JPH0532705B2 - - Google Patents
Info
- Publication number
- JPH0532705B2 JPH0532705B2 JP58047834A JP4783483A JPH0532705B2 JP H0532705 B2 JPH0532705 B2 JP H0532705B2 JP 58047834 A JP58047834 A JP 58047834A JP 4783483 A JP4783483 A JP 4783483A JP H0532705 B2 JPH0532705 B2 JP H0532705B2
- Authority
- JP
- Japan
- Prior art keywords
- antigen
- sensitized
- monoclonal antibody
- latex
- carrier
- 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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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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
本発明は、完全抗原の新しいタイプの免疫化学
的測定方法及びその方法を利用するのに適した新
しいタイプの測定試薬に関する。
とくに、本発明は例えば血液、尿その他の体液
や体液成分などの如き被検体中の微量な抗原を免
疫化学的に測定する測定方法及びその試薬に関
し、優れた確実性、信頼度、精度、敏感性及び高
い特異性をもつて、偽反応生起のトラブルを伴う
ことなしに、再現性良く安定且つ容易な操作で、
短時間に被検体中の微量な抗原を免疫化学的に測
定できる新しいタイプの測定方法及びその方法に
利用するに適した新しいタイプの測定試薬に関す
る。
更に詳しくは、本発明は、
(A) 完全抗原感作担体及び
(B) 上記抗原に対する単一種のモノクロナル抗体
感作担体
を用い、被検体中の該抗原による上記(A)及び(B)両
試薬成分の凝集阻止反応を測定することを特徴と
する上記抗原の免疫化学的測定方法に関する。本
発明はまた、
(A) 完全抗原感作担体及び
(B) 上記抗原に対する単一種のモノクロナル抗体
感作担体
から成ることを特徴とする上記抗原の免疫化学的
測定試薬にも関する。
従来、例えば血液、尿その他の体液や体液成分
などの如き検体中に存在する生物学的活性を有す
る微量物質を、免疫化学的手段で測定する方法は
古くから知られている。かかる免疫化学的測定法
として、例べば赤血球を担体として用い、これを
抗原又は抗体で感作し、これを被検液中の抗体又
は抗原と反応させ、その際、免疫化学的凝集又は
凝集阻止反応を生起させて、該微量物質を測定す
る方法も既に知られている。また、担体として赤
血球の代りに、非生物学的粒子として例えば合成
樹脂ラテツクス、ベントナイト、コロジオン、コ
レステロール結晶、水晶等を免疫化学反応におけ
る固体担体として用いることも知られている（以
上例えば特開昭50−82230号公開公報）。
このような抗原抗体反応を利用した免疫化学的
手法によつて被検体中の抗原の存在（定性）もし
くはその濃度（定量）を測定検出するのに従来も
つとも普通に利用されてきた抗体感作担体は、ポ
リクロナル抗体感作担体であつた。
1975年C.Milsteinらがマウスのミエローマ細胞
と脾臓中の抗体産生細胞とを細胞融合し、該細胞
からモノクロナル抗体の産生に成功して以来、細
胞融合技術分野における著るしい技術の進歩に伴
つて、モノクロナル抗体産生細胞株の形成、それ
を利用したモノクロナル抗体の産生が容易とな
り、このようなモノクロナル抗体を利用した抗原
の免疫化学的測定方法に関する提案がなされるよ
うになつた。
このような提案として、特開昭57−86051号の
提案が知られている。この提案に於ては、２個又
はそれ以上の異種類のモノクロナル抗体が同一の
抗原に対応して用いられることを特徴とし、少く
とも２個の抗体分子に抗原を結合させる免疫化学
的反応を利用した抗原定量法が提案されている。
そして、この提案には、従来の抗体すなわちポリ
クロナル抗体が対応抗原と反応して沈殿物を生ず
る公知現象とは全く異なつて、モノクロナル抗体
は対応抗原と結合して沈殿物を生成することがな
く、モノクロナル抗体で被覆された例えば赤血
球、ラテツクス球、金属粒子などの如き担体粒子
が対応抗原の存在下で凝集しないことが記載され
ている。
この提案に於ては、従来公知の知見とは異つ
て、モノクロナル抗体は対応抗原と結合して沈殿
物を生成しないという事実があるにも拘わらず、
複数種の異種モノクロナル抗体を使用すると抗原
と結合して沈殿物を生成できるという新しい知見
が得られたことを記載し、それゆえに、この提案
においては、上述のとおり、少なくとも二種の異
種モノクロナル抗体の使用を必須とする抗原定量
法に特定されている。
更に、この提案には、このような複数種の異種
モノクロナル抗体の使用によつてもなお、測定の
感度と特異性に関して必ずしも好結果が得られる
わけではなく、全く効果的ではないことさえあり
得るため、可能な限りの結合の仕方を試みた上で
検討選択しなければならないことを記述してい
る。
このような複数種の異種モノクロナル抗体の使
用を必須とする類似の提案として、特開昭57−
118159号の提案も知られている。
そして、これらモノクロナル抗体を利用する抗
体原の免疫化学的測定方法に関する従来提案に於
ては、従来のポリクロナル抗体の場合とは異なつ
て、単一種のモノクロナル抗体は対応抗原と結合
して沈殿物を生成せず、複数種の異種モノクロナ
ル抗体の使用によつてはじめて沈殿物を生成でき
るという事実から当然のことながら、複数種の異
種モノクロナル抗体の使用が必須であるという点
で共通している。
しかしながら、前者の提案に記載されているよ
うに、モノクロナル抗体と対応抗原との凝集を生
じさせるために複数種の異種モノクロナル抗体を
担体に感作し、その感作担体を使用してもなお、
測定の感度と特異性に関して必ずしも好結果が達
成できるとはかぎらない欠陥があり、更には、利
用し得るような凝集さえ生ぜず、全く効果的でな
いことさえあるという重大な技術的欠点があつ
た。そして、満足すべき凝集性を与えるために、
例えばより多種の異種モノクロナル抗体を使用す
ればするほど、当然のことながら、モノクロナル
抗体の利用による高い特異性の利点はより多く失
われていく不都合を伴うことが回避できなういと
いう両立し難い技術的課題が生じていた。
すなわち、モノクロナル抗体は抗原分子中の一
つの特定部位を特異的に認識する能力を有する抗
体であるが、二種以上より多種のモノクロナル抗
体を利用すればするほど、二種以上より多種の特
定部位を同時に認識する結果となり、モノクロナ
ル抗体の利用による高い特異性の利点がより多く
失われていくという両立し難い技術的課題を伴
う。更に又、抗原分子の多数の部位を認識できる
従来のポリクロナル抗体利用の場合とは異なつ
て、上述したように、モノクロナル抗体は一つの
特定部位しか認識しないので、ポリクロナル抗体
利用の場合に比して、凝集反応における凝集性は
著るしく弱いことが予期され、事実、上述したよ
うに、モノクロナル抗体利用の従来提案において
は単一のモノクロナル抗体の利用では対応抗原と
結合して沈殿物を生成しないので複数種の異種モ
ノクロナル抗体の利用が必須であることを教え、
更に、前述した前者の提案においては、複数種の
異種モノクロナル抗体を利用してもなお、凝集が
生じない場合があるという技術的欠陥のあること
を開示している。
本発明者等は、モノクロナル抗体を免疫化学的
な抗原測定法に利用する際の上述の如き両立し難
い技術的課題ないし技術的欠陥を解決できる方法
を開発すべく研究を行つてきた。
その結果、モノクロナル抗体利用における前記
従来知見とは全く異なつて、完全抗原を担体に感
作させた完全抗原感作担体の場合には、該抗原に
対する単一種のモノクロナル抗体を担体に感作さ
せた単一種のモノクロナル抗体感作担体との間
に、満足すべき凝集反応が生起し、斯くて、モノ
クロナル抗体利用における従来知見に必須であつ
た複数種の異種モノクロナル抗体を利用する必要
が全くないという予想外の新しい知見を得た。
このモノクロナル抗体利用における全く新しい
知見に基いて更に研究を進めた結果、(A)完全抗原
感作担体及び(B)上記抗原に対する単一種のモノク
ロナル抗体感作担体の組み合わせから成る新しい
タイプの試薬を用い、被検体中の該抗原による上
記(A)及び(B)両試薬成分の凝集反応に対する凝集阻
止反応を測定するという新しいタイプの免疫化学
的な完全抗原測定方法が提供でき、顕著に優れた
確実性、信頼度、精度、感度及び顕著に高い抗原
特異性をもつて、偽反応生起のトラブルを伴うこ
となしに、優れた再現性、安定性且つ容易な操作
で、短時間に被検体中の微量な抗原を免疫化学的
に測定できることを見い出した。
更に又、上記の顕著に優れた作用効果は、対象
完全抗原に対するモノクロナル抗体の種類に実質
的な影響を受けない利点を有し、斯くて広汎な任
意の抗原の免疫化学的測定方法に適用可能である
ことがわかつた。
従つて、本発明の目的はモノクロナル抗体を利
用した完全抗原の新しいタイプの免疫化学的測定
方法及びその方法に利用するのに適した新しいタ
イプの測定試薬を提供するにある。
本発明の上記目的及び更に多くの他の目的なら
ばに利点は、以下の記載から一層明らかなとなる
であろう。
本発明に於いて完全抗原とは、生体内に於てそ
れ自体で抗体産性能を有する抗原を意味し、生体
内に於てそれ自体では抗体産生能を有しないハプ
テン（不完全抗原）を除外する意味である。換言
すると、それ自体で抗原性を有する抗原を指す。
このような完全抗原として任意の抗原が利用で
き、例えば、以下の如き抗原を例示することがで
きる。
（） ペプチドホルモンとしては、例えば
(i) 成長ホルモン（GH）、副腎皮質刺激ホル
モン（ACTH）、メラミン細胞刺激ホルモン
（MSH）、プロラクチン、甲状腺刺激ホルモ
ン（TSH）、黄体形成ホルモン（LH）、卵胞
刺激ホルモン（FSH）、オキシトシン等の下
垂体ホルモン、
(ii) カルシトニン、副甲状腺ホルモン等のカル
シウム代謝調節ホルモン、
(iii) インシユリン、プロインシユリン、膵グル
カゴン等の膵ホルモン、
(iv) ガストリン、セレクチン等の消化管ホルモ
ン、
(v) アンデオテンシン、ブラジキニン等の血管
に作用するホルモン、
(vi) ヒト胎盤性性腺刺激ホルモン（hCG）、ヒ
ト脱盤催乳ホルモン（hPL）等の胎盤ホルモ
ン、
等をあげることができ、また、
（） ホルモン以外の物質としては、例えば
(i) 前立腺性酸性フオスフアターゼ（PAP）、
アルカリ性フオスフアターゼ、トランスアミ
ナーゼ、乳酸脱水素酵素、トランスアミナー
ゼ、トリプシン、ペプシノーゲン等の酵素
(ii) α−フエトプロテイン（AFP）、ガン胎児
性抗原（CEA）等のガン特異物質
(iii) 免疫グロプリンＧ（IgG）、フイブリン−フ
イブリノゲン分解産物（FDP）、抗トロンビ
ン（AT）、トランスフエリン等の血清
タンパク成分、
(iv) その他として、リユマチ因子、セロトニ
ン、ウロキナーゼ、フエリチン、サブスタン
スＰ等の物質
等々の生体内に存在する成分及び代謝産物の如く
多くの完全抗原類をあげることができる。
本発明の完全抗原類は、しかし、上記例示の完
全抗原類に限定されるものではない。
本発明で利用する上記例示の如き完全抗原に対
するモノクロナル抗体は、細胞融合技術分野にお
いてそれ自体公知の手法を適宜に選択組み合わせ
てモノクロナル抗体産生融合細胞株を形成し、該
細胞株を利用して産生、取得することができる。
その一態様によれば、上記例示の如き完全抗原
を用いて、これを適当な動物たとえばマウス、ラ
ツト、ウサギ、ヒツギ、ウマ、ウシなどの如き動
物に、投与たとえばアジユバントと共に皮下注射
の如き手法で投与して、該動物を免疫したのち、
この免疫動物たとえば免疫マウスの該抗原に対す
る抗体産生細胞たとえば脾細胞、胸腺細胞、リン
パ節細胞および／または末梢血細胞の如き細胞を
採取し、該細胞と自己増殖性を有するが抗体産生
能を実質的に有しない適当な株化細胞たとえばマ
ウス骨髄腫株化細胞とを、それ自体公知の手法に
より細胞融合処理する。
単クローン性抗体を得るためのミエローマ細胞
と抗体産性細胞との組合せは、各細胞が融合して
増殖しつつ抗体を産生することが可能であれば、
それぞれの細胞の由来する動物の種類は限定され
ず、任意の組合せでよい。
使用されるミエローマ細胞は特に限定はなく、
多くのマウス、ラツト、ウサギ、ヒトなどの動物
の細胞体を使用することができる。好ましい株化
細胞は薬剤抵抗性のものであり、かつ末融合のミ
エローマ細胞が選択培地で生存せず、一方融合細
胞のみが生存するようなものが良い。最も普通に
用いられるものは８−アザグアニジン抵抗性の株
化細胞で、これはヒポキサンチン・グアニン・ホ
スホリボシル・トランスフエラーゼを欠損し、ヒ
ボキサンチン・アミンプテリン・チミジン
（HAT）培地に育生できない性質を有している。
さらに使用する株化細胞は「非分泌型」のもので
あることが好ましい。例えばマウスミエローマ
MOPC−21株由来のP3／×63−Ag8U1（P3U1）、
P3／×63−Ag8・６・５・３、P3／NSI−１−
Ag4−１、Sp2／Ｏ−Ag14、ラツトミエローマ
210・RCY3・Ag1・２・３などが好適に用いら
れる。
該細胞融合処理は、例えば、通常イーグル最少
基本培地（MEM）、RPMI−1640などの培地中で
上記免疫マウスの脾細胞１〜５×108個と上記マ
ウス骨髄腫株化細胞１〜５×107個とを、混合し
て行なうことができる。融合促進剤としては、平
均分子量1000〜6000のポリエチレングリコール
（PEG）が好ましく、他の融合促進剤、例えば、
ポリビニルアルコール、ウイルスなどを使用する
ことができる。PEGの使用濃度は約30〜50％で
用いることができる。
上述のようにして得ることのできる融合細胞含
有系から融合細胞を、それ自体公知の手法を利用
して、選別処理、抗体活性スクリーニング処理及
びクローニング処理して、免疫マウスと形成に用
いた完全抗原に対するモノクロナル抗体産生能を
有し且つ自己増殖能を持つ融合細胞株を取得する
ことができる。
上記融合細胞の選別処理は、例えば、20％ウシ
胎児血清含有RPMI−1640培地なで細胞融合を終
えた細胞を適当に希釈し、96穴マイクロプレート
に105〜106／ウエル程度に分注し、各ウエルに選
択培地（たとえばHAT培地）を加え、以後選択
培地交換を行いながら、５％CO2培養器（37℃）
で培養を続けることにより行うことができる。ミ
エローマ細胞として８−アザグアニン抵抗性株を
用いれば、末融合のミエローマ細胞はHAT培地
で死滅し、また抗体産生細胞は正常細胞なのでin
vitro培養では長期間生育できない。したがつて
培養後10〜14日ぐらいから生育してくる細胞は全
く融合細胞である。
上述のようにして得ることのできる融合細胞株
の抗体活性スクリーニング処理及びクローニング
処理は、常法により行うことができるが例えば、
以下のようにして行うことができる。
融合細胞の生育したウエルの培養上清の一部を
採取し、一定量の標識抗原とインキユベーシヨン
し、標識抗原との結合能を測定することにより、
目的とする抗体を分泌しているウエルを検索する
ことができる。即ち、125Ｉ、131Ｉなどのラジオア
イソトープあるいは酵素などで標識した抗原と培
養上清を反応させた後、各反応液について抗原−
抗体結合物を分離し、標識量を測定することによ
り、目的とする抗体の存在および結合能を検索す
ることができる。
目的とする抗体活性の認められる各ウエル中に
は２種以上の融合細胞が生育している可能性があ
るので、限界希釈法や軟寒天によるコロニー形成
法によりクローニングを行い、モノクロナル抗体
産生融合細胞株を得ることができる（このような
モノクロナル抗体産生融合細胞株は微工研の寄託
受託拒否対象である。）
上述のようにして得ることのできるモノクロナ
ル抗体産生細胞株を用いて、前記免疫動物の形成
に用いた完全抗原に対するモノクロナル抗体を取
得するには、該モノクロナル抗体産生細胞株を、
例えば適当な培地に培養し、培地からモノクロナ
ル抗体を採取する方法、ミエローマ細胞由来動物
と同系の動物に該細胞株を移植し腹水中のモノク
ロナル抗体を採取する方法など、それ自体公知の
手法を利用して取得することができる。
上記前者の態様によれば、例えば、モノクロナ
ル抗体産生融合細胞株を10％ウシ胎児血清含有
RPMI1640培地などの培養液で培養し、その培養
上清液を硫安分画、抗原を結合させたセフアロー
ス4Bなどのアフイニテイークロマトグラフイー
などによつて精製することにより目的とするモノ
クロナル抗体を採取することができる。
又、上記後者の態様によれば、例えば、同系動
物にプリスタン（２，６，10，14−テトラメチル
ペンタデカン）などの鉱物油を腹腔内投与した
後、融合細胞を腹腔内投与することによりin
vivoで融合細胞を大量に増殖させる。その結果、
形成される腹水には高濃度のモノクロナル抗体が
含まれている。この腹水から硫安分画及び必要に
応じて前記アフイニテイークロマトグラフイーな
どにより、目的とするモノクロナル抗体を取得す
ることができる。
上述のようにして取得できるようなモノクロナ
ル抗体は市販品として入手することも可能であ
り、利用できる。
本発明によれば、前記例示の如き完全抗原を適
当な担体に感作させた(A)完全抗原感作担体と、上
述のようにして取得できる上記抗原に対するモノ
クロナル抗体の単一種を適当な担体に感作させた
(B)上記抗原に対する単一種のモノクロナル抗体感
作担体との組み合わせから成ることを特徴とする
上記抗原の免疫化学的測定試薬が提供できる。
このような感作担体の調製に利用する担体とし
ては、従来より免疫化学的凝集反応および凝集阻
止反応において一般的に用いられる微粒子の担体
を使用することができ、例えばヒト、羊、ウサギ
などの赤血球、細菌の細胞などの生物学的粒子、
高分子ラテツクス、ベントナイト、コロジオン、
コレステロール結晶、シリカ、カオリンなどの非
生物学的粒子などの如き担体を挙げることができ
る。
このような担体の中で、高分子ラテツクス担体
の例としては、ポリスチレンラテツクス、スチレ
ン−ブタジエンコポリマーラテツクス、ポリビニ
ルトルエンラテツクス、ビニルトルエン・ｔ−ブ
チルスチレンコポリマーラテツクス、スチレン−
メタアクリレートコポリマーラテツクスなどおよ
び官能基としてカルボキシル基、第１級アミノ基
又はカルボアミド基（−CONH2）を有し、且つ
基体が前記ラテツクスから成る反応性高分子ラテ
ツクスなどを挙げることができる。
上記例示の如き高分子ラテツクス担体の粒子サ
イズは適宜に選択できるが、例えば、平均粒径が
約0.01〜約２ミクロンのものが使用でき、特に平
均粒径が約0.05〜約1.5ミクロンのものが好まし
い。又、他の非生物学的担体についても上記高分
子ラテツクス担体と同様に平均粒径約0.01〜約２
ミクロンのものが使用できる。
上記例示の如き担体に、完全抗原もしくは該抗
原に対するモノクロナル抗体を感作させる手法は
種々知られており、本発明において適宜に選択利
用できる。このような感作手法としては、担体に
これらを吸着させる手法及び化学的に結合させる
手法のいずれの手法も利用することができ、本発
明に於て、完全抗原感作担体もしくはモノクロナ
ル抗体感作担体と称するものは、これらの任意の
手法を適宜に選択利用して担体に完全抗原もしく
はモノクロナル抗体を担持させたすべての感作担
体を意味する。
以下、その数態様について更に詳しく説明す
る。
完全抗原及びモノクロナル抗体感作担体の調製
態様：−
完全抗原またはモノクロナル抗体を吸着により
担体に感作するには、完全抗原またはモノクロナ
ル抗体の溶液と担体の懸濁液を混合することによ
り、容易に完全抗原感作担体あるいはモノクロナ
ル抗体感作担体を得ることができる。
完全抗原及び抗体は多くの場合カルボキシル基
と第１級アミノ基の双方を有する。抗原またはモ
ノクロナル抗体を化学的に担体に結合させるに
は、例えば前記官能基を有する反応性高分子ラテ
ツクスと完全抗原又はモノクロナル抗体をカルボ
ジイミド法、カルボニルジイミダゾール法、混合
酸無水物法、活性エステル法などの一つを適宜選
択して用い、結合することにより完全抗原感作担
体あるいはモノクロナル抗体感作担体を得ること
ができる。
また赤血球を担体として用い、完全抗原又はモ
ノクロナル抗体を感作するには、例えば赤血球を
ホルマリン、グルタルアルデヒド又はピルビンア
ルデヒド等の適切なもので固定化した固定赤血球
を用い、必要に応じタンニン酸あるいはビスジア
ゾベンチジン（BDB）やグルタルアルデヒド等
の縮合剤を用いて感作させ、完全抗原感作血球あ
るいはモノクロナル抗体感作血球を得ることがで
きる。
たとえば上述のようにして調製できる(A)完全抗
原感作担体と(B)上記抗原に対する単一種のモノク
ロナル抗体感作担体の組み合せから成る本発明の
免疫化学的測定試薬としては、下記の如き測定試
薬を例示することができる。
(1)Ａ：前立腺性酸性フオスフアターゼ（PAP）
感作ラテツクスと
Ｂ：抗PAPモノクロナル抗体感作ラテツクス
よりなる免疫化学的測定試薬、
(2)Ａ：α−フエトプロテイン（AFP）感作ラテ
ツクスと
Ｂ：抗AFPモノクロナル抗体感作ラテツクス
よりなる免疫化学的測定試薬、
(3)Ａ：ヒト胎盤催乳ホルモン（hPL）感作ラテツ
クスと
Ｂ：抗hPLモノクロナル抗体感作ラテツクス
よりなる免疫化学的測定試薬、
(4)Ａ：ヒト絨毛性性腺刺激乳ホルモン（hCG）感
作ラテツクスと
Ｂ：抗hCGモノクロナル抗体感作ラテツクスよ
りなる免疫化学的測定試薬、
(5)Ａ：ヒト生長ホルモン（hGH）感作ラテツク
スと
Ｂ：抗hGHモノクロナル抗体感作ラテツクス
よりなる免疫化学的測定試薬、
(6)Ａ：ガン胎児性抗原（CEA）感作ラテツクス
と
Ｂ：抗CEAモノクロナル抗体感作ラテツクス
よりなる免疫化学的測定試薬、
(7)Ａ：フイブリン・フイブリノゲン分解産物
（FDP）感作ラテツクスと
Ｂ：抗FDPモノクロナル抗体感作ラテツクス
よりなる免疫化学的測定試薬、
(8)Ａ：抗トロンビン（AT）感作ラテツクス
と
Ｂ：抗ATモノクロナル抗体感作ラテツクス
よりなる免疫化学的測定試薬、
(9)Ａ：PAP感作赤血球と
Ｂ：抗PAPモノクロナル抗体感作赤血球より
なる免疫化学的測定試薬、
(10)Ａ：AFP感作赤血球と
Ｂ：抗AFPモノクロナル抗体感作赤血球より
なる免疫化学的測定試薬、
(11)Ａ：hCG感作赤血球と
Ｂ：抗hCGモノクロナル抗体感作赤血球よりな
る免疫化学的測定試薬、
(12)Ａ：AFP感作血球と
Ｂ：抗AFPモノクロナル抗体感作赤血球より
なる免疫化学的測定試薬、
等々を挙げることができるが、本発明の試薬は上
記に掲げる具体例の例示に限定されるものではな
い。
本発明によれば、上述の如き新しいタイプの測
定試薬を利用した完全抗原の新しいタイプの免疫
化学的測定方法が提供できる。この測定方法によ
れば、(A)完全抗原感作担体及び(B)上記抗原に対す
る単一種のモノクロナル抗体感作担体の組み合わ
せを用い、被検体中の該抗原による上記(A)及び(B)
両試薬成分の凝集を阻止する凝集阻止反応を測定
することにより、上記抗原を免疫化学的に測定す
ることができる。測定は、被検体中の抗原の存在
を測定検出する定性的な測定及び被検体中の抗原
の濃度を測定検出する定量的な測定のいずれの態
様によつても行なうことができる。被検体の例と
しては、尿、血清、血漿、羊水などの如き体液も
しくは体液成分を例示することができる。以下、
本発明の免疫化学的測定方法について更に詳しく
説明する。
血液、尿その他の体液中に存在する微量物質は
前記試薬を用いて凝集阻止反応により定量するこ
とができる。具体的な測定法は実施例として示し
たが、一般的な測定法は次に述べる通りである。
原理的には完全抗原感作担体とモノクロナル抗
体感作担体の凝集反応系を用いた凝集阻止反応で
ある。すなわち、(1)例えば、清浄なスライド板上
に１滴の試薬検体（適宜希釈）を置き、その上に
１滴の前記モノクロナル抗体感作ラテツクスを滴
下し、次いで完全抗原感作ラテツクス１滴を滴下
した後、三者を混合し、２分間揺動することによ
り、試験の結果が観察され、凝集像を陰性、凝集
阻止像（非凝集像）を陽性と判定する。
(2)清浄な丸底試験管に検体（適宜希釈）の一定
量を入れ、これにモノクロナル抗体感作血球懸濁
液の一定量を加えて振盪混和後、抗原感作血球懸
濁体の一定量を加えて振盪混和し、ミラー付スタ
ンドに静置し、２時間後に管底像を観察する。こ
の場合、凝集阻止像（陽性像）は沈降リングを形
成し、凝集像（陰性像）はマツト状を呈する。
検体中の抗原濃度は、検体を適宜希釈して試験
を行い、陽性像を呈する最高希釈倍数に測定感度
を乗ずることにより求めることができる。
本発明の(A)完全抗原感作担体及び(B)上記抗原に
対する単一種のモノクロナル抗体感作担体の組み
合わせからなる該抗原の免疫化学的測定試薬及び
これを用いた該抗原のの免疫化学的測定方法によ
れば、モノクロナル抗体利用の従来技術において
は、単一種のモノクロナル抗体の利用によつては
対応抗原と結合して沈殿物を生成しないので二以
上複数種の異種モノクロナル抗体の使用が必須で
あつたにも拘わらず、全く意外なことに、上記(A)
及び(B)両試薬の間には満足すべき凝集反応が生ず
る。斯くて、本発明によれば、抗原分子中の一つ
の特定部位を特異的に認識する能力を有するモノ
クロナル抗体の単一種と抗原との組み合わせによ
る凝集反応であるため、顕著に高い特異性が達成
できる特色がある。更に、上記(A)及び(B)両試薬の
間の凝集反応で形成される凝集像は、予想外なこ
とにも、従来のポリクロナル抗体を用いたものよ
りも強い凝集性を示す鮮明な凝集像となる特色が
ある。又更に、モノクロナル抗体利用の従来技術
においては、二以上の異種モノクロナル抗体を使
用してもなお、全く効果的でないことさえあると
いうトラブルがあつたにも拘わらず、本発明によ
ればそのようなトラブルから解放され、広汎な任
意の抗原の免疫学的測定方法に適用可能となる特
色を有する。
本発明によれば、上述の如き特色を持つたモノ
クロナル抗体利用の新しいタイプの免疫化学的測
定試薬及び測定方法が提供でき、顕著に優れた確
実性、信頼度、精度、感度及び顕著に高い特異性
をもつて、偽反応生起のトラブルを伴うことなし
に、優れた再現生、安定性で且つ容易な操作で、
短時間に被検体中の微量の抗原を免疫化学的に測
定することができる。
実施例 １
前立腺性酸性フオスフアターゼ（PAP）の測
定（）
(a) PAP感作ラテツクスの製造
PAP50μｇを５mlのグリシン緩衝化食塩液
（PH8.2）を溶解し、これに10％ポリスチレンラ
テツクス１mlを加えて混合し、室温で２時間処
理した。次いで遠心分離し、得られた沈殿をグ
リシン緩衝化食塩液にて遠心洗浄し、沈殿を
0.05％にBSA（ウシ血清アルブミン）を含むグ
リシン緩衝化食塩液10mlに懸濁し、PAP感作
ポリスチレンラテツクスを製造した。
(b) 抗PAPモノクロナル抗体感作ラテツクスの
製造
抗PAPモノクロナル抗体0.2mgを５mlのグリ
シン緩衝化食塩液（PH8.2）を溶解し、これに
10％ポリスチレンラテツクス１mlを加えて混合
し、56℃で30分間処理した。次いで遠心分離し
て得た沈殿をグリシン緩衝化食塩得にて遠心洗
浄し、沈殿を0.05％にBSAを含むグリシン緩衝
化食塩液20mlにて懸濁させ、抗PAPモノクロ
ナル抗体感作ポリスチレンラテツクスを製造し
た。
(c) PAPの測定
前立腺癌患者血清５検体および健常人血清３
検体について原血清及びグリシン緩衝化食塩液
（PH8.2）で５、10、20、40及び80倍に希釈し、
各希釈検体の１滴（0.03ml）を反応スライド板
上に滴下し、これに(d)で製造した抗PAPモノ
クロナル抗体感作ラテツクスを各１滴ずつ滴下
し、次いで(c)で製造したPAP感作ラテツクス
を各１滴ずつ滴下する。この三者を均一に混合
し、２分間揺動後、肉眼で凝集像、凝集阻止像
を観察した、尚、本実施例においてはその試薬
の感度を10nｇ／ml調整してあるので各検体の
PAP濃度は第１表に示す値であつた。
The present invention relates to a new type of immunochemical measurement method for a complete antigen and a new type of measurement reagent suitable for using the method. In particular, the present invention relates to a method and reagent for immunochemically measuring trace amounts of antigens in a specimen such as blood, urine, other body fluids, and body fluid components, with excellent certainty, reliability, precision, and sensitivity. It is stable and easy to operate with good reproducibility, without the trouble of false reactions, and has high specificity.
The present invention relates to a new type of measurement method that can immunochemically measure a minute amount of antigen in a specimen in a short time, and a new type of measurement reagent suitable for use in the method. More specifically, the present invention uses (A) a complete antigen-sensitized carrier and (B) a single type of monoclonal antibody-sensitized carrier against the above antigen, and the above (A) and (B) by the antigen in a subject. The present invention relates to a method for immunochemically measuring the above-mentioned antigen, which comprises measuring the agglutination inhibition reaction of both reagent components. The present invention also relates to a reagent for immunochemical measurement of the above-mentioned antigen, characterized in that it consists of (A) a complete antigen-sensitized carrier and (B) a single type of monoclonal antibody-sensitized carrier against the above-mentioned antigen. BACKGROUND ART Conventionally, methods have been known for a long time for measuring trace amounts of biologically active substances present in specimens such as blood, urine, and other body fluids and body fluid components using immunochemical means. In such an immunochemical measurement method, for example, red blood cells are used as a carrier, sensitized with an antigen or antibody, and reacted with the antibody or antigen in the test solution. A method of measuring the trace substance by causing a blocking reaction is also already known. It is also known to use non-biological particles such as synthetic resin latex, bentonite, collodion, cholesterol crystals, and quartz crystals as solid carriers in immunochemical reactions instead of red blood cells as carriers (for example, Publication No. 50-82230). Antibody-sensitized carriers have traditionally been commonly used to measure and detect the presence (qualitative) or concentration (quantitative) of antigens in a specimen by immunochemical techniques that utilize such antigen-antibody reactions. was a polyclonal antibody sensitized carrier. Since 1975, when C. Milstein and his colleagues fused mouse myeloma cells with antibody-producing cells in the spleen and succeeded in producing monoclonal antibodies from the cells, significant technological progress has been made in the field of cell fusion technology. Along with this, it has become easier to form monoclonal antibody-producing cell lines and use them to produce monoclonal antibodies, and proposals have been made regarding methods for immunochemical measurement of antigens using such monoclonal antibodies. . As such a proposal, the proposal in Japanese Patent Application Laid-open No. 86051/1983 is known. This proposal is characterized by the use of two or more different types of monoclonal antibodies in response to the same antigen, and involves an immunochemical reaction that binds the antigen to at least two antibody molecules. An antigen quantification method using the method has been proposed.
This proposal also requires that monoclonal antibodies do not bind to the corresponding antigen and produce a precipitate, which is completely different from the well-known phenomenon in which conventional antibodies, that is, polyclonal antibodies, react with the corresponding antigen and produce a precipitate. It has been described that carrier particles such as red blood cells, latex spheres, metal particles etc. coated with monoclonal antibodies do not agglutinate in the presence of the corresponding antigen. In this proposal, despite the fact that, contrary to conventional knowledge, monoclonal antibodies do not bind to the corresponding antigen and produce a precipitate,
We describe the new finding that multiple types of heterologous monoclonal antibodies can be used to bind to antigens and generate precipitates, and therefore, in this proposal, at least two types of heterologous monoclonal antibodies are used, as described above. Specific for antigen quantification methods that require the use of null antibodies. Furthermore, this proposal suggests that the use of such multiple heterologous monoclonal antibodies does not always yield good results in terms of sensitivity and specificity of the assay, and may even be completely ineffective. It states that in order to obtain this, one must consider and select as many possible combinations as possible. As a similar proposal that requires the use of multiple types of heterologous monoclonal antibodies,
Proposal No. 118159 is also known. In the conventional proposals for immunochemical measurement methods of antibody antigens using these monoclonal antibodies, unlike the case of conventional polyclonal antibodies, a single type of monoclonal antibody binds to the corresponding antigen and precipitates. Naturally, from the fact that a precipitate can be generated only by using multiple types of heterologous monoclonal antibodies without producing a monoclonal antibody, the common point is that the use of multiple types of heterologous monoclonal antibodies is essential. ing. However, as described in the former proposal, it is possible to sensitize a carrier with multiple types of heterologous monoclonal antibodies and use the sensitized carrier to cause agglutination of the monoclonal antibody and the corresponding antigen. In addition,
There are deficiencies in the sensitivity and specificity of the measurements that do not always allow good results to be achieved, and there are also serious technical shortcomings in that they do not even result in usable aggregation and may even be completely ineffective. . And to give satisfactory cohesiveness,
For example, the more different types of monoclonal antibodies are used, the more the advantage of high specificity that can be achieved by using monoclonal antibodies is inevitably lost. Difficult technical issues arose. In other words, monoclonal antibodies are antibodies that have the ability to specifically recognize one specific site in an antigen molecule, but the more monoclonal antibodies are used, the more monoclonal antibodies are used. This results in the simultaneous recognition of specific sites, which poses an irreconcilable technical problem in that the advantage of high specificity provided by the use of monoclonal antibodies is further lost. Furthermore, unlike the conventional use of polyclonal antibodies, which can recognize multiple sites on an antigen molecule, monoclonal antibodies only recognize one specific site, so compared to the use of polyclonal antibodies. Therefore, the agglutination in the agglutination reaction is expected to be extremely weak, and in fact, as mentioned above, in the conventional proposals for using monoclonal antibodies, when a single monoclonal antibody is used, it binds to the corresponding antigen and forms a precipitate. teaches that it is essential to use multiple types of heterologous monoclonal antibodies because they do not produce
Furthermore, the former proposal described above discloses that there is a technical defect in that even if multiple types of heterologous monoclonal antibodies are used, aggregation may not occur in some cases. The present inventors have conducted research to develop a method capable of solving the above-mentioned incompatible technical problems or technical deficiencies when using monoclonal antibodies in immunochemical antigen measurement methods. As a result, in the case of a complete antigen-sensitized carrier in which the carrier is sensitized with a complete antigen, which is completely different from the above-mentioned conventional knowledge regarding the use of monoclonal antibodies, the carrier is sensitized with a single type of monoclonal antibody against the antigen. A satisfactory agglutination reaction occurs between the single type of monoclonal antibody sensitized carrier, thus making it possible to utilize multiple types of different types of monoclonal antibodies, which was essential to the conventional knowledge in the use of monoclonal antibodies. I learned something new and unexpected that I didn't need it at all. As a result of further research based on this completely new knowledge in the use of monoclonal antibodies, a new type of carrier consisting of a combination of (A) a complete antigen-sensitized carrier and (B) a single type of monoclonal antibody-sensitized carrier against the above antigen was developed. It is possible to provide a new type of immunochemical complete antigen measurement method in which a reagent is used to measure the agglutination inhibition reaction against the agglutination reaction of both the reagent components (A) and (B) by the antigen in the specimen, and it is significantly With excellent certainty, reliability, precision, sensitivity and exceptionally high antigen specificity, it can be applied in a short time with excellent reproducibility, stability and ease of operation, without the trouble of generating false reactions. We discovered that trace amounts of antigens in specimens can be measured immunochemically. Furthermore, the above-mentioned remarkable effects have the advantage that they are not substantially affected by the type of monoclonal antibody against the target complete antigen, and thus can be applied to a wide range of immunochemical measurement methods for any antigen. It turns out it's possible. Therefore, an object of the present invention is to provide a new type of immunochemical measurement method for a complete antigen using a monoclonal antibody and a new type of measurement reagent suitable for use in the method. The above objects and many other objects and advantages of the present invention will become more apparent from the following description. In the present invention, a complete antigen means an antigen that has the ability to produce antibodies by itself in a living body, and excludes haptens (incomplete antigens) that do not have the ability to produce antibodies by themselves in a living body. It means to do. In other words, it refers to an antigen that itself has antigenicity. Any antigen can be used as such a complete antigen, and examples thereof include the following antigens. () Examples of peptide hormones include (i) growth hormone (GH), adrenocorticotropic hormone (ACTH), melamine cell stimulating hormone (MSH), prolactin, thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle pituitary hormones such as stimulating hormone (FSH) and oxytocin; (ii) calcium metabolism regulating hormones such as calcitonin and parathyroid hormone; (iii) pancreatic hormones such as insulin, proinsulin and pancreatic glucagon; (iv) gastrin and selectin. (v) Hormones that act on blood vessels, such as andeotensin and bradykinin; (vi) Placental hormones, such as human placental gonadotropin (hCG) and human lactating hormone (hPL), etc. () Substances other than hormones include (i) prostatic acid phosphatase (PAP);
Enzymes such as alkaline phosphatase, transaminase, lactate dehydrogenase, transaminase, trypsin, and pepsinogen (ii) Cancer-specific substances such as α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) (iii) Immunoglobulin G (IgG) ), serum protein components such as fibrin-fibrinogen degradation products (FDP), antithrombin (AT), and transferrin; (iv) other substances such as rheumatoid arthritis factor, serotonin, urokinase, ferritin, and substance P, There are many complete antigens as well as components and metabolites present. The complete antigens of the present invention, however, are not limited to the complete antigens exemplified above. Monoclonal antibodies against complete antigens such as those exemplified above to be used in the present invention can be obtained by appropriately selecting and combining methods known per se in the field of cell fusion technology to form a monoclonal antibody-producing fused cell line, and then utilizing this cell line. can be produced and obtained. According to one embodiment, a complete antigen as exemplified above is used and administered to a suitable animal such as a mouse, rat, rabbit, sheep, horse, cow, etc., for example, by subcutaneous injection together with an adjuvant. After administering and immunizing the animal,
Cells such as splenocytes, thymocytes, lymph node cells, and/or peripheral blood cells that produce antibodies against the antigen from the immunized animal, such as immunized mice, are collected, and cells that have self-propagation properties but substantially no antibody-producing ability are collected from the immunized animal, such as immunized mice. Cell fusion treatment is performed using a suitable established cell line, such as a mouse myeloma cell line, which is not present in the cell line, by a method known per se. The combination of myeloma cells and antibody-producing cells to obtain monoclonal antibodies is possible if each cell can fuse and proliferate while producing antibodies.
The types of animals from which each cell is derived are not limited, and any combination may be used. The myeloma cells used are not particularly limited;
Cell bodies from many animals including mice, rats, rabbits, and humans can be used. Preferred cell lines are those that are drug resistant, and in which terminally fused myeloma cells do not survive in the selective medium, while only fused cells survive. The most commonly used cell line is 8-azaguanidine-resistant cell lines, which lack hypoxanthine guanine phosphoribosyl transferase and cannot grow in hypoxanthine-aminepterin-thymidine (HAT) medium. have.
Furthermore, the established cell line used is preferably of a "non-secreting type". For example, mouse myeloma
P3 /×63- Ag8U1 ( P3U1 ) derived from MOPC-21 strain,
P 3 /×63−Ag8・6・5・3, P 3 /NSI−1−
Ag4-1, Sp2/O-Ag14, rat myeloma
210, RCY3, Ag1, 2, 3, etc. are preferably used. The cell fusion treatment is carried out, for example, by combining 1 to 5 x 10 8 splenocytes of the above-mentioned immunized mouse and 1 to 5 x of the above mouse myeloma cell line in a medium such as Eagle's minimal essential medium (MEM) or RPMI-1640. 10 7 pieces can be mixed together. The fusion promoter is preferably polyethylene glycol (PEG) with an average molecular weight of 1000 to 6000, and other fusion promoters, such as
Polyvinyl alcohol, viruses, etc. can be used. The concentration of PEG used can be about 30-50%. The fused cells from the fused cell-containing system obtained as described above are subjected to selection, antibody activity screening, and cloning using techniques known per se to obtain immunized mice and the complete antigen used to form them. It is possible to obtain a fused cell line that has the ability to produce monoclonal antibodies against the cell line and has the ability to self-propagate. The above-mentioned fusion cell selection process can be carried out, for example, by appropriately diluting the fused cells in RPMI-1640 medium containing 20% fetal bovine serum, and dispensing the cells into a 96-well microplate at approximately 10 5 to 10 6 per well. Then, add a selective medium (for example, HAT medium) to each well, and then incubate in a 5% CO 2 incubator (37°C) while changing the selective medium.
This can be done by continuing the culture. If an 8-azaguanine-resistant strain is used as myeloma cells, the terminally fused myeloma cells will die in HAT medium, and the antibody-producing cells will be normal cells, so in
Cannot grow for long periods in vitro culture. Therefore, cells that grow from about 10 to 14 days after culture are completely fused cells. The antibody activity screening treatment and cloning treatment of the fused cell line obtained as described above can be carried out by conventional methods, but for example,
This can be done as follows. By collecting a portion of the culture supernatant of a well in which fused cells have grown, incubating it with a certain amount of labeled antigen, and measuring the binding ability with the labeled antigen.
Wells secreting the antibody of interest can be searched. That is, after reacting a culture supernatant with an antigen labeled with a radioisotope such as 125 I or 131 I or an enzyme, the antigen-
By separating the antibody conjugate and measuring the amount of label, the presence and binding ability of the antibody of interest can be detected. Since there is a possibility that two or more types of fused cells are growing in each well in which the desired antibody activity is observed, cloning is performed by limiting dilution method or colony formation method using soft agar, and monoclonal antibody-producing fusion cells are used. (Such monoclonal antibody-producing fusion cell lines are subject to refusal of deposits by FAIKI.) Using the monoclonal antibody-producing cell lines that can be obtained as described above, To obtain a monoclonal antibody against the complete antigen used to form the immunized animal, the monoclonal antibody-producing cell line is
For example, methods known per se can be used, such as culturing in an appropriate medium and collecting monoclonal antibodies from the medium, or transplanting the cell line into an animal of the same gene as the myeloma cell-derived animal and collecting monoclonal antibodies in ascites. It can be obtained using . According to the former embodiment, for example, a monoclonal antibody-producing fusion cell line containing 10% fetal bovine serum may be used.
The desired monoclonal antibody is collected by culturing in a culture medium such as RPMI1640 medium and purifying the culture supernatant using ammonium sulfate fractionation or affinity chromatography using antigen-bound Sepharose 4B. can do. According to the latter aspect, for example, after intraperitoneally administering mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) to a syngeneic animal, the fused cells are intraperitoneally administered.
Large numbers of fused cells are grown in vivo. the result,
The ascites that forms contains high concentrations of monoclonal antibodies. The desired monoclonal antibody can be obtained from this ascites by ammonium sulfate fractionation and, if necessary, the aforementioned affinity chromatography. Monoclonal antibodies that can be obtained as described above are also commercially available and can be used. According to the present invention, (A) a complete antigen sensitized carrier in which a suitable carrier is sensitized with a complete antigen as exemplified above, and a single type of monoclonal antibody against the antigen obtained as described above are combined in an appropriate manner. sensitized to carrier
(B) It is possible to provide a reagent for immunochemical measurement of the above-mentioned antigen, which is characterized in that it consists of a combination with a carrier sensitized with a single type of monoclonal antibody against the above-mentioned antigen. As carriers used in the preparation of such sensitized carriers, fine particle carriers commonly used in immunochemical agglutination reactions and agglutination inhibition reactions can be used, such as human, sheep, rabbit, etc. biological particles such as red blood cells, bacterial cells,
polymer latex, bentonite, collodion,
Mention may be made of carriers such as cholesterol crystals, silica, non-biological particles such as kaolin, and the like. Among such carriers, examples of polymeric latex carriers include polystyrene latex, styrene-butadiene copolymer latex, polyvinyltoluene latex, vinyltoluene/t-butylstyrene copolymer latex, and styrene-butadiene copolymer latex.
Examples include methacrylate copolymer latex, etc., and reactive polymer latex having a carboxyl group, primary amino group, or carboxamide group ( -CONH2 ) as a functional group, and a base consisting of the above-mentioned latex. The particle size of the polymer latex carrier as exemplified above can be selected as appropriate, but for example, those with an average particle size of about 0.01 to about 2 microns can be used, particularly those with an average particle size of about 0.05 to about 1.5 microns. preferable. In addition, other non-biological carriers also have an average particle size of about 0.01 to about 2, similar to the polymer latex carrier mentioned above.
Micron ones can be used. Various techniques are known for sensitizing carriers such as those exemplified above with complete antigens or monoclonal antibodies against the antigens, and can be appropriately selected and used in the present invention. As such a sensitization method, either a method of adsorbing these to a carrier or a method of chemically bonding them can be used.In the present invention, a complete antigen-sensitized carrier or a monoclonal antibody-sensitized carrier The term sensitized carrier refers to all sensitized carriers prepared by appropriately selecting and utilizing any of these methods to support a complete antigen or monoclonal antibody on the carrier. Hereinafter, several aspects thereof will be explained in more detail. Preparation mode of complete antigen and monoclonal antibody sensitized carrier:- To sensitize the complete antigen or monoclonal antibody to the carrier by adsorption, by mixing a solution of the complete antigen or monoclonal antibody and a suspension of the carrier. , a complete antigen-sensitized carrier or a monoclonal antibody-sensitized carrier can be easily obtained. Complete antigens and antibodies often have both carboxyl groups and primary amino groups. In order to chemically bond an antigen or a monoclonal antibody to a carrier, for example, a reactive polymer latex having the above-mentioned functional groups and a complete antigen or a monoclonal antibody can be bonded using a carbodiimide method, a carbonyldiimidazole method, a mixed acid anhydride method, a reactive polymerization method, etc. A complete antigen-sensitized carrier or a monoclonal antibody-sensitized carrier can be obtained by appropriately selecting and using one of the ester methods and the like. Furthermore, in order to sensitize complete antigens or monoclonal antibodies using red blood cells as carriers, for example, fixed red blood cells prepared by fixing red blood cells with a suitable substance such as formalin, glutaraldehyde or pyruvaldehyde are used, and if necessary, tannic acid or Complete antigen-sensitized blood cells or monoclonal antibody-sensitized blood cells can be obtained by sensitization using a condensing agent such as bisdiazobenzidine (BDB) or glutaraldehyde. For example, the immunochemical assay reagent of the present invention comprising a combination of (A) a complete antigen-sensitized carrier prepared as described above and (B) a carrier sensitized with a single type of monoclonal antibody against the above-mentioned antigen is as follows. A measurement reagent can be exemplified. (1) A: Prostatic acid phosphatase (PAP)
An immunochemical assay reagent consisting of a sensitized latex and B: an anti-PAP monoclonal antibody sensitized latex, (2) A: an α-fetoprotein (AFP) sensitized latex, and B: an anti-AFP monoclonal antibody sensitized latex. (3) A: Human placental lactating hormone (hPL) sensitized latex; B: Anti-hPL monoclonal antibody sensitized latex; (4) A: Human chorionic gonad. Stimulating milk hormone (hCG) sensitized latex and B: Immunochemical assay reagent consisting of anti-hCG monoclonal antibody sensitized latex, (5) A: Human growth hormone (hGH) sensitized latex and B: anti-hGH monoclonal antibody. Immunochemical measurement reagent consisting of sensitized latex, (6) A: Immunochemical measurement reagent consisting of carcinoembryonic antigen (CEA) sensitized latex and B: anti-CEA monoclonal antibody sensitized latex, (7) A: Fibrin/fibrinogen degradation product (FDP) sensitized latex and B: Immunochemical measurement reagent consisting of anti-FDP monoclonal antibody sensitized latex, (8) A: Anti-thrombin (AT) sensitized latex and B: Anti-AT monoclonal Immunochemical measurement reagent consisting of antibody-sensitized latex, (9) A: PAP-sensitized red blood cells B: Immunochemical measurement reagent consisting of anti-PAP monoclonal antibody-sensitized red blood cells, (10) A: AFP-sensitized red blood cells B: Immunochemical assay reagent consisting of anti-AFP monoclonal antibody-sensitized red blood cells, (11) A: hCG-sensitized red blood cells and B: Immunochemical assay reagent consisting of anti-hCG monoclonal antibody-sensitized red blood cells, (12) A : AFP-sensitized blood cells and B: an immunochemical measurement reagent consisting of anti-AFP monoclonal antibody-sensitized red blood cells, etc. However, the reagent of the present invention is not limited to the specific examples listed above. do not have. According to the present invention, a new type of immunochemical measurement method for a complete antigen can be provided using a new type of measurement reagent as described above. According to this measurement method, a combination of (A) a complete antigen-sensitized carrier and (B) a single type of monoclonal antibody-sensitized carrier against the above antigen is used, and the above (A) and (B) are sensitized by the antigen in the subject. )
The above antigen can be immunochemically measured by measuring an agglutination inhibition reaction that inhibits agglutination of both reagent components. The measurement can be carried out either qualitatively, which measures and detects the presence of the antigen in the subject, or quantitatively, which measures and detects the concentration of the antigen in the subject. Examples of the analyte include body fluids or body fluid components such as urine, serum, plasma, amniotic fluid, and the like. below,
The immunochemical measurement method of the present invention will be explained in more detail. Trace amounts of substances present in blood, urine, and other body fluids can be quantified by an agglutination inhibition reaction using the above-mentioned reagent. A specific measuring method was shown as an example, but a general measuring method is as described below. The principle is an agglutination inhibition reaction using an agglutination reaction system of a complete antigen-sensitized carrier and a monoclonal antibody-sensitized carrier. That is, (1) For example, place one drop of reagent specimen (appropriately diluted) on a clean slide plate, drop one drop of the monoclonal antibody sensitized latex thereon, and then add one drop of complete antigen sensitized latex. After dripping, the three components are mixed and shaken for 2 minutes to observe the test results, and an agglutination image is determined to be negative, and an agglutination inhibition image (non-aggregation image) is determined to be positive. (2) Put a certain amount of the sample (appropriately diluted) into a clean round-bottomed test tube, add a certain amount of monoclonal antibody-sensitized blood cell suspension to it, mix by shaking, and prepare the antigen-sensitized blood cell suspension. Add a certain amount, mix by shaking, leave to stand on a stand with a mirror, and observe the image of the bottom of the tube after 2 hours. In this case, the agglutination-inhibited image (positive image) forms a sedimentation ring, and the agglutination image (negative image) has a mat-like shape. The antigen concentration in a specimen can be determined by suitably diluting the specimen, conducting a test, and multiplying the highest dilution factor that gives a positive image by the measurement sensitivity. A reagent for immunochemical measurement of the antigen of the present invention comprising a combination of (A) a carrier sensitized with a complete antigen and (B) a carrier sensitized with a single type of monoclonal antibody against the antigen, and an immunochemical measurement reagent for the antigen using the same. According to conventional measurement methods using monoclonal antibodies, monoclonal antibodies of two or more different types do not bind to the corresponding antigen and form a precipitate when using a single type of monoclonal antibody. Although the use of (A) above was required, quite surprisingly,
and (B) a satisfactory agglutination reaction occurs between both reagents. Thus, according to the present invention, the agglutination reaction is a combination of a single type of monoclonal antibody that has the ability to specifically recognize one specific site in an antigen molecule and an antigen, and therefore has a significantly high specificity. There are characteristics that can be achieved. Furthermore, the agglutination image formed by the agglutination reaction between both reagents (A) and (B) above unexpectedly shows clear agglutination that shows stronger agglutination than that using conventional polyclonal antibodies. It has characteristics that make it an image. Moreover, in the conventional technology using monoclonal antibodies, there was a problem that even if two or more different types of monoclonal antibodies were used, they were not even effective at all, but the present invention can solve the problem. This method is free from such troubles and has the feature of being applicable to a wide range of immunological measurement methods for any antigen. According to the present invention, it is possible to provide a new type of immunochemical measurement reagent and measurement method using monoclonal antibodies having the above-mentioned characteristics, which have significantly superior certainty, reliability, precision, sensitivity, and significantly high With specificity, excellent reproducibility, stability, and easy operation without the trouble of false reactions,
A minute amount of antigen in a subject can be immunochemically measured in a short time. Example 1 Measurement of prostatic acid phosphatase (PAP) (a) Production of PAP sensitized latex 50 μg of PAP was dissolved in 5 ml of glycine buffered saline (PH8.2), and 1 ml of 10% polystyrene latex was dissolved in this. The mixture was added and mixed for 2 hours at room temperature. Next, centrifugation is performed, and the resulting precipitate is centrifugally washed with glycine-buffered saline.
PAP-sensitized polystyrene latex was prepared by suspending it in 10 ml of glycine-buffered saline containing 0.05% BSA (bovine serum albumin). (b) Production of anti-PAP monoclonal antibody sensitized latex 0.2 mg of anti-PAP monoclonal antibody was dissolved in 5 ml of glycine buffered saline (PH8.2), and dissolved in this.
1 ml of 10% polystyrene latex was added, mixed, and treated at 56°C for 30 minutes. Next, the precipitate obtained by centrifugation was centrifugally washed with glycine buffered saline solution, the precipitate was suspended in 20 ml of glycine buffered saline solution containing 0.05% BSA, and anti-PAP monoclonal antibody sensitized polystyrene latex was prepared. was manufactured. (c) Measurement of PAP 5 serum samples from prostate cancer patients and 3 serum samples from healthy volunteers
Dilute the sample 5, 10, 20, 40 and 80 times with original serum and glycine buffered saline (PH8.2),
One drop (0.03 ml) of each diluted sample was dropped onto the reaction slide plate, one drop of each of the anti-PAP monoclonal antibody sensitized latex produced in (d) was added, and then one drop of each of the anti-PAP monoclonal antibody sensitized latex produced in (c) was added. Add one drop each of PAP sensitized latex. These three materials were mixed uniformly, and after shaking for 2 minutes, the agglutination image and agglutination inhibition image were observed with the naked eye.In this example, the sensitivity of the reagent was adjusted to 10ng/ml, so each sample was
The PAP concentrations were as shown in Table 1.
【表】
実施例 ２
PAPの測定（）
(a) PAP感作血球の製造
ホルマリン固定羊血球の４％懸濁液（リン酸
緩衝化食塩液、PH6.4）に等量の0.01％タンニ
ン酸溶液を加えて56℃30分反応させた後、リン
酸緩衝化食塩液にて血球を洗浄し、８％懸濁液
とした。次いでPAP0.005％溶液を等量加え、
37℃１時間反応させた。反応終了後、リン酸緩
衝化食塩液にて血球を遠心洗浄し、0.2％に
NRS（正常ウサギ血清）、５％に乳糖を含むリ
ン酸緩衝化食塩液にて0.75％血球濃度に希釈
し、0.1mlずつ分注した後、凍結乾燥してPAP
感作血球を製造した。
(b) 抗PAPモノクノナル抗体感作血球の製造
ホルマリン固定羊血球の４％懸濁液（リン酸
緩衝化食塩液、PH6.4）に等量の0.01％タンニ
ン酸溶液を加えて、56℃30分反応させた後、リ
ン酸緩衝化食塩液にて血球を洗浄して８％懸濁
液とした。次いで抗PAPモノクロナル抗体の
0.001％溶液を等量加え56℃で２時間反応させ
る。反応終了後、リン酸緩衝化食塩液にて血球
を遠心洗浄し、0.2％にＮ乙Ｓ、５％に乳糖を
含むリン酸緩衝化食塩液にて0.75％血球濃度に
希釈し、0.1mlずつ分注した、次いで凍結乾燥
して抗PAPモノクロナル抗体感作血球を製造
した。
(c) PAPの測定
前立腺癌患者血清及び健常人血清について、
それぞれをリン酸緩衝化食塩液で５、25、50、
100、200、400倍に希釈し、各希釈検体を丸底
小試験管に0.1mlずつ分注し、次いで、上記(b)
で製造した抗PAPモノクロナル抗体感作血球
１アンプルをリン酸緩衝化食塩液0.2mlで懸濁
させた全量を、それぞれに添加し、混合した
後、前記(a)で製造したPAP感作血球１アンプ
ルをリン酸緩衝化食塩液0.2mlで懸濁させた全
量をそれぞれに添加してよく撹拌し、ミラー付
スタンドに静置し、２時間後の管底像により判
定した。本実施例では測定感度を2nｇ／mlに
調整してあるので、各検体のPAP濃度は第２
表に示す値であつた。[Table] Example 2 Measurement of PAP () (a) Production of PAP-sensitized blood cells Add an equal volume of 0.01% tannic acid to a 4% suspension of formalin-fixed sheep blood cells (phosphate buffered saline, PH6.4). After adding the solution and reacting for 30 minutes at 56°C, the blood cells were washed with phosphate buffered saline to form an 8% suspension. Then add an equal amount of PAP 0.005% solution,
The reaction was carried out at 37°C for 1 hour. After the reaction, the blood cells were centrifuged and washed with phosphate buffered saline to 0.2%.
NRS (normal rabbit serum), diluted with phosphate buffered saline containing 5% lactose to a blood cell concentration of 0.75%, dispensed in 0.1 ml portions, lyophilized and PAP
Sensitized blood cells were produced. (b) Production of anti-PAP monoclonal antibody-sensitized blood cells Add an equal volume of 0.01% tannic acid solution to a 4% suspension of formalin-fixed sheep blood cells (phosphate buffered saline, pH 6.4), and incubate at 56℃30. After reacting for several minutes, the blood cells were washed with phosphate buffered saline to form an 8% suspension. Next, anti-PAP monoclonal antibody
Add an equal amount of 0.001% solution and react at 56°C for 2 hours. After the reaction, centrifugally wash the blood cells with phosphate-buffered saline, dilute to 0.75% blood cell concentration with phosphate-buffered saline containing 0.2% N2S and 5% lactose, and add 0.1ml each. The mixture was dispensed and then lyophilized to produce anti-PAP monoclonal antibody-sensitized blood cells. (c) Measurement of PAP For prostate cancer patient serum and healthy human serum,
5, 25, 50, respectively, with phosphate buffered saline.
Dilute 100, 200, and 400 times, dispense 0.1 ml of each diluted sample into a small round-bottom test tube, and then proceed as described in (b) above.
One ampoule of anti-PAP monoclonal antibody-sensitized blood cells prepared in step (a) was suspended in 0.2 ml of phosphate-buffered saline, and the entire amount was added to each ampoule and mixed. The entire amount of 1 ampoule suspended in 0.2 ml of phosphate buffered saline was added to each, stirred well, and placed on a stand with a mirror. After 2 hours, judgment was made based on the image of the bottom of the tube. In this example, the measurement sensitivity was adjusted to 2 ng/ml, so the PAP concentration of each sample was
The values were as shown in the table.
【表】【table】
【表】
実施例 ３
ヒト絨毛性ゴナドトロピン（hCG）の測定
(a) 抗hCGモノクロナル抗体感作ラテツクスの製
造
抗hCGモノクロナル抗体を用い、実施例１−
(b)と同様にして、抗hCGモノクロナル抗体感作
ラテツクスを製造した。
(b) hCG感作ラテツクスの製造
hCG（比活性9600IU／mg）100μｇを５mlの精
製水に溶解した。該溶液にカルボキシモデイフ
アイドラテツクスの10％懸濁液１mlを加えて混
合し、次いで10mgの１−エチル−３（３−ジメ
チルアミノプロピル）カルボジイミド・ハイド
ロクロライドを加え、撹拌下に一夜反応を行つ
た。反応終了後、遠心分離して得た沈殿をグリ
シン緩衝化食塩液で洗浄し、沈殿を0.1％に
BSAを含むグリシン緩衝化食塩液10mlで懸濁
させ、56℃で30分間反応させた。反応後、遠心
分離して得た沈殿をグリシン緩衝化食塩液で洗
浄し、同緩衝液10mlに懸濁させてhCG感作ラテ
ツクスを製造した。
(c) hCGの測定
妊婦尿５検体及び正常婦人尿３検体につい
て、前記(a)で製造した抗hCGモノクロナル抗体
感作ラテツクスと上記(b)で製造したhCG感作ラ
テツクスとを用いて実施例１−(c)と同様な操作
により、hCGを測定した。
尚、この実施例で用いた試薬の感度を
0.5IU／mlに調整してあるので、各検体のhCG
濃度は第３表に示す値であつた。[Table] Example 3 Measurement of human chorionic gonadotropin (hCG) (a) Production of anti-hCG monoclonal antibody sensitized latex Using anti-hCG monoclonal antibody, Example 1-
An anti-hCG monoclonal antibody-sensitized latex was produced in the same manner as in (b). (b) Production of hCG sensitized latex 100 μg of hCG (specific activity 9600 IU/mg) was dissolved in 5 ml of purified water. To the solution was added 1 ml of a 10% suspension of Carboximo Diff Id Latex and mixed, then 10 mg of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride was added and the reaction was allowed to proceed overnight with stirring. I went. After the reaction, the precipitate obtained by centrifugation was washed with glycine buffered saline and the precipitate was adjusted to 0.1%.
The suspension was suspended in 10 ml of glycine buffered saline containing BSA and reacted at 56°C for 30 minutes. After the reaction, the precipitate obtained by centrifugation was washed with glycine buffered saline and suspended in 10 ml of the same buffer to produce hCG sensitized latex. (c) Measurement of hCG Performed on 5 pregnant women's urine samples and 3 normal women's urine samples using the anti-hCG monoclonal antibody sensitized latex produced in (a) above and the hCG sensitized latex produced in (b) above. hCG was measured by the same procedure as in Example 1-(c). In addition, the sensitivity of the reagent used in this example is
The hCG of each sample is adjusted to 0.5IU/ml.
The concentrations were as shown in Table 3.
【表】
実施例 ４
ヒト胎盤性ラクトゲン（hPL）の測定
(a) 抗hPLモノクロナル抗体感作ラテツクスの製
造
抗hPLモノクロナル抗体を用い、実施例１(b)
と同様な方法で、抗hPLモノクロナル抗体感作
ラテツクスを製造した。
(b) hPL感作ラテツクスの製造
実施例１−(a)と同様な方法で、hPL感作ラテ
ツクスを製造した。
(c) hPLの測定
妊婦血清５検体及び対照として正常婦人血清
３検体について、前記(a)で製造した抗hPLモノ
クロナル抗体感作ラテツクスと上記(b)で製造し
たhPL感作ラテツクスとを用いて、実施例１−
(c)と同様な操作により、検体中のhPLを測定し
た。
尚、本実施例においては試薬の感度を0.5μ
ｇ／mlに調整してあるので、各検体のhPL濃度
は第４表に示す値であつた。[Table] Example 4 Measurement of human placental lactogen (hPL) (a) Production of anti-hPL monoclonal antibody sensitized latex Example 1 (b) using anti-hPL monoclonal antibody
An anti-hPL monoclonal antibody-sensitized latex was produced in the same manner as above. (b) Production of hPL-sensitized latex An hPL-sensitized latex was produced in the same manner as in Example 1-(a). (c) Measurement of hPL Five pregnant serum samples and three normal female serum samples were used as a control using the anti-hPL monoclonal antibody sensitized latex produced in (a) above and the hPL sensitized latex produced in (b) above. So, Example 1-
hPL in the specimen was measured by the same procedure as in (c). In this example, the sensitivity of the reagent was set to 0.5μ.
g/ml, the hPL concentration of each sample was the value shown in Table 4.
【表】
参考例
上記実施例で用いるモノクロナル抗体は例えば
次の様な操作により製造することができる。
(a) 精製PAPの製造
ヒト精漿200mlから硫酸アンモニウムによる
塩析（40％上清、75％沈殿）によりPAP粗抽
出物6.75ｇを得た。これをｐ−セルロースクロ
マトグラフイー（2.7×19cm）に対し、次いで
未吸着分画を濃縮し、0.1M酢酸緩衝液（１ｍ
MCaCl2、MgCl2、MnCl2、0.1M NaCl、PH
5.0）で透析後、上記緩衝液で平衡化したコン
カナバリンＡ−セフアロースカラム（２×45
cm）に付した後、４℃で一晩インキユベートし
た。次いで上記緩衝液で蛋白質の流出がなくな
るまで洗浄し、α−メチル−Ｄ−マンノシドの
グラジユエントクロマトグラフイー（0.1M→
0.5M）を行い、酵素活性を有する分画を集め
濃縮した。次いで同画分を0.02Mリン酸緩衝液
（PH7.0）で平衡化したDEAE−セルロースカラ
ム（３×40cm）に付し、同緩衝液で蛋白質の流
出がなくなるまで洗浄後、0.02Mリン酸緩衝液
（PH7.0）及び0.5M NaCl含有0.02Mリン酸緩衝
液（PH6.0）を用いてグラジユエントクロマト
グラフイーを行い、酵素活性分画を集め濃縮し
た。次いで、0.02Mクエン酸緩衝液（PH6.0）
で平衡化したセフアクリルＳ−200（26×84cm）
及びセフアデツクスＧ−100（2.6×88cm）によ
るゲル過で精製して、精製PAP128mgを得
た。
(b) 抗PAPモノクロナル抗体の製造
上記(a)で製造した精製PAP50μｇを完全フロ
イドアジユバンドと共にBALB／Ｃマウス
（６〜８週令）に３週間おきに皮下投与し、最
後に80μｇを静注した。
最後免疫から３日後にマウスの脾臓を摘出し
て、脾細胞を採取し、デルベコのモデイフアイド
最少基本培地（以下D′MEMと称す）で３回洗浄
した後、細胞数を算出し、その２×108個をマウ
スミエローマ細胞P3−NS−１／１−Ag4・１（以
下NS−１と称す）１×107個と混合して遠心し細
胞を集めた。このペレツトに37℃に温めておいた
ポリエチレングリコール溶液（PEG−100：4.25
％、DMSO：1.5％含有D′MEM）を１ml加え、
１分間遠心管をゆつくり回転させて細胞融合を行
つた。37℃のD′MEMを30秒ごとに２mlずつ10回
加えたのち遠心分離し、ペレツトを20％ウシ胎児
血清含有RPMI−1640培地でNS−01として５×
104個／0.2mlとなるように懸濁し、96ウエルマイ
クロプレートに0.2mlずつ分注し、５％CO2培養
器で培養した。24時間後各ウエルの上清の半量を
すて、HAT培地（ヒポキサンチン・アミノプテ
リン・チミジン、10％ウシ胎児血清含有RPMI−
1640培地）を0.1ml加え、その後３〜４日ごとに
半量をHAT培地交換を行いながら２週間培養し
たのち、増殖したウエル中の培養上清の抗体活性
を測定した。
活性の認められたウエルの細胞をBALB／Ｃ
マウス胸腺細胞を含む10％ウシ胎児血清含有RPI
−1640培地で希釈し、限界希釈法によりクローニ
ングを行つて12株の抗モノクロナル抗体産生融合
細胞を得た。これを大量培養し、その培養上清か
らPAPを結合させたセフアロース4Bを用いたア
フイテイークロマトグラフイーに付し、モノクロ
ナル抗体を得た。また２×106個以上の細胞をプ
リスタン0.5mlを予め投与したBALB／Ｃマウス
に腹腔内投与し、腹水腫瘍を作せて腹水を得たの
ち、この腹水をPAPを結合させたセフアロース
4Bを用いたアフイニテイークロマトグラフイー
によりモノクロナル抗体を得た。[Table] Reference Example The monoclonal antibodies used in the above examples can be produced, for example, by the following procedure. (a) Production of purified PAP 6.75 g of PAP crude extract was obtained from 200 ml of human seminal plasma by salting out with ammonium sulfate (40% supernatant, 75% precipitation). This was subjected to p-cellulose chromatography (2.7 x 19 cm), the unadsorbed fraction was concentrated, and 0.1 M acetate buffer (1 m
MCaCl2 , MgCl2 , MnCl2 , 0.1M NaCl, PH
After dialysis with 5.0) concanavalin A-Sepharose column (2 x 45
cm) and then incubated at 4°C overnight. Next, the protein was washed with the above buffer solution until no protein flowed out, and subjected to gradient chromatography of α-methyl-D-mannoside (0.1M→
0.5M), and fractions with enzyme activity were collected and concentrated. Next, the same fraction was applied to a DEAE-cellulose column (3 x 40 cm) equilibrated with 0.02M phosphate buffer (PH7.0), washed with the same buffer until no protein flowed out, and then treated with 0.02M phosphate buffer (PH7.0). Gradient chromatography was performed using a buffer solution (PH7.0) and a 0.02M phosphate buffer solution (PH6.0) containing 0.5M NaCl, and enzyme active fractions were collected and concentrated. Then 0.02M citrate buffer (PH6.0)
Sephacryl S-200 (26 x 84 cm) equilibrated with
The resulting product was purified by gel filtration using Sephadex G-100 (2.6 x 88 cm) to obtain 128 mg of purified PAP. (b) Production of anti-PAP monoclonal antibody 50 μg of purified PAP produced in (a) above was administered subcutaneously to BALB/C mice (6-8 weeks old) together with complete Floyd adjuvant every 3 weeks, and finally 80 μg was administered subcutaneously to BALB/C mice (6-8 weeks old). I gave it an intravenous injection. Three days after the last immunization, the spleen of the mouse was removed and splenocytes were collected. After washing three times with Delbeco's Modified Minimal Basic Medium (hereinafter referred to as D'MEM), the number of cells was calculated, and the 10 8 cells were mixed with 1×10 7 mouse myeloma cells P 3 -NS-1/1-Ag4.1 (hereinafter referred to as NS-1) and centrifuged to collect the cells. Add polyethylene glycol solution (PEG-100: 4.25
%, DMSO: Add 1 ml of D′MEM containing 1.5%,
Cell fusion was performed by gently rotating the centrifuge tube for 1 minute. After adding 2 ml of D'MEM at 37°C every 30 seconds 10 times, centrifuging, and dissolving the pellet 5x as NS-01 in RPMI-1640 medium containing 20% fetal bovine serum.
The cells were suspended at 10 4 cells/0.2 ml, dispensed into 96-well microplates in 0.2 ml portions, and cultured in a 5% CO 2 incubator. After 24 hours, half of the supernatant from each well was discarded and added to HAT medium (RPMI containing hypoxanthine, aminopterin, thymidine, and 10% fetal bovine serum).
1640 medium) was added, and after culturing for 2 weeks while replacing half of the HAT medium every 3 to 4 days, the antibody activity of the culture supernatant in the wells in which the cells had grown was measured. BALB/C cells from wells where activity was observed
RPI containing 10% fetal bovine serum containing mouse thymocytes
-1640 medium and cloning was performed by limiting dilution method to obtain 12 strains of anti-monoclonal antibody-producing fused cells. This was cultured in large quantities, and the culture supernatant was subjected to affinity chromatography using Sepharose 4B bound to PAP to obtain monoclonal antibodies. In addition, 2 × 10 6 or more cells were intraperitoneally administered to BALB/C mice that had been pre-administered with 0.5 ml of pristane to generate ascites tumors and ascites were obtained.
Monoclonal antibodies were obtained by affinity chromatography using 4B.
Claims (1)
感作担体 を用い、被検体中の該抗原による上記(A)及び(B)両
試薬成分の凝集阻止反応を測定することを特徴と
する上記抗原の免疫化学的測定方法。 ２ (A) 完全抗原感作担体及び (B) 上記抗原に対する単一種のモノクロナル抗体
感作担体 から成ることを特徴とする上記抗原の免疫化学的
測定試薬。[Scope of Claims] 1. By using (A) a complete antigen-sensitized carrier and (B) a monoclonal antibody-sensitized carrier of a single type against the above antigen, both (A) and (B) above are sensitized by the antigen in the subject. The method for immunochemically measuring an antigen described above, which comprises measuring an agglutination inhibition reaction of reagent components. 2. An immunochemical measurement reagent for the above-mentioned antigen, comprising (A) a complete antigen-sensitized carrier and (B) a single type of monoclonal antibody-sensitized carrier against the above-mentioned antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4783483A JPS59173760A (en) | 1983-03-24 | 1983-03-24 | Method and reagent for immunochemical measurement |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4783483A JPS59173760A (en) | 1983-03-24 | 1983-03-24 | Method and reagent for immunochemical measurement |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS59173760A JPS59173760A (en) | 1984-10-01 |
| JPH0532705B2 true JPH0532705B2 (en) | 1993-05-17 |
Family
ID=12786383
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4783483A Granted JPS59173760A (en) | 1983-03-24 | 1983-03-24 | Method and reagent for immunochemical measurement |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS59173760A (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6358262A (en) * | 1986-08-29 | 1988-03-14 | Yatoron:Kk | Analysis of biocomponent by using specific monoclonal antibody |
| JP2615024B2 (en) * | 1986-10-03 | 1997-05-28 | 株式会社 ヤトロン | Highly sensitive method for analysis of human hemoglobin using monoclonal antibodies |
| JPH03118471A (en) * | 1989-09-29 | 1991-05-21 | Sekisui Chem Co Ltd | Insulin assay and reagent therefor |
| EP2068148B1 (en) | 2006-07-24 | 2014-03-26 | Sekisui Medical Co., Ltd. | Method of measuring anticoagulation and reagent for measuring anticoagulation |
| EP2560005B1 (en) * | 2010-04-14 | 2016-12-07 | Nitto Boseki Co., Ltd | Test instrument for measuring analyte in sample, and method for measuring analyte using same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5341420A (en) * | 1976-09-29 | 1978-04-14 | Mochida Pharm Co Ltd | Immunochemically measuring methoa of hapten |
| JPS58127169A (en) * | 1982-01-25 | 1983-07-28 | Tokyo Tatsuno Co Ltd | Tachometer for internal combustion engine |
-
1983
- 1983-03-24 JP JP4783483A patent/JPS59173760A/en active Granted
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5341420A (en) * | 1976-09-29 | 1978-04-14 | Mochida Pharm Co Ltd | Immunochemically measuring methoa of hapten |
| JPS58127169A (en) * | 1982-01-25 | 1983-07-28 | Tokyo Tatsuno Co Ltd | Tachometer for internal combustion engine |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS59173760A (en) | 1984-10-01 |
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