.5×40cm)のカラムでゲル過し
て、その素通り画分を得た[CEA抗体のアミノ
基に3−(2′−ベンゾチアゾリル−ジチオ)プロ
ピオニル基を導入した誘導体を含む]。次いで、
この3−(2′−ベンゾチアゾリル−ジチオ)プロ
ピオニル基を導入した誘導体1.5mgを含有する50
mMリン酸緩衝液(PH7.0)2mlにβ−ガラクト
シダーゼ3.33mg含有50mMリン酸緩衝液(PH7.0)
2mlを加え、室温で1時間反応した。反応液をセ
フアデツクスG−150(1.5×90cm)のカラムでゲ
ル過してその素通り画分を回収してβ−ガラク
トシダーゼ標識CEA抗体結合体(純度98%)(β
−Gal標識CEA抗体)を得た。
〔EIAによるCEAとフエリチンの同時測定〕
固相体[試験管]−Aに免疫反応用緩衝液(前
記)200μ、および標準液(CEAとして2.5〜
160ng/ml、およびフエリチンとして、5〜
320ng/mlを含有)100μを加え、これを固相体
[エレメント]−Bを1個加え、37℃で5時間反応
を行なつた。終了後内溶液を吸引除却し、生理食
塩水5mlを加え、固相体−A、Bを洗滌した。こ
れにβ−Gal標識フエリチン抗体、およびβ−
Gal標識CEA抗体を含有する免疫反応用緩衝液
300μを含え、室温で18時間反応を行なつた。
終了後、生理食塩水5mlを加え、まず固相体−B
を洗滌しながら引き抜き、あらかじめβ−ガラク
トシダーゼ活性測定液(前記)500μを加えて
ある小試験管に加え、37℃で3時間反応させた。
反応停止液(100mMグリシン−NaOH緩衝液
(PH11.0)2mlを加え、反応を停止させ、固相体
−Bを除いた後、その液の420nmの吸光度を測
定した。また別に固相体−Aは、洗滌液をデカン
テーシヨンで取り除いた後β−ガラクトシダーゼ
活性測定液(前記)2mlを加え、37℃で5時間反
応させた。反応停止液(400mMグリシン−
NaOH緩衝液(PH11.0)]0.5mlを加え、反応を停
止させ、反応液の420nmの吸光度を測定した。
第9図に示す通りで、また第9図中、〇−〇は
CEAの定量曲線を示し、△−△はフエリチンの
定量曲線を示すもので、本発明のCEA、フエリ
チン同時測定系はきわめて良好な定量曲線を示す
ものであつた。
実施例 4
サイロキシン[T4]とサイロニン[T3]の
EIAによる同時測定
〔固相体[エレメント]−Aの調製法〕
第6図に示すポリスチレン製エレメントフラグ
メント(第5図にしエレメントを4等分割したエ
レメントフラグメント)を、4個合体(分割可
能)させたエレメントを硫酸で活性化し、SO3 -
基を導入し、これに抗ウサギIgGヤギ血清のIgG
画分20μgを含有する100mMリン酸緩衝液(PH
8.0)1mlに浸し、5℃で1夜反応せしめ、1本
当り8.4μgのIgGを固定せしめたポリスチレン製
エレメント固相体を得た。
次いでこのエレメント固相体を抗T4−OMe血
清(ウサギ)の8000倍希釈液(前項記載)1mlに
浸し、5℃で一夜反応せしめて[エレメント−抗
ウサギIgG−T4抗体]結合物の固相体−Aを得
た。
〔固相体[エレメント]−Bの調製法〕
第6図に示す形状と全く同じポリスチレン製エ
レメント(成型時にフタロシアニンブルーを混入
し、淡青色に着色)を前記と同様に処理し抗ウサ
ギIgG−固定化エレメント固相体を得た。次いで
固相体をT3−OEt血清(ウサギ)の20000倍希釈
液1mlに浸し、5℃で一夜反応せしめて[エレメ
ント−抗ウサギIgG−T3抗体〕結合物の固相体−
Bを得た。
〔固相体[エレメント]−Cの調製〕
再び分割した固相体エレメント−Aのフラグメ
ント1個と、固相体エレメント−Bのフラグメン
ト3個を両端縁が鋭利状で、かつ中央部にストツ
パーを有する連結機にて合体させ、T4、T3−同
時測定用固相体[エレメント]−Cを調製した。
〔T4−OMeの調製法〕
T4(シグマ社製)600mgをメタノール15mlに懸
濁し、−10℃で乾燥塩化水素ガスを飽和するまで
吹きこんだ後、室温で20時間撹拌した。さらに0
℃で乾燥塩化水素ガスを吹きこんだ後、析出した
沈殿をろ取し、塩化水素ガス含有メタノールで洗
滌後固体水酸化ナトリウムを入れたデシケーター
で、減圧乾燥後、メタノール−エーテルで再結晶
して、目的のT4−OMe582.5mg(収率91.2%)を
得た。
〔β−Gal標識T4−OMeの調製法〕
T4−OMe2mgを90%ジメチルホルムアミド、10
%100mMリン酸緩衝液(PH8.0)2mlに溶解し、
3−(2′−ベンゾチアゾリルジチオ)プロピオン
酸スクシンシイミドエステル1.78mg含有ジメチル
ホルムアミド1.78mlを加えて、冷却下1時間反応
せしめた。反応液をLH−20(1.5×40cm)のカラ
ムに添加し、90%ジメチルホルムアミド、10%
100mMリン酸緩衝液(PH5.0)の溶媒でゲル過
を行なつた。溶出液を2mlづつ分画し、T4のア
ミノ基に3−(2′−ベンゾチアゾリル−ジチオ)
プロピオニル基を導入した誘導体を含む画分(12
〜15本目)を集めた。
次いで、この3−(2′−ベンゾチアゾリル−ジ
チオ)プロピオニル基を導入した誘導体8μgを
含有する90%リン酸緩衝液(PH8.0)10%ジメチ
ルホルムアミド液2mlにβ−ガラクトシダーゼ2
mgを加え、室温で2時間反応せしめた。反応液を
セフアデツクスG−150(1.5×90cm)のカラムに
添加し、0.15M NaClを含有する10mMリン酸緩
衝液(PH7.4)でゲル過を行ない、その素通り
画分を回収して、T4のアミノ基とβ−ガラクト
シダーゼのチオール基とをもつて結合せしめたβ
−ガラクトシダーゼ−T4−OMe結合体(純度93
%)(β−Gal標識T4−OMe)を得た。
〔T3−OEtの調製法〕
T3(シグマ社製)500mgをエタノール15mlに懸
濁し、−10℃で乾燥塩化水素ガスを飽和するまで
吹きこんだ後、室温で20時間撹拌した。反応液を
ろ過し、ろ液を減圧濃縮し、ベンゼンを加え、析
出した沈殿を集め乾燥した。さらにベンゼンに懸
濁洗滌して、目的のT3−OEt515mg(収率93.7%)
を得た。
〔β−Gal標識T3−OEtの調製法〕
T3−OEt2mgを90%ジメチルホルムアミド、10
%リン酸緩衝液(PH8.0)2mlに溶解し、3−
(2′−ベンゾチアゾリルジチオ)プロピオン酸ス
クシンシイミドエステル2.06mg含有ジメチルホル
ムアミド2.06mlを加えて、冷却下1時間反応せし
めた。反応液をLH−20(1.5×40cm)のカラムに
添加し、90%ジメチルホルムアミド、10%100m
M酢酸緩衝液(PH5.0)の溶媒でゲル過を行な
つた。溶出液を2mlづつ分画し、T3のアミノ基
に3−(2′−ベンゾチアゾリル−ジチオ)プロピ
オニル基を導入した誘導体を含む画分(12〜15本
目)を集めた。
次いで、この3−(2′−ベンゾチアゾリル−ジ
チオ)プロピオニル基を導入した誘導体7.2μgを
含有する90%リン酸緩衝液(PH8.0)10%ジメチ
ルホルムアミド溶液2mlにβ−ガラクトシダーゼ
2mgを加え、室温で2時間反応せしめた。反応液
をセフアデツクスG−150(1.5×90cm)のカラム
に添加し、0.15M NaClを含有する10mMリン酸
緩衝液(PH7.4)でゲル過を行ない、その素通
り画分を回収して、T3のアミノ基とβ−ガラク
トシダーゼのチオール基とをもつて結合せしめた
β−ガラクトシダーゼ−T3−OEt結合体(純度
95%)(β−Gal標識T3−OEt)を得た。
〔T4−BSA結合物の調製法〕
T4−OMe25mgをジメチルホルムアミド17.5ml
に溶解し、撹拌下、グルタルアルデヒド6mg含む
10mMリン酸緩衝液(PH8.0)16mlを加え室温で
30分間反応させた。次いでこれにBSA113.6mgを
含む10mMリン酸緩衝液(PH8.0)20mlを加え、
さらに室温で2時間反応させた。反応液を蒸留水
に対し十分に透析を行ない、透析内液を凍結乾燥
して、BSA−T4−OMe結合体(結合モル比1:
8.6)を得た。
〔T3−BSA結合物の調製法〕
T3−OEt25mgをジメチルホルムアミド17.5mlに
溶解し、撹拌下グルタルアルデヒド7mg含む10m
Mリン酸緩衝液(PH8.0)16mlを加え、室温で30
分間反応させた。次いでこれにBSA118mgを含む
10mMリン酸緩衝液(PH8.0)20mlを加え、さら
に室温で2時間反応させた。反応液を蒸留水に対
し透析内液を凍結乾燥してBSA−T3−OEt結合
体(結合モル比1:12.6)を得た。
〔抗体の作製〕
各々BSA−T4−OMeおよびBSA−T3−OEt結
合体1mg(T4−OMeとして95μg、T3−OEtと
して116μg)を0.15M NaCl含有10mMリン酸緩
衝液(PH7.4)1mlに溶解し、これにフロイン
ト・コンプリート・アジユバント(Freund′s
Complete adjuvant)1.0mlを加えて充分混和し
て乳剤を得た。次いでウサギ1匹に乳剤2mlを四
肢指の皮中、および背中の皮下数ケ所に注射し
た。2週間おきに同量の乳剤を8回皮下注射し、
最終免疫より10日後にその全血を採取し、60分間
室温に放置して凝固せしめた後に、3000rpm、10
分間遠心して、各々抗T4−OMe血清(ウサギ)、
抗T3−OEt血清(ウサギ)を得た。
〔T4とT3のEIAによる同時測定〕
試験管(15×105mm、内径12.8±0.1mm)に免疫
反応用緩衝液(前記)100μ、β−Gal標識T4−
OMe(T4として約2.0ng)50μ、β−Gal標識T3
−OEt(T3として約0.1mg)50μ、および標準液
(T4として10〜640mg/ml、T3として0.5〜32ng/
mlを含有する液)100μを加え、これに固相体
[エレメント]−Cを1個加え、37℃で30分間隔で
固相を回転させながら、3時間反応を行なつた。
終了後、蒸留水を5ml加え、固相体−Cを洗滌し
ながら引き抜いた。洗滌固相体をT4用固相体フ
ラグメント1個とT3用固相体フラグメント3個
とに分割し、あらかじめβ−ガラクトシダーゼ活
性測定液2mlを加えてある試験管にそれぞれ浸積
し、T4用固相体フラグメントは37℃で30分、T3
固相体フラグメントは2時間反応させた。反応終
了後、反応停止液[400mMグリシン−NaOH緩
衝液(PH11.0)]を0.5ml加え、反応を停止させ、
固相体を取り除いた後、それぞれの液の420nm
における吸光度を測定した。その結果第10図に
示す通りで、第10図中、〇−〇はT4の定量曲
線を示し、△−△はT3の定量曲線を示すもので、
本発明のT4とT3の同時測定系は、きわめて良好
な定量曲線を示すものであつた。なお、被検試料
(血清、血漿等)中のT4、T3を測定する場合は、
標準液100μの代わりに試料100μ加え、前記
と同様の操作を行ない、定量曲線と対照すること
により試料中のT4、T3量を求めた。 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for simultaneously measuring trace amounts of biological components using a medium element carrier that expands the surface area of a reaction medium. Many methods have been known for quantitative measurement, such as immune reactions based on antigens (including papten) and antibodies, and receptor reactions based on substances such as drugs and hormones and receptors (hereinafter abbreviated as immune reactions). ing. Such methods mainly involve serum, urine,
Quantification of components present in trace amounts in body fluids such as saliva,
It has been applied for qualitative purposes. Trace components include various hormones as in-vivo components, receptors for them, in-vivo enzymes, components specific to pathological conditions, administered drugs and their receptors, and their metabolites, as well as bacteria and viruses. Microorganisms such as
Examples include trace components that exist in various living organisms, such as antibodies against these. Such trace components are mainly measured by applying immune reactions such as antigen-antibody reactions, for example, hormones and antibodies or receptors against them. Methods that utilize the fact that, as a result of immune reactions, these conjugates become insoluble or that large aggregates consisting of a large number of antigen-antibody molecules are formed are already known as classical methods. These are measured by various methods such as directly measuring the amount precipitated in a reaction tube, immunodiffusion method using precipitation in a gel, or method using complement. Furthermore, various methods are well known in which antigens, antibodies, or receptors are labeled by some method, subjected to an immune reaction, and then the labeling activity is measured. Among these, radioimmunoassay (RIA), which uses radioactive substances as a labeling substance, is well known as a widely used highly sensitive measurement method.Although RIA uses radioactive substances, it is safer. As an alternative method, enzyme immunoassay (EIA), which uses an enzyme label instead of a radioactive substance label, has been widely used in recent years. EIA is a method in which an enzyme is bound to an antigen or a receptor such as an antibody or receptor, the degree of immune reaction is measured using the enzyme activity as a label, and the amount of antigen or receptor is measured from the results. It is. In such a case, a receptor or antigen having specific binding ability for the component to be measured is used, and the antigen or receptor as the component to be measured is caused to compete with the enzyme-labeled antigen or receptor, Then, when the bound and non-bound substances generated by the immune reaction are separated or their enzyme activity is measured without separation, or when a receptor or antigen that has specific binding ability for the substance to be measured is used. is used to react with the antigen or receptor to be measured, and then the conjugate is reacted with a compound capable of binding to the enzyme-labeled antigen or a compound capable of binding to the receptor, thereby producing a sandwich. Thereafter, the enzyme activity may be measured. Conventionally, when utilizing these various immune reactions, one of the components to be measured is
The method only carried out one reaction, and there were many drawbacks to measuring a wide variety of components. That is,
Measuring multiple components requires a large amount of samples such as serum equivalent to the number of target components, and a wide variety of reagents are used for each measurement, which also requires a long time. It was hot. In contrast, a solid phase body in which two types of antibodies are immobilized on a carrier is used, and samples containing the two types of antigens to be measured are added and reacted, and a labeled compound of one type of antigen between them is first reacted. A method in which the labeling activity of the antigen is measured, followed by reacting with a labeling compound of another antigen and measuring the labeling activity (JP-A-56-78598, No. 52-15815, No. 56)
-96248) is known. In these measurement methods, beads and test tubes, which have traditionally been used as solid phase substances, are used as antibodies, microparticles with various distinguishable particle sizes are used as carriers, or beads and microparticles are used instead of beads or microparticles. Because a carrier with multiple fins is used and a solid phase is used on which two or more types of antibodies are immobilized, it is difficult to react trace components with extremely small amounts of samples. However, it is extremely difficult to calculate the titer of this immobilized antibody, and the measurement requires a reaction to sequentially measure each component.
This had the disadvantage of requiring a longer measurement time. The present inventors have researched and developed a method that can easily and accurately measure various trace components in a living body simultaneously over multiple components,
The purpose of this is to use a reaction tube for immune reaction as a carrier, and to use it as a carrier to be added to the reaction medium of immune reaction, the carrier has a shape that can be inserted into the immune reaction part of the reaction tube, and is external. It is formed so that the diameter maintains a slight distance from the inner diameter of the reaction tube, and the distance between the reaction tube and the element carrier is the same.It is an element carrier for the medium that expands the surface of the reaction medium. By using this carrier as one or more carriers, when reacting a small amount of sample, it is possible to expand the surface area of the reaction medium containing a small amount of sample liquid using the medium element carrier. This method provides a simple and good multi-component simultaneous measurement method that can be measured using a very small amount of sample liquid. The present invention can also be applied to EIA and RIA, as well as competitive reactions and sandwich immunoreactions. First, a reaction tube as a carrier used in the present invention may be a reaction test tube for immune reactions conventionally used in various EIAs and RIAs; for example, a reaction tube made of glass or plastic may be used. is used in any form. In addition, the element carrier has a shape that can be inserted into the immune reaction part of the reaction tube, and is formed so that its outer diameter maintains a slight distance from the inner diameter of the reaction tube, and the element carrier is formed so that it can be inserted into the immune reaction part of the reaction tube. It may be a medium element carrier that is formed so that the space is the same, expands the surface area of the reaction medium, and has a shape that can be divided into one or more parts as appropriate.
In addition, in the case of an immune reaction, any type of material that can immobilize a receptor or antigen that has specific binding ability with the component to be measured, and which can be added to the reaction tube, is usually made of glass or an insoluble material. Various things such as polysaccharides and insoluble proteins are granular, plate-shaped, bullet-shaped,
It is used in shapes such as a spear. Such carriers used in the present invention include:
An insoluble carrier used in general EIA and RIA is used. especially antibodies or antibody fragments
When insolubilizing Fab' etc., the target to be bound to an insoluble carrier is generally an antibody protein, so a carrier similar to that used in the production of immobilized enzymes to immobilize enzyme proteins is used. is used. Examples of this carrier include insoluble protein carriers such as albumin and gelatin treated with glutaraldehyde, agarose, epichlorohydrin treatment or cyanogen bromide treatment such as cellulose and dextrin, and insoluble insoluble protein carriers treated with an amination reagent. Semi-synthetic polymer carrier (polysaccharide), acrylonitrile, acrylic acid, acrylic ester, methacrylic acid, methyl methacrylic acid, vinyl alcohol, vinyl acetate, styrene, aminostyrene, divinylbenzene, acrylamide, ethylene, maleic anhydride, Polymers or copolymers such as crotonic acid, insoluble synthetic polymer carriers (polymer resins) obtained by treating these with aminating reagents, and other insoluble inorganic carriers such as silane compounds treated with aminating reagents. It may also be used by combining them. In addition, various known means may be used to introduce amino groups into this insoluble carrier, such as amination by reduction of nitrile groups, γ of hydroxyl groups, etc.
-Introduction of aminoalkyl groups by reaction with aminopropyltriethoxysilane, or conversion of hydroxymethyl groups to aldehyde groups by periodic acid oxidation, and introduction of amino groups by reacting the aldehyde groups with diamines, polysaccharide hydroxyl groups is converted to an imidocarbonate group by cyanogen bromide reaction, the amide group is converted to an iminohalogenide group by a phosphorus halide compound, and this functional group is further treated with a functional group-introducing reagent to introduce a new functional group as appropriate. It's okay. Among these insoluble carriers, suitable for processing include polymer resins such as nylon, polystyrene, polyacrylamide, and vinyl chloride, insolubilized polysaccharides such as cellulose and dextran, and inorganic materials such as glass. The element carrier in such a carrier may be bullet-shaped, spear-shaped, or similar in shape, but it may be attached to the surface of another solid molded object such as metal or plastic in such a way that the shape of the element of the present invention is maintained. Preferably, the medium element is formed to expand the surface area of the reaction medium and has the same shape as the immunoreaction tube used. Next, the element carrier of the present invention will be explained with reference to the drawings. In FIG. 1, a medium element carrier as an element carrier is generally indicated by reference numeral 1. The element carrier 1 is made of a polymeric resin material such as nylon 6,6, and is composed of an element body 2 and a mounting leg 3 protruding from the center of the upper surface 2a of the body 2. Although another operating rod (not shown) is attached to the mounting leg 3, the mounting leg 3 itself may have an extended shape for use as an operating rod. The element main body 2 is molded into a bullet shape, and its outer diameter is formed to maintain a slight clearance from the inner diameter of the reaction tube 4. A wedge-shaped projection 5 is provided at the top of the lower peripheral surface of the element body 2.
The protrusion 5 maintains a certain gap 6 between the arcuate outer circumferential surface 2b of the element body 2 and the arcuate inner circumferential surface 4a of the reaction tube 4. Further, instead of this wedge-shaped projection 5, a convex projection may be used. In short, any shape may be used as long as the projection 5 can prevent the element body 2 from coming into close contact with the arcuate inner circumferential surface 4a of the reaction tube 4. The height of this protrusion may be such that the width of the element main body 2 and the inner wall surface of the reaction tube 4 are the same. As shown in FIG. 2, the upper surface 2a of the element main body 2 may be provided with a square cut groove 8 centered on the mounting leg 3. The end of the cut groove 8 is opened at the peripheral edge of the element main body 2 in order to weaken the surface tension of the reaction liquid on the extension of the upper surface 2a. Further, the groove shape of the cut groove 8 may have any cross-sectional shape such as a semicircular shape, an elliptical shape, a trapezoidal shape, a triangular shape, or a quadrangular shape. Since the element carrier of the present invention has the above-described structure, when the element body is immersed in the reaction liquid in the reaction tube, the protrusions of the element body come into contact with the inner bottom surface of the reaction tube, and the element body A slight gap is formed between the outer peripheral surface and the corresponding inner peripheral surface of the reaction tube (see FIG. 1). Therefore,
Even if the amount of reaction liquid is small, by immersing the element body, the liquid is guided into the gap and the reaction liquid that rises to the top of the element body flows in the cut groove and reaches the surface of the element itself. Loss of tension action. Therefore, the reaction liquid comes into contact with the outer peripheral surface of the element main body under uniform conditions (in the form of a layer of constant thickness). As a modification of the cut grooves 8 as described above, they may be arranged radially in the radial direction of the upper surface portion 2a of the element main body 2, as shown in FIG. 3A. Moreover, as another modification, as shown in FIG. As another modification, as shown in FIG. 3C, the elements may be arranged symmetrically in the circumferential direction of the upper surface 2a of the element main body 2. In this manner, the cut grooves 8 may have any shape as long as their terminal ends are opened at the peripheral edge of the upper surface portion 2a. FIG. 4 shows another modification of the element 1. The element body 2 in this example is shaped like a spear. Therefore, the reaction tube 4 used has a tapered shape along the element body 2, and the element can be modified and processed according to the shape of various commercially available reaction tubes. The rest of the structure of the element main body 2 is the same as that of the example shown in FIG. 1, so the same numbers are given to the parts having the same structure, and the explanation thereof will be omitted. Furthermore, as mentioned above, in order to perform multiple immune reactions without also increasing the surface area of a small amount of reaction solution, a carrier having multiple elements in the shape of a plate or rod may be used as a solid phase. . Insoluble antibodies, etc. (antigen, receptor ) and prepare a solid phase. The solid phase may be prepared by introducing a functional group into the carrier in advance to bind antibodies, etc., and processing it into a specific shape, or by introducing the functional group after processing. For example, if the reaction tube or element carrier is nylon 6,6,
It is processed in advance into the above-mentioned shape, and then activated by dimethyl sulfate or immicroscopic method, treated with hexamethylene diamine and glutaraldehyde to bind an antibody, such as a second antibody, and then immunized with the first antibody. A solid phase body may be obtained by immobilization by reaction. Furthermore, when the antibody is a dextran gel, such as Sephadex, it is processed in advance into the above-mentioned shape, then activated with BrCN, and the antibody is bound to and immobilized thereon. Furthermore, in the case of polystyrene, etc., after processing, it may be fixed after being chemically treated with sulfuric acid, or it may be fixed by physical adsorption. In the case of glass, etc., it may be aminoalkylsilylated using an organic amino group-introducing silane reagent such as a γ-aminoalkyltriethoxysilane compound, and then immobilized based on this amino group by a known immobilization means. Furthermore, in general, when immobilizing chemically, a functional group in the molecule of the carrier or an introduced functional group and a functional group in the molecule of the antibody, such as an amino group, a hydroxyl group,
A polyfunctional compound capable of bonding the two based on a functional group such as a carboxyl group, a thiol group, or an aldehyde group is used. Examples of various known polyfunctional compounds include diisocyanate compounds such as hexamethylene diisocyanate and 2,4-toluene diisocyanate, diisothiocyanates such as hexamethylene diisothiocyanate, and succinic aldehyde. , dialdehydes such as glutaraldehyde and adipaldehyde, N,N'-ethylene bismaleimide, N,N'-0-phenylene dimaleimide, bisdiazobenzine, N,N'-
Polymethylene biiodoacetamide, diethylenemalonimidate, dimethyladipineimidate, 3-(2'-benzothiazolyldithio)-propionic acid, 3-(2'-pyridyl-N-oxide-dithio)propionic acid, 6 Sulfide carboxylic acids such as -N[3-(2'-benzothiazolyl-dithio)propionyl]caproic acid or its reactive derivatives such as succinimide ester, p-nitrophenyl ester, acid chloride, imidate, etc. -85900), maleimidocarboxylic acids such as maleimidobenzoic acid, maleimidophenyl acetic acid, maleimidophenylpropionic acid, or reactive derivatives thereof. This polyfunctional compound can also be used as a functional group introduction reagent for introducing a functional group into a carrier. Furthermore, in general, when obtaining a solid phase, an inert medium such as a buffer solution of pH 6 to 8, acetone, methanol, ethanol, dioxane, dimethyl sulfoxide, dimethylacetamide, tetrahydrofuran, or a mixed medium thereof is used. The carrier and the polyfunctional compound are reacted using a medium at cooling or room temperature. Thereafter, the insoluble carrier is washed if necessary, and then an antibody or the like is further added and reacted using the same inert medium to cause the insoluble carrier to react with the antibody or the like. Furthermore, in order to obtain an antibody-second antibody-first antibody conjugate obtained by binding a first antibody with a second antibody, for example, using an antibody processed into a desired shape, Based on the means of obtaining the counterpart,
First, a second antibody is bound. Next, the first antibody may be added to this and bound based on an immune reaction, and the immunoactivity of the first antibody obtained in this way is hardly deteriorated, so it is a particularly preferable solid phase. When using two or more of the solid phase bodies obtained in this way, for example, a reaction tube solid phase body and one element solid phase body or an element solid phase body that can be separated into two or more. or a combination of multiple element solid phase bodies that can be separated into two or more. In particular, as an element solid phase that can be separated into two or more, for example, a plurality of element carriers shown in Fig. 1 are used, and one element is immobilized with a target antibody, etc., and another element is used with a different purpose. An antibody or the like is immobilized to obtain a solid phase body on which a plurality of different antibodies or the like are immobilized. These solid phase bodies are then cut into respective sizes. For example, when making a solid phase of two types of elements, if the result of combining them constitutes one element, you can cut them to an appropriate size, and simply cut them into symmetrical halves. In addition, if the density of the faces differs, the element may be constructed using each fragment cut according to the ratio according to the density, for example, a fragment of 1/4 size. By using an element fragment and an element fragment of 3/4 size, the low-density part is made into a 3/4-sized fragment, and the high-density part is made into a 1/4-sized fragment, which are then glued together to form an element. Further, when preparing a solid phase body of three or more types of elements, each fragment may be cut according to the number thereof, and then these fragments may be combined and used as an element in the original shape. Furthermore, when combining the fragments of the element solid phase, they may be simply integrated into a shape that can be separated according to the cut surface of the element body, or the element may be combined at the joining site of the cut surface of each element fragment. Each fragment may be integrated with a joint portion for maintaining the gap distance between the main body and the reaction tube. In particular, in the latter case, it is preferable to use a fragment of the element main body as a carrier in advance and immobilize an antibody or the like onto it to form a solid phase. Furthermore, when using a solid phase consisting of a plurality of plate-shaped or rod-shaped elements that are separable as described above, antibodies, etc. are immobilized on the entire surface of each carrier so that the entire surface can be used as a solid phase. It is preferable to form a bonding area in the vicinity of the contact area of each solid phase body in order to maintain the gap distance. Furthermore, the components to which enzymes are bound, such as antibodies, antigens, haptens, etc., and the components to which radioactive isotopes are bound, which are used in measurements, are prepared using known EIA or
The means for labeling used in RIAs are utilized. In addition, various known enzymes such as those belonging to oxidoreductases, hydrolases, transposases, lyases, isomerases, and ligases can be used as enzymes as labeling substances. In order to measure two or more components to be measured, such as antigens and antibodies, which have specific binding properties to the antigen, etc., by using two or more insoluble antibodies etc. immobilized on a carrier in this way, Known EIA or
Measurement method by RIA is applied. For example, the small test tube used is 15 x 105 mm (inner diameter
12.8mm±0.1mm), elements having the respective dimensions shown by symbols A to I in FIG. 5 are used. That is, A: hemispherical part with a radius of 6 mm, B: the height of the cylindrical part is 10 mm, C: the height of the conical protrusion part is 0.4 mm,
D: radius of cone of conical projection 0.5mm, E: radius
0.75mm semicircular groove, F: Center distance between opposing grooves 7.5mm, G: Diameter of convex part for inserting the operating rod 4mm, H: Diameter of operating rod insertion hole 2mm, I: The element body consists of a cylindrical part with a height of 3 mm (height 10 mm, diameter 12 mm) and a hemispherical part with a radius of 6 mm, and can be separated or divided. Under these conditions, the amount of immunoreaction buffer is 100 μl.
The liquid containing the component bound to the labeled substance is
Approximately 100μ and test liquid (serum, urine, etc.) 100μ
It is sufficient to use degree. Further, at this time, the width (thickness) of the reaction liquid was uniformly formed at 0.4 mm.
In the above-mentioned element bodies, an insulin antibody is immobilized on one element body, a glucagon antibody is immobilized on the other element body having the same shape, and then each element has the same shape, that is, the first The top surface shape cut into the cut shape shown in the figure is made into semicircular element fragments, and the obtained semicircular fragments of the insulin antibody solid phase body and semicircular fragments of the glucagon antibody solid phase body are obtained. It is used as a solid phase for the separable element body. Examples of other simultaneous measurements include simultaneous measurement of α-fetoprotein, CEA, and ferritin, simultaneous measurement of T 3 and T 4 , and other appropriate combinations of various steroid hormones and peptide hormones. This can be done by using a divisible solid phase that incorporates various specific antibodies. It is also clear from the above description that the wall of the reaction tube may be used as one solid phase that can be divided. The reaction temperature and time vary depending on the object, but for example, in the case of simultaneous measurement of insulin and glucagon, the reaction time is 5°C and 8 to 48 hours. Generally, the time period is appropriate depending on the component to be measured.
The temperature and the concentration of the component to be measured can be selected. Furthermore, the amount of each liquid used may be adjusted based on the small test tube used and the size of the element used therein. Examples of methods for measuring insulin and glucagon content in serum are as follows. Buffer solution in a small reaction tube (15 x 105 mm, inner diameter 12.8 mm ± 0.1 mm), which is a solid phase substance for glucagon antibody.
Add 100μ of enzyme-labeled insulin solution, 50μ of enzyme-labeled glucagon solution, and 100μ of a solution containing insulin and glucagon at a constant concentration.
Next, an element which is a solid phase body of an insulin antibody having the size and shape described above was added to this, and the mixture was incubated at 5°C for 15 minutes.
Perform time incubation. After the reaction is complete, add 5 ml of distilled water or physiological saline to wash this element, and then add 500~500ml of enzyme substrate solution in advance.
Transfer the element to another small test tube in which 1000μ of the sample has been dispensed, and perform the enzyme reaction at 37°C for 30-120 minutes. Add 1.5 to 2.0 ml of reaction stop solution to stop the reaction, remove the elements, and then measure the components consumed and produced based on the enzymatic reaction. After the washing solution is removed from the reaction tube using an aspirator or decantation, about 2 to 3 ml of the enzyme substrate solution is added in the same manner as above to allow the enzyme reaction to occur, and then the absorbance of the solution is measured. The amounts of insulin and glucagon in the subject sample are determined from the labeling curve determined from the absorbance of a labeling solution of known concentration. Although it is measured as described above, a more detailed explanation will be added. 6,6-nylon having the shape of the element of the present invention (see Figure 5) (hereinafter referred to as 6,6 nylon measuring element) was activated by the immicro method using phosphorus pentachloride, and hexamethylene diamine and glutaraldehyde were activated. Processing yields a spacer-introduced 6,6 nylon measuring element. This is reacted with the IgG fraction of rabbit anti-guinea pig IgG serum to immobilize the IgG. Next, this was reacted with guinea pig insulin antibody serum, and 6.
6-Nylon element-anti-guinea pig IgG-
Prepare insulin antibody. Similarly, the IgG fraction of rabbit anti-guinea pig IgG serum was reacted with
A 6,6-nylon element-anti-guinea pig IgG-glucagon antibody is prepared by reacting the guinea pig glucagon antibody serum with the antibody immobilized with IgG. Then this 6,6-nylon element-anti-guinea pig IgG-insulin antibody, and 6,
6-Nylon element-anti-guinea pig IgG-
Each solid phase body of glucagon antibody is cut in half using a stainless steel cutter to form a semicircular shape. Each element fragment is then combined to form an element body. Next, this element body is reacted with serum containing a β-galactosidase-insulin conjugate, a β-galactosidase-glucagon conjugate, and various concentrations of labeled insulin and glucagon, or both,
After washing, the element body is removed, each fragment is separated, and β-galactosidase activity is measured to prepare a standard curve. The insulin and glucagon contents in the sample are simultaneously measured using a standard curve using standard insulin and glucagon. As described above, by using the distinguishable labeled compound of the present invention, two or more immune reactions can be performed in a short time with an extremely small amount of sample reaction solution, and as a result, trace amounts of components can be accurately detected with a small amount of sample. And it can be measured in a short time. Next, examples of the measurement method will be described, but it goes without saying that the present invention is not limited thereto. Example 1 Simultaneous measurement of insulin and glucagon by EIA [Preparation method of solid phase [test tube]-A] Polystyrene test tube (15 x 105 mm, inner diameter 12.8 ±
2 ml of sulfuric acid was added to 2 ml of sulfuric acid (pH 8.0) and reacted at 5°C for 6 hours to introduce SO 3 C - groups. ) and reacted overnight at 5°C to obtain test tubes in which 8 μg was immobilized per tube.
Next, add a 30,000-fold dilution of anti -16-29 glucagon fragment serum (guinea pig) to this test tube (0.25
%BSA, 5mM EDTA, 3mM MgCl2 , 0.15
Add 2 ml of diluted solution consisting of 0.01 M phosphate buffer (PH7.4) containing mM NaOl, 0.1% NaN3 ,
After reacting overnight at °C,
16-29 glucagon fragment antibody] conjugate was obtained. [Preparation method of solid phase body [element]-B] A polystyrene element having the size and shape shown in Fig. 5 is activated with sulfuric acid, SO 3 - groups are introduced, and this is injected with anti-guinea pig IgG rabbit serum.
0.1M phosphate buffer (PH) containing 20μg of IgG fraction
8.0) Polystyrene element solid support was immersed in 1 ml and reacted overnight at 5°C to obtain a polystyrene element solid support on which 10 μg of IgG was immobilized each. Next, this element solid phase was immersed in 1 ml of a 23,200-fold diluted serum containing guinea pig insulin antibody (described in the previous section) and allowed to react overnight at 5°C to form a [element-anti-guinea pig IgG-insulin antibody] conjugate. A solid phase was obtained. [Preparation method of β-galactosidase-insulin] 60 mg of insulin was added to 0.1 M phosphate buffer (PH
8.0) Dissolve in 17 ml, add 1.7 ml of dimethylformamide containing 6.5 mg of 3-(2'-benzothiazolyl-dithio)propionic acid succinimide ester, react for 1 hour under cooling, and after the reaction, the pH becomes 5.0 and precipitates. This precipitate was dissolved in 4.0ml of 0.1M phosphate buffer (PH7.0) (8.9mg/ml as insulin), 10.0μ of this was collected, and 6mg of β-galactosidase was added to it. The mixture was added and reacted for 1 hour, and then the reaction solution was transferred to Cephadex G.
In a column (1.5 x 120 cm) packed with −100
Elute with 0.01M phosphate buffer (PH7.4) containing 0.15M NaCl, collect 65-73ml fractions [(insulin)-CO-( CH2 ) 2 -S-(β-galactosidase)] Insulin-β-galactosidase conjugate (β-galactosidase) consisting of the amino group of insulin and the thiol group of β-galactosidase is
-Gal-labeled insulin) (insulin 2.4 μg/ml and a conjugate of 1 molecule of insulin per molecule of β-galactosidase, and has 95% of the activity of the insulin conjugate against insulin antibodies) obtained). [Method for preparing β-galactosidase- 19-29 glucagon fragment] 0.4 mg of glucagon fragment (19-29) at 50
Dissolve in 0.4 ml of mM phosphate buffer (PH8.0), add 10μ of 100 mM EDTA aqueous solution and 0.1% 3-
625μ of a dimethylformamide solution containing (2'-benzothiazolyl-dithio)propionic acid succinimide ester was added and reacted at 5°C for 1 hour. After the reaction, this was transferred to Cephadex G-15 (1.5 x 40
The pass-through fraction was obtained by gel filtration with a 3-cm column (containing a derivative in which a 3-(2'-benzothiazolyl-dithio)propionyl group was introduced at the N-terminus of glucagon fragment (19-29)). Then,
50 containing 20 μg of this 3-(2′-benzothiazolyl-dithio)propionyl group-introduced derivative.
Add 50mM phosphate buffer (PH7.0) containing 6.19mg of β-galactosidase to 2ml of mM phosphate buffer (PH7.0).
7.0) was added and reacted at room temperature for 1 hour. The reaction solution was gel-filtered through a Cephadex G-150 (1.5 x 90 cm) column, and the fraction that passed through was collected and bonded to the N-terminus of the glucagon fragment (19-29) and the thiol group of β-galactosidase. A β-galactosidase-glucagon fragment (19-29) conjugate (purity 95%) (β-Gal labeled glucagon) was obtained. Simultaneous measurement method of insulin and glucagon by EIA Immune reaction buffer (0.25% BSA, 5mM EDTA, 3mM MgCl 2 ,
100μ of 0.15M NaCl, 0.01M phosphate buffer (PH7.4) containing 0.1% NaN3 , 50μ of β-Gal labeled insulin (20.0pg as insulin), 50μ of β-Gal labeled glucagon (as glucagon fragment)
2.0pg) 50μ, and standard solution (5-640μ/ml as insulin and 25-640μ/ml as glucagon)
Add 100 μ of a liquid containing 3200 pg/ml, add 1 piece of solid phase-B (element), and incubate at 5°C.
The reaction was carried out at night. After completion, 5 ml of distilled water was added and solid phase B was first extracted while being washed.
0.1% BSA, 0.15M NaCl, 1mM
0.01M phosphate buffer containing MgCl2 , 0.1% NaN3 (PH
6.7) Solution consisting of 85 parts and 15 parts of methanol solution containing 200mM mercaptoethanol] Solid phase substance-B was added to a small test tube containing 500μ, and reacted at 37°C for 1 hour. After the reaction is complete, remove the reaction stop solution [100 m
After adding 2 ml of M glycine-NaOH buffer (PH 11.0) to stop the reaction and removing solid phase-B, the absorbance of the solution at 420 nm was measured. Next, after removing the washing solution by decantation, the solid phase body A was prepared using the β-galactosidase activity measurement solution (described above).
2 ml was added and reacted at 37℃ for 2.5 hours. After the reaction was completed, 500μ of reaction stop solution [400mM glycine-NaOH buffer (PH11.0)] was added to stop the reaction, and the absorbance at 420 nm of the reaction solution was measured. A quantitative curve was created. The results are shown in Figure 7, and in Figure 7, 〇-〇 indicates the quantitative curve of insulin, and △
−△ indicates the quantitative curve of glucagon;
The simultaneous measurement system for insulin and glucagon of the present invention showed an extremely good quantitative curve.
In addition, when measuring insulin and glucagon in the test sample (serum, plasma, etc.), use a standard solution of 100μ
Instead, 100μ of the sample was added, the same operation as above was performed, and the amounts of insulin and glucagon in the sample were determined by comparing with the quantitative curve. Example 2 Simultaneous measurement of insulin and glucagon by RIA [Preparation method of solid phase [test tube]-A] Prepared in the same manner as above. [Preparation method of solid phase [element]-B] 50 6,6 nylon elements having the size and shape shown in Figure 5 were stirred for 2 days in 60 ml of benzene containing 4 g of phosphorus pentachloride and 4 g of pyridine. Afterwards, it was washed four times with benzene to obtain an immicrolided 6,6 nylon element.
Add 50 ml of an aqueous solution (PH 11.5) containing 100 mM hexamethylene diamine, react at room temperature for 1 hour, wash thoroughly with 0.1 M phosphate buffer (PH 8.0), and add 4% glutaraldehyde to this solution.
Add 50ml of 0.1M phosphate buffer (PH8.0) and react at room temperature for 1 hour.
8.0), and spacers were added to 6,6 nylon elements (50 pieces) in 50 ml of 0.1 M phosphate buffer (PH 8.0) containing 5 mg of the IgG fraction of anti-guinea pig IgG serum (rabbit). The mixture was reacted overnight at 5° C. to obtain a nylon element solid phase on which 50 μg of IgG was immobilized per piece. Next, a 23200-fold dilution of serum containing guinea pig insulin antibodies [0.25% BSA, 5mM EDTA, 3mM
50 ml ( 250
mg of insulin antibody) and reacted overnight at 5°C to form a solid phase of [nylon element-anti-guinea pig IgG-insulin antibody] conjugate.
I got a B. [Method for preparing 125 I-insulin] 125 I-insulin can be prepared using WMHunter,
FC Greenwood et al.'s method [Nature (London)]
194, 495 (1962)] and gel filtration was performed using a Sephadex G-25 column (0.9 x 20 cm) to obtain 125 I-insulin. [ 125 I-[Tyr 18 ]-Glucagon (18-29) preparation method] (A) Production of [Tyr 18 ]-Glucagon (18-29) H-Tyr-Ala-Gln-Asp-Phe-Val-
Production of Gln-Trp-Leu-Met-Asn-Thr-OH Boc-Tyr-(Bzl- Cl2 )-Ala-Gln-Asp
(OBzl) −Phe−Val−Gln−Trp−Leu−Met−
Asn-Thr(Bzl)-OBzl 2.04g (1mM), anisole 6ml, ethanedithiol 0.94mm (10m
M), 131 mg (1 mM) of skatole, and 0.62 ml (10 mM) of dimethyl sulfide were added. to this,
Introduce 70 ml of anhydrous hydrogen fluoride (HF) and heat at 0°C.
After stirring for an hour, HF was quickly distilled off under reduced pressure. 100 ml of Et 2 O was added to the residue and the resulting precipitate was collected. This is dissolved in 100 ml of 50% acetic acid, centrifuged, and the supernatant liquid is lyophilized. Dissolve this in 20 ml of 8M urea solution and 20 ml of acetic acid, add 2 ml of thioglycolic acid, reduce at 45°C for 20 hours, and then use a Sephadex LH-20 column (4.2 x 137 cm).
and eluted with 50% acetic acid. The eluate was fractionated into 7 ml portions, and the 74th to 80th fractions were collected and lyophilized to obtain [Tyr 18 ]-glucagon (18-29).
Yield 120mg, Rf 2 0.73 (cellulose), amino acid analysis values: Asp1.97(2), Thr0.95(1), Gln2.07(2),
Ala0.98(1), Val0.94(1), Met0.98(1), Leu1(1),
Phe0.97(1), Tyr0.94(1), Trp1.05(1), the above raw materials were produced as follows. (1) P(19−29)Boc−Ala−Gln−Asp(OBzl)
−Phe−Val−Gln−Trp−Leu−Met−Asn
-Thr(Bzl)-OBzl [1] P(20-29) (Synthesis reported in Example 1 of JP-A-56-81540) 12.4 g (7.5 mM)
TFA 40ml, dimethyl sulfide 20ml, ethanedithiol 20ml, and skatole 983
After stirring at room temperature for 50 minutes, TFA was distilled off under reduced pressure. Ether was added to the residue, and the resulting precipitate was dissolved in a mixture of 150 ml of dry DMF and 50 ml of N-methylpyrrolidone. to this
Boc-Ala-OH2.0g (1.4 times equivalent) HOBt1.4
Add g (1.4 times equivalent) and WSCD1.92ml (1.4 times equivalent), adjust the pH to 7 with NMM, stir overnight, then add DMF50ml, HOBt0.51g,
0.71 g of Boc-Ala-OH and 0.69 ml of WSCD were added and stirred overnight. After distilling off DMF under reduced pressure, it was poured into ice water, and the resulting precipitate was washed twice with hot ethanol to obtain 11.35 g of the target product (yield: 87.8%).
I got it. TLC; Rf 1 0.75 (silica gel) Amino acid analysis; Asp1.92(2), Thr0.88(1),
Glu1.94(2), Ala0.88(1), Val0.91(1), Met0.95
(1), Leu1, Phe0.93(1), Trp0.71(1), (2) [Tyr 18 ]P(18−29)Boc−Tyr(Bzl−
Cl 2 )−Ala−Gln−Asp(OBzl)−Phe−Val−
Gln−Trp−Leu−Met−Asn−Thr(Bzl)−
OBzl[2] P(19-29)[1] 8.61g (5mM)
40ml TFA, 20ml dimethyl sulfide, 20ml ethanedithiol, and 983mg skatole
After stirring at room temperature for 50 minutes, under reduced pressure.
TFA was distilled off. Ether was added to the residue, and the resulting precipitate was dissolved in a mixed solution of 150 ml of DMF and 50 ml of N-methylpyrrolidone. To this, Boc−Tyr
(Bzl- Cl2 )-OH3.08g (1.4 times equivalent)
HOBt0.95g (1.4 times equivalent), WSCD1.28ml
(1.4 times equivalent) was added, the pH was adjusted to 7 with NMM, and the mixture was stirred at room temperature for 48 hours. After distilling off DMF under reduced pressure, ice water was added, and the resulting precipitate was washed three times with hot ethanol to obtain 9.00 g of the target product (yield:
88.0%). TLC; Rf 1 0.76 (silica gel) Amino acid analysis value; Asp1.90(2), Thr0.91(1),
Glu1.96(2), Ala0.89(1), Val0.90(1), Met0.93
(1), Leu(1), Phe0.95(1), Tyr0.92(1),
Tvp0.68.(1), In addition, the abbreviations described in this specification have the following meanings. Ala; L-alanine Gln; L-glutamine Asp; L-aspartic acid Asn; L-asparagine Phe; L-phenylalanine Val; L-valine Trp; L-tryptophan Leu; L-leucine Met; L-methionine Thr; L-threonine Boc; t-butoxycarbonine Bzl; benzyl OBzl; benzyl ester TFA; trifluoroacetic acid NMM; N-methylmorpholine DMF; dimethylformamide HOBt; 1-hydroxybenzotriazole WSCD; N-ethyl, N'-3- Dimethylaminopropyl-carbodiimide Bzl-Cl 2 ; 2,6-dichlorobenzyl The following carriers and developing solvents were used for thin layer chromatography (TLC). 1; Chloroform-methanol-acetic acid (80:25:2) Silica gel manufactured by Merck & Co.
Avt5721, 2; 1-butanol-pyridine-acetic acid-
Water (15:10:3:12) Merck cellulose
Avt5716 (B) Preparation of 125 I-[Tyr 18 ]-glucagon (18-29) Using [Tyr 18 ]-glucagon (18-29) obtained in (A) above, WMHunter, FC
The method of Greenwood et al. [Nature (London) 194 ,
495 (1962)] and gel-filtered with a Sephadex G-25 column (0.9 x 20 cm).
125 I-[Tyr 18 ]-glucagon (18-29) was obtained. [Simultaneous measurement of insulin and glucagon by RIA] Solid phase body [test tube]-A contains 100μ of immune reaction buffer (above), 125I -insulin (10.0pg as insulin) 50μ 125I- [Tyr 18 ]-glucagon ( 18-29) (4.5pg as glucagon) 50μ,
and standard solution (5-640μU/as insulin)
ml), contains 25-3200 pg/ml as glucagon)
Add 100μ and add solid phase (element)-B to this.
One piece of was added and the reaction was carried out at 5°C overnight. After completion of the test, 5 ml of physiological saline was added, and solid phase material B was first extracted while being washed, transferred to a new test tube, and its radioactivity was measured. Separately, the radioactivity of solid phase material A was measured after the washing liquid was removed by decantation. The results are shown in Figure 8.
Further, in FIG. 8, ○-○ indicates the quantitative curve of insulin, and Δ-△ indicates the quantitative curve of glucagon.The simultaneous insulin and glucagon measurement system of the present invention showed an extremely good quantitative curve. In FIG. 8, the horizontal axis shows insulin and glucagon concentrations, and the vertical axis shows B/Bo (%). Example 3 CEA and ferritin by Sanderuch method
Simultaneous measurement with EIA [Preparation method of solid phase substance [test tube]-A] Polystyrene test tube (15 x 105 mm, inner diameter 12.8±
Add 2 ml of concentrated sulfuric acid to 0.1 mm), react at 5°C for 6 hours to introduce -SO 3 - group, and add 0.1 M phosphate buffer (PH8. 0) Add 2 ml and react at 5℃ overnight.
[Test tube - EIA antibody] Solid phase body A of the conjugate was obtained. [Preparation method of solid phase body [element]-B] 50 elements made of 6,6 nylon at 30% (V/
V) Add to 50 ml of a methanol solution containing dimethyl sulfate, react at 37°C for 50 minutes, wash thoroughly with methanol, add 50 ml of an aqueous solution (PH 11.5) containing 100 mM hexamethylene diamine, and react for 1 hour at room temperature. Wash thoroughly with M phosphate buffer (PH8.0), add 50 ml of 0.1M phosphate buffer (PH8.0) containing 4% (V/V) glutaraldehyde, and react at room temperature for 1 hour. After washing with 0.1M phosphate buffer (PH8.0), a spacer was introduced into a 6,6 nylon element. These elements (50 pieces) were added to 50 ml of 0.1 M phosphate buffer (PH8.0) containing 5 mg of the IgG fraction of anti-ferritin rabbit serum.
[Element-
Ferritin antibody] A solid phase body B of the conjugate was obtained. [Preparation method of β-Gal-labeled ferritin antibody] An IgG fraction was obtained from ferritin antiserum by ammonium sulfate fractionation, and the antibody was further purified by affinity chromatography using Sepharose 4B antibody on which ferritin was immobilized. 4 mg of purified antibody
Dissolve in 1.8ml of 0.1M phosphate buffer (PH8.0) and add 3-
200 μg of a dimethylformamide solution containing 14.88 μg of (2'-benzothiazolyl-dithio)propionic acid succinimide ester and 20 μg of a 100 mM EDTA aqueous solution were added, and the mixture was reacted at 5° C. for 1 hour. After the reaction, this was mixed with Cephadex G-15 (1.5×
The flow-through fraction was obtained by gel filtration with a 40cm column (3-(2'-
[Including derivatives with benzothiazolyl-dithio)propionyl groups introduced]. Next, 3.33 mg of β-galactosidase was added to 2 ml of 50 mM phosphate buffer (PH7.0) containing 1.5 mg of this 3-(2′-benzothiazolyl-dithio)propionyl group-introduced derivative.
2 ml of 50 mM phosphate buffer (PH7.0) was added, and the mixture was reacted at room temperature for 2 hours. Transfer the reaction solution to Sephadex G-
The gel was filtered through a 150 (1.5 x 90 cm) column and the flow-through fraction was collected to obtain a β-galactosidase labeled ferritin antibody conjugate (purity 98%) (β-Gal labeled ferritin antibody). [Preparation method of β-Gal-labeled CEA antibody] An IgG fraction was obtained from CEA antiserum by ammonium sulfate fractionation, and the antibody was further purified by affinity chromatography using Sepharose 4B antibody on which CEA was immobilized. 4 mg of purified antibody in 0.1M phosphate buffer (PH8.0) 1.8
3-(2′-benzothiazolyl-dithio)pyropionic acid succinimide ester dissolved in
200μ of dimethylformamide containing 14.88μg, and
20μ of 100mM EDTA aqueous solution was added and reacted at 5°C for 1 hour. After the reaction, this was gel-filtered through a Cephadex G-15 (1.5 x 40 cm) column to obtain the flow-through fraction [3-(2'-benzothiazolyl-dithio)propionyl group was introduced into the amino group of the CEA antibody. [including derivatives]. Then,
50 containing 1.5 mg of this 3-(2′-benzothiazolyl-dithio)propionyl group-introduced derivative.
50mM phosphate buffer (PH7.0) containing 3.33mg of β-galactosidase in 2ml of mM phosphate buffer (PH7.0)
2 ml was added and reacted at room temperature for 1 hour. The reaction solution was gel-filtered through a Cephadex G-150 (1.5 x 90 cm) column, the flow-through fraction was collected, and β-galactosidase labeled CEA antibody conjugate (purity 98%) (β
-Gal-labeled CEA antibody) was obtained. [Simultaneous measurement of CEA and ferritin by EIA] In solid phase body [test tube]-A, add 200 μl of immune reaction buffer (above) and standard solution (2.5 to 2.5 μl as CEA).
160ng/ml, and as ferritin, 5~
320ng/ml) was added thereto, one solid phase [element]-B was added thereto, and the reaction was carried out at 37°C for 5 hours. After completion, the internal solution was removed by suction, and 5 ml of physiological saline was added to wash the solid phase bodies-A and B. This was combined with β-Gal-labeled ferritin antibody and β-Gal-labeled ferritin antibody.
Immunoreaction buffer containing Gal-labeled CEA antibody
The reaction was carried out for 18 hours at room temperature.
After finishing, add 5 ml of physiological saline and first add solid phase-B.
The sample was taken out while being washed, and added to a small test tube to which 500μ of the β-galactosidase activity measurement solution (above) had been added in advance, and allowed to react at 37°C for 3 hours.
2 ml of reaction stop solution (100mM glycine-NaOH buffer (PH11.0)) was added to stop the reaction, and after removing solid phase B, the absorbance of the solution at 420 nm was measured. In A, after the washing solution was removed by decantation, 2 ml of the β-galactosidase activity measurement solution (above) was added, and the reaction was allowed to proceed at 37°C for 5 hours.
0.5 ml of NaOH buffer (PH 11.0) was added to stop the reaction, and the absorbance of the reaction solution at 420 nm was measured.
As shown in Figure 9, and in Figure 9, 〇-〇 are
A quantitative curve of CEA is shown, and Δ-Δ is a quantitative curve of ferritin. The CEA and ferritin simultaneous measurement system of the present invention showed an extremely good quantitative curve. Example 4 Thyroxine [T 4 ] and thyronine [T 3 ]
Simultaneous measurement by EIA [Preparation method of solid phase body [element]-A] Four polystyrene element fragments shown in Fig. 6 (element fragments obtained by dividing the element into four equal parts in Fig. 5) are combined (dividable) into four pieces. The element was activated with sulfuric acid and SO 3 -
IgG of anti-rabbit IgG goat serum
100mM phosphate buffer (PH) containing 20μg of fractions
8.0) Polystyrene element solid phase was immersed in 1 ml and reacted overnight at 5°C to obtain 8.4 μg of IgG immobilized on each polystyrene element solid phase. Next, this element solid phase was immersed in 1 ml of an 8000-fold dilution of anti-T 4 -OMe serum (rabbit) (described in the previous section) and allowed to react at 5°C overnight to determine the [element-anti-rabbit IgG-T 4 antibody] conjugate. Solid phase body-A was obtained. [Preparation method of solid phase body [element]-B] A polystyrene element having exactly the same shape as shown in Fig. 6 (phthalocyanine blue was mixed in during molding and colored pale blue) was treated in the same manner as above to obtain anti-rabbit IgG- An immobilized element solid phase body was obtained. Next, the solid phase body was immersed in 1 ml of a 20,000-fold dilution of T3 - OEt serum (rabbit) and reacted overnight at 5°C to form a solid phase body of the [element-anti-rabbit IgG- T3 antibody] conjugate.
I got a B. [Preparation of solid phase element [element]-C] One fragment of the solid phase element-A that was divided again and three fragments of the solid phase element-B were separated into two pieces with sharp edges and a stopper in the center. They were combined in a coupling machine having a T 4 , T 3 -solid phase for simultaneous measurement [element]-C was prepared. [Preparation method of T 4 -OMe] 600 mg of T 4 (manufactured by Sigma) was suspended in 15 ml of methanol, dried hydrogen chloride gas was blown into the suspension at -10°C until saturation, and the suspension was stirred at room temperature for 20 hours. 0 more
After blowing in dry hydrogen chloride gas at °C, the precipitate precipitated was collected by filtration, washed with methanol containing hydrogen chloride gas, dried under reduced pressure in a desiccator containing solid sodium hydroxide, and then recrystallized with methanol-ether. , the desired T 4 -OMe 582.5 mg (yield 91.2%) was obtained. [Preparation method of β-Gal-labeled T 4 -OMe] 2 mg of T 4 -OMe was dissolved in 90% dimethylformamide, 10
%100mM phosphate buffer (PH8.0) 2ml,
1.78 ml of dimethylformamide containing 1.78 mg of 3-(2'-benzothiazolyldithio)propionic acid succinimide ester was added, and the mixture was allowed to react for 1 hour under cooling. The reaction solution was added to a LH-20 (1.5 x 40 cm) column, and 90% dimethylformamide, 10%
Gel filtration was performed using a 100 mM phosphate buffer (PH5.0) as a solvent. The eluate was fractionated into 2 ml portions, and 3-(2'-benzothiazolyl-dithio) was added to the amino group of T4 .
Fraction containing a propionyl group-introduced derivative (12
~15th item) was collected. Next, β-galactosidase 2 was added to 2 ml of a 10% dimethylformamide solution in a 90% phosphate buffer (PH8.0) containing 8 μg of this 3-(2′-benzothiazolyl-dithio)propionyl group-introduced derivative.
mg was added thereto, and the mixture was allowed to react at room temperature for 2 hours. The reaction solution was added to a Sephadex G-150 (1.5 x 90 cm) column, gel filtration was performed with 10 mM phosphate buffer (PH7.4) containing 0.15 M NaCl, and the flow-through fraction was collected. 4 and the thiol group of β-galactosidase.
-Galactosidase- T4 -OMe conjugate (purity 93
%) (β-Gal labeled T4 -OMe) was obtained. [Preparation method of T 3 -OEt] 500 mg of T 3 (manufactured by Sigma) was suspended in 15 ml of ethanol, dried hydrogen chloride gas was blown into the suspension at -10°C until saturation, and the suspension was stirred at room temperature for 20 hours. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, benzene was added, and the deposited precipitate was collected and dried. Furthermore, the target T 3 -OEt was 515 mg (yield 93.7%) by suspending and washing in benzene.
I got it. [Preparation method of β-Gal-labeled T 3 -OEt] 2 mg of T 3 -OEt was dissolved in 90% dimethylformamide, 10
Dissolve in 2 ml of 3-% phosphate buffer (PH8.0) and
2.06 ml of dimethylformamide containing 2.06 mg of (2'-benzothiazolyldithio)propionic acid succinimide ester was added, and the mixture was allowed to react for 1 hour under cooling. Add the reaction solution to a LH-20 (1.5 x 40cm) column, add 90% dimethylformamide, 10% 100m
Gel filtration was performed using M acetate buffer (PH5.0) as a solvent. The eluate was fractionated into 2 ml portions, and fractions (12th to 15th fractions) containing a derivative in which a 3-(2'-benzothiazolyl-dithio)propionyl group was introduced into the amino group of T3 were collected. Next, 2 mg of β-galactosidase was added to 2 ml of a 10% dimethylformamide solution in 90% phosphate buffer (PH 8.0) containing 7.2 μg of this 3-(2'-benzothiazolyl-dithio)propionyl group-introduced derivative, and the mixture was heated at room temperature. The mixture was allowed to react for 2 hours. The reaction solution was added to a Sephadex G-150 (1.5 x 90 cm) column, gel filtration was performed with 10 mM phosphate buffer (PH7.4) containing 0.15 M NaCl, and the flow-through fraction was collected. β-galactosidase-T 3 -OEt conjugate (purity:
95%) (β-Gal labeled T 3 -OEt) was obtained. [Preparation method of T 4 -BSA conjugate] Add 25 mg of T 4 -OMe to 17.5 ml of dimethylformamide.
Contains 6 mg of glutaraldehyde, dissolved in
Add 16ml of 10mM phosphate buffer (PH8.0) and stir at room temperature.
The reaction was allowed to proceed for 30 minutes. Next, 20 ml of 10 mM phosphate buffer (PH8.0) containing 113.6 mg of BSA was added to this.
The mixture was further reacted at room temperature for 2 hours. The reaction solution was sufficiently dialyzed against distilled water, the dialyzed solution was freeze-dried, and the BSA-T 4 -OMe conjugate (binding molar ratio 1:
8.6) was obtained. [Preparation method of T 3 -BSA conjugate] 25 mg of T 3 -OEt was dissolved in 17.5 ml of dimethylformamide, and 10 molar solution containing 7 mg of glutaraldehyde was added under stirring.
Add 16ml of M phosphate buffer (PH8.0) and incubate for 30 minutes at room temperature.
Allowed to react for minutes. This then contains 118mg of BSA.
20 ml of 10 mM phosphate buffer (PH8.0) was added, and the mixture was further reacted at room temperature for 2 hours. The reaction solution was dialyzed with distilled water, and the dialyzed solution was freeze-dried to obtain a BSA-T 3 -OEt conjugate (bond molar ratio 1:12.6). [Preparation of antibodies] 1 mg of each BSA-T 4 -OMe and BSA-T 3 -OEt conjugate (95 μg as T 4 -OMe, 116 μg as T 3 -OEt) was added to a 10 mM phosphate buffer containing 0.15 M NaCl (PH7. 4) Dissolve in 1 ml and add Freund's Complete Adjuvant (Freund's
Complete adjuvant) 1.0 ml was added and thoroughly mixed to obtain an emulsion. Next, each rabbit was injected with 2 ml of the emulsion into the skin of the fingers and toes and subcutaneously at several locations on the back. The same amount of emulsion was subcutaneously injected 8 times every 2 weeks,
Whole blood was collected 10 days after the final immunization, left at room temperature for 60 minutes to coagulate, and then incubated at 3000 rpm for 10 minutes.
Anti- T4 -OMe serum (rabbit), respectively, by centrifugation for min.
Anti- T3 -OEt serum (rabbit) was obtained. [Simultaneous measurement of T 4 and T 3 by EIA] In a test tube (15 x 105 mm, inner diameter 12.8 ± 0.1 mm), 100 μl of immunoreaction buffer (described above) and β-Gal labeled T 4 −
OMe (approximately 2.0ng as T4 ) 50μ, β-Gal labeled T3
−OEt (approximately 0.1 mg as T 3 ) 50μ, and standard solution (10-640 mg/ml as T 4 , 0.5-32 ng/ml as T 3 )
ml of solution) was added, one solid phase [Element]-C was added thereto, and the reaction was carried out at 37°C for 3 hours while rotating the solid phase at 30 minute intervals.
After completion of the reaction, 5 ml of distilled water was added and the solid phase -C was removed while being washed. Divide the washed solid phase into one solid phase fragment for T 4 and three solid phase fragments for T 3 , and immerse each in a test tube to which 2 ml of β-galactosidase activity measurement solution has been added in advance. Solid phase fragments for 30 min at 37 °C, T 3
The solid phase fragments were allowed to react for 2 hours. After the reaction is complete, add 0.5ml of reaction stop solution [400mM glycine-NaOH buffer (PH11.0)] to stop the reaction.
After removing the solid phase, 420nm of each liquid
The absorbance at was measured. The results are as shown in Figure 10, in which 〇-〇 indicates the quantitative curve of T 4 , △-△ indicates the quantitative curve of T 3 ,
The simultaneous measurement system for T 4 and T 3 of the present invention showed an extremely good quantitative curve. In addition, when measuring T 4 and T 3 in a test sample (serum, plasma, etc.),
The same operation as above was carried out by adding 100μ of the sample instead of 100μ of the standard solution, and the amounts of T 4 and T 3 in the sample were determined by comparing with the quantitative curve.
図面は本発明の好適な数個の実施例を示したも
のであり、第1図はエレメントの拡大部分断面
図、第2図はその平面図、第3図イ,ロ,ハはエ
レメント本体の上面部に形成する切込み溝のそれ
ぞれ異つた実施例を示す平面図、第4図は第2実
施例のエレメントの拡大断面図、第5図はエレメ
ント製作のための1例の実測図、第6図は4等分
したエレメントフラグメント、第7図はEIAによ
るインシユリンとグルカゴンの同時測定の定量曲
線、第8図はRIAによるインシユリンとグルカゴ
ンの同時測定の定量曲線、第9図はEIAによる
CEAとフエリチンの同時測定の定量曲線、第1
0図はEIAによるT4とT3の同時測定の定量曲線
を示す。
符号の説明、1はエレメント担体、2はエレメ
ント本体、4は反応管、5は突起、6は隙間部、
8は切込み溝である。
The drawings show several preferred embodiments of the present invention; FIG. 1 is an enlarged partial sectional view of the element, FIG. 2 is a plan view thereof, and FIGS. FIG. 4 is an enlarged sectional view of the element of the second embodiment; FIG. 5 is an actual measurement diagram of one example for manufacturing the element; FIG. The figure shows an element fragment divided into four equal parts, Figure 7 is a quantitative curve for simultaneous measurement of insulin and glucagon by EIA, Figure 8 is a quantitative curve for simultaneous measurement of insulin and glucagon by RIA, and Figure 9 is by EIA.
Quantitation curve for simultaneous measurement of CEA and ferritin, 1st
Figure 0 shows the quantitative curve of simultaneous measurement of T 4 and T 3 by EIA. Explanation of the symbols: 1 is the element carrier, 2 is the element body, 4 is the reaction tube, 5 is the protrusion, 6 is the gap,
8 is a cut groove.
Claims (1)
疫反応部に挿入可能な形状であつて、外径が反応
管の内径と僅かな間〓を保つように形成され、こ
の間〓と同一間〓を形成する突起部を底部に構成
して反応管とエレメント担体と間〓が同一となる
ように形成された1以上の分別可能な担体として
用いることできる媒体用エレメント担体と反応管
担体を用いて、その担体の各々に測定すべき成分
に対して特異的結合能を有する物質を固定化せし
めてなる固相体の2以上を用い、反応において反
応媒体の表面積を拡大せしめて反応せしめること
を特徴とする多成分同時測定法。 2 1以上の分別可能な担体として用いることで
きる媒体用エレメント担体が、2以上に分別可能
な媒体用エレメント担体である特許請求の範囲第
1項記載の多成分同時測定法。 3 反応媒体中、反応媒体の表面積を拡大せし
め、反応管の免疫反応部に挿入可能な形状であつ
て、外径が反応管の内径と僅かな間〓を保つよう
に形成され、この間〓と同一間〓を形成する突起
部を底部に構成して反応管とエレメント担体と間
〓が同一となるように形成された1以上の分別可
能な担体として用いることできる媒体用エレメン
ト担体と反応管担体を用いて、その担体の各々に
測定すべき成分に対して特異的結合能を有する物
質を固定化せしめてなる固相体の2以上に、測定
すべき成分を含有する液および結合能を有する標
識化合物を反応せしめ、次いでそれらの固相体を
回収し、それらの固相体に結合した標識化合物の
標識活性を測定することを特徴とする特許請求の
範囲第1項記載の多成分同時測定法。 4 測定すべき成分に対して特異的結合能を有す
る物質が受容体であり、測定すべき成分が抗原で
あり、結合能を有する標識化合物が標識抗原であ
るか、または、測定すべき成分に対して特異的結
合能を有する物質が抗原であり、測定すべき成分
が受容体であり、結合能を有する標識化合物が標
識受容体であるか、または、測定すべき成分に対
して特異的結合能を有する物質が受容体であり、
測定すべき成分が抗原であり、結合能を有する標
識化合物が受容体と抗原とが反応した後に反応せ
しめるための抗原に対して結合能を有する標識受
容体であるか、または、測定すべき成分に対して
特異的結合能を有する物質が抗原であり、測定す
べき成分が受容体であり、結合能を有する標識化
合物が抗原と受容体が反応した後に反応せしめる
ための受容体に対して結合能を有する標識抗原で
ある、同一または異なる測定にて測定してなる特
許請求の範囲第3項記載の多成分同時測定法。 5 標識が、酵素または放射性物質である特許請
求の範囲第3項または第4項記載の多成分同時測
定法。 6 固相体が、担体−第2抗体−第1抗体である
特許請求の範囲第1項、第3項または第4項記載
の多成分同時測定法。[Scope of Claims] 1. The reaction medium has a shape that expands the surface area and can be inserted into the immune reaction part of the reaction tube, and is formed so that the outer diameter maintains a slight distance from the inner diameter of the reaction tube. Reaction with one or more media element carriers that can be used as a separable carrier, which are formed with protrusions on the bottom that form the same gap as the reaction tube and the element carrier. The surface area of the reaction medium is expanded in the reaction by using a tubular carrier and using two or more solid phase bodies each having a substance that has specific binding ability for the component to be measured immobilized on each of the carriers. A multi-component simultaneous measurement method characterized by reaction. 2. The multi-component simultaneous measurement method according to claim 1, wherein the media element carrier that can be used as one or more separable carriers is a media element carrier that can be separable into two or more. 3 The reaction medium has a shape that expands the surface area of the reaction medium and can be inserted into the immune reaction part of the reaction tube, and is formed so that the outer diameter maintains a slight distance from the inner diameter of the reaction tube, and during this time A media element carrier and a reaction tube carrier that can be used as one or more separable carriers that are formed with protrusions forming the same gap on the bottom so that the gap between the reaction tube and the element carrier is the same. A liquid containing the component to be measured and a solid phase body having the binding ability to immobilize a substance having specific binding ability for the component to be measured on two or more of the carriers. Simultaneous multi-component measurement according to claim 1, characterized in that the labeled compounds are reacted, their solid phase bodies are then collected, and the labeling activity of the labeled compounds bound to the solid phase bodies is measured. Law. 4. The substance that has specific binding ability for the component to be measured is a receptor, the component to be measured is an antigen, and the labeled compound that has binding ability is a labeled antigen, or The substance that has specific binding ability to the antigen is the antigen, the component to be measured is the receptor, and the labeled compound that has the binding ability is the labeled receptor, or the substance that has specific binding ability to the component to be measured is the labeled receptor. The substance that has the ability is the receptor,
The component to be measured is an antigen, and the labeled compound having binding ability is a labeled receptor having binding ability to the antigen to be reacted after the receptor and antigen have reacted, or the component to be measured is The substance that has specific binding ability is the antigen, the component to be measured is the receptor, and the labeled compound that has the binding ability binds to the receptor for reaction after the antigen and receptor have reacted. 4. The method for simultaneous multi-component measurement according to claim 3, wherein the multi-component simultaneous measurement method is performed using the same or different measurements. 5. The method for simultaneous measurement of multiple components according to claim 3 or 4, wherein the label is an enzyme or a radioactive substance. 6. The method for simultaneous multi-component measurement according to claim 1, 3 or 4, wherein the solid phase body is a carrier-second antibody-first antibody.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP57058612A JPS58174851A (en) | 1982-04-08 | 1982-04-08 | Method of simultaneously examining multiple components |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP57058612A JPS58174851A (en) | 1982-04-08 | 1982-04-08 | Method of simultaneously examining multiple components |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS58174851A JPS58174851A (en) | 1983-10-13 |
JPH0251151B2 true JPH0251151B2 (en) | 1990-11-06 |
Family
ID=13089357
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP57058612A Granted JPS58174851A (en) | 1982-04-08 | 1982-04-08 | Method of simultaneously examining multiple components |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS58174851A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0546128U (en) * | 1991-11-13 | 1993-06-18 | 株式会社精工舎 | Electric field strength display |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE9302423U1 (en) * | 1993-02-19 | 1993-04-15 | Müller, Frank Joachim, 4150 Krefeld | Microtitration unit |
JPH08178926A (en) * | 1994-10-25 | 1996-07-12 | Sumitomo Pharmaceut Co Ltd | Immunoassay plate and use thereof |
JP5465758B2 (en) * | 2011-09-26 | 2014-04-09 | 富士フイルム株式会社 | Thyroxine immunoassay using fluorescent particles |
-
1982
- 1982-04-08 JP JP57058612A patent/JPS58174851A/en active Granted
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0546128U (en) * | 1991-11-13 | 1993-06-18 | 株式会社精工舎 | Electric field strength display |
Also Published As
Publication number | Publication date |
---|---|
JPS58174851A (en) | 1983-10-13 |
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