JPH02107967A - Immunoassay - Google Patents
ImmunoassayInfo
- Publication number
- JPH02107967A JPH02107967A JP26093088A JP26093088A JPH02107967A JP H02107967 A JPH02107967 A JP H02107967A JP 26093088 A JP26093088 A JP 26093088A JP 26093088 A JP26093088 A JP 26093088A JP H02107967 A JPH02107967 A JP H02107967A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- protein
- measured
- substance
- monoclonal antibody
- 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.)
- Pending
Links
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Landscapes
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Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は、被検試料液中の被測定物質を被測定物質と特
異的に結合するモノクローナル抗体などのタンパク質お
よびこのタンパク質を認識する抗体を使用した免疫測定
法により測定する方法に関する。さらに詳しくは、種々
の身体的疾患に伴う、例えば血清もしくは他の体液中の
被測定物質の検出および／または濃度を測定することが
できる方法に関するものである。Detailed Description of the Invention [Industrial Application Field] The present invention relates to a protein such as a monoclonal antibody that specifically binds an analyte in a test sample solution and an antibody that recognizes this protein. The present invention relates to a method of measuring using the immunoassay method used. More specifically, the present invention relates to a method capable of detecting and/or measuring the concentration of a substance to be measured in, for example, serum or other body fluids associated with various physical diseases.
［従来の技術］
従来、モノクローナル抗体のような被測定物質と特異的
に結合するタンパク質を使用した免疫測定法としては、
不溶性固体に結合したモノクローナル抗体、抗原性物質
、標識モノクローナル抗体を結合させて得た免疫複合体
における標識物質量を測定する方法などが知られている
（特開昭５７−８６０５１号公報、特開昭５７−１１８
１５９号公報、特開昭５７−７９４５５号公報）。[Prior Art] Conventionally, immunoassay methods using proteins that specifically bind to the analyte, such as monoclonal antibodies, include:
There are known methods for measuring the amount of a labeled substance in an immune complex obtained by binding a monoclonal antibody, an antigenic substance, and a labeled monoclonal antibody bound to an insoluble solid (Japanese Unexamined Patent Publication No. 57-86051, Showa 57-118
No. 159, JP-A-57-79455).
［発明が解決しようとする問題点コ
しかしながら、従来の測定法は、被測定物質とタンパク
質との親和力が弱い場合、十分な感度が得られないなど
の問題点がある。[Problems to be Solved by the Invention] However, conventional measurement methods have problems such as insufficient sensitivity when the affinity between the analyte and the protein is weak.
［問題点を解決するための手段］
本発明者らは、上記問題点に鑑みて、モノクローナル抗
体のような被測定物質と特異的に結合するタンパク質の
親和力にかかわらず、十分な感度を持つ測定法を得るべ
く鋭意検討した結果、本発明に到達した。[Means for Solving the Problems] In view of the above problems, the present inventors have developed a measurement method with sufficient sensitivity regardless of the affinity of a protein that specifically binds to a analyte, such as a monoclonal antibody. As a result of intensive study to obtain a method, the present invention was arrived at.
すなわち、本発明は、被測定物質、被測定物質と特異的
に結合するタンパク質（第一結合タンパク質）、不溶性
固体上の第一結合タンパク質の一部分を切断除去したタ
ンパク質（第二結合タンパク質）、および第一結合タン
パク質の切断除去部分の全部又は一部を認識する標識抗
体（標識抗体）を結合させて得た複合体における標識物
質量を測定することにより、該被測定物質を測定するこ
とを特徴とする被測定物質の免疫測定法、並びに、被測
定抗原性物質、被測定抗原性物質を認識するモノクロー
ナル抗体（第一抗体）、不溶性固体上の第一抗体の一部
分を切断除去したモノクローナル抗体（第二抗体）、お
よび第一抗体の切断除去部分の全部又は一部を認識する
標識抗体（第三抗体）を結合させて得た免疫複合体にお
ける標識物質量を測定することにより、該被測定抗原性
物質量を測定することを特徴とする抗原性物質の免疫測
定法である。That is, the present invention provides a substance to be measured, a protein that specifically binds to the substance to be measured (first binding protein), a protein obtained by cutting and removing a portion of the first binding protein on an insoluble solid (second binding protein), and The analyte is measured by measuring the amount of a labeled substance in a complex obtained by binding a labeled antibody (labeled antibody) that recognizes all or part of the cleaved portion of the first binding protein. An immunoassay method for a substance to be measured, as well as an antigenic substance to be measured, a monoclonal antibody (first antibody) that recognizes the antigenic substance to be measured, and a monoclonal antibody obtained by cutting off a part of the first antibody on an insoluble solid ( By measuring the amount of labeled substance in the immune complex obtained by binding a labeled antibody (second antibody) that recognizes all or a part of the cleaved portion of the first antibody (third antibody), This is an immunoassay method for antigenic substances, which is characterized by measuring the amount of antigenic substances.
本発明における被測定抗原性物質または被測定物質とし
ては、ホルモン（インシュリン、ＨＣＧ−β、成長ホル
モン、ＴＳ■、サイロキシン、トリヨードサイロニン、
ガストリン、グルカゴン、ソマトスタチンなど）、酵素
（エラスターゼ、アミラーゼ、プロテアーゼ、リパーゼ
、リボヌクレアーゼなど）、血清タンパク質（ＩｇＧ、
ＩｇＡｌｌｇＬ ＩｇＥｌｌｇＤＮ 糖タンパ
ク質、β２−マイクログロブリン、ＴＧＢｌ 糖脂質
など）、腫瘍関連抗原（ＣＥＡ、 α−フェトプロテ
ィン、フェリチン、ＰＯＡ１ＣＡ１９−９、ＣＡ１２５
など）、ＤＮＡ結合性タンパク質因子、サイトカイン（
インターフェロン、インターロイキン１、インターロイ
キン２など）または種々の細菌、ウィルス、原虫（真菌
、連鎖球菌、肝炎ウィルス、ヘルペスウィルス、エイズ
ウィルス、 トキソプラズマ原虫、マラリア原虫、赤痢
アメーバ−など）などがあげられる。これらのうち、好
ましくは、特に高感度の測定系が要求されるホルモンお
よび腫瘍関連抗原である。The antigenic substances or substances to be measured in the present invention include hormones (insulin, HCG-β, growth hormone, TS, thyroxine, triiodothyronine,
gastrin, glucagon, somatostatin, etc.), enzymes (elastase, amylase, protease, lipase, ribonuclease, etc.), serum proteins (IgG,
IgAllgL IgEllgDN Glycoprotein, β2-microglobulin, TGBl Glycolipid, etc.), tumor-associated antigen (CEA, α-fetoprotein, ferritin, POA1CA19-9, CA125
), DNA-binding protein factors, cytokines (
interferon, interleukin 1, interleukin 2, etc.) or various bacteria, viruses, protozoa (fungi, streptococcus, hepatitis virus, herpes virus, AIDS virus, Toxoplasma gondii, malaria parasite, Entamoeba histolytica, etc.). Among these, hormones and tumor-related antigens, which require particularly sensitive measurement systems, are preferred.
第一結合タンパク質または第一抗体としては、被測定物
質と特異的に結合するモノクローナル抗体、ポリクロー
ナル抗体、酵素、酵素インヒビタホルモン、サイトカイ
ン、レセプター レクチン、などを用いることができる
。たとえば、被測′定物質が被測定抗原性物質なら第一
結合タンパク質としてモノクローナル抗体、被測定物質
が酵素インヒビターなら第一結合タンパク質としては酵
素、被測定物質が酵素なら第一結合タンパク質として酵
素インヒビター 被測定物質がホルモンレセプターなら
第一結合タンパク質としてホルモン、被測定物質がサイ
トカインレセプターなら第一結合タンパク質としてサイ
トカイン、被測定物質がホルモンなら第一結合タンパク
質としてホルモンレセプター 被測定物質が複合糖質な
ら第一結合タンパク質としてレクチンなどを用いること
ができる。このうち好ましくは、特異性に優れているモ
ノクローナル抗体である。モノクローナル抗体は、ミエ
ローマ細胞を文献に記載の方法［たとえば、ジー・ケラ
−シー・ミルスタイン；ネイチャー （Ｇ、Ｋｏｈｌｅ
ｒ、Ｃ，Ｍｉｌｓｔｅｌｎ；Ｎａｔｕｒ） Ｖｏｌ、
２５Ｅｉ。As the first binding protein or first antibody, monoclonal antibodies, polyclonal antibodies, enzymes, enzyme inhibitor hormones, cytokines, receptor lectins, etc. that specifically bind to the substance to be measured can be used. For example, if the substance to be measured is an antigenic substance, the first binding protein is a monoclonal antibody, if the substance to be measured is an enzyme inhibitor, the first binding protein is an enzyme, and if the substance to be measured is an enzyme, the first binding protein is an enzyme inhibitor. If the substance to be measured is a hormone receptor, the first binding protein is the hormone. If the substance to be measured is a cytokine receptor, the first binding protein is the cytokine. If the substance to be measured is a hormone, the first binding protein is the hormone receptor. If the substance to be measured is a complex carbohydrate, the first binding protein is the hormone receptor. A lectin or the like can be used as one binding protein. Among these, monoclonal antibodies with excellent specificity are preferred. Monoclonal antibodies can be used to target myeloma cells using methods described in the literature [e.g., G. Kelsey, Milstein;
r, C, Milsteln; Nature) Vol.
25Ei.
ＰＰ、４９５（１９７５）］によって融合させた細胞由
来の抗体であり、同一クローンの抗体産生細胞から産生
された抗体であるので、その抗体は完全に同一であり、
被測定抗原性物質に対する特異性も完全に同一であると
いう特徴をもつことが知られている。PP, 495 (1975)] and produced from antibody-producing cells of the same clone, the antibodies are completely identical;
It is known that the specificity for the antigenic substance to be measured is also completely the same.
第二結合タンパク質又は第二抗体としては、第−結合タ
ンバク質又は第一抗体をプロテアーゼ（ペプシン、パパ
イン、 トリプシン、プラスミンなど）を用いる方法［
たとえば続生化学実験講座５免疫生化学研究法 Ｐ８９
〜ＰＩＯＩ］により一部分切断した後、ゲルろ過クロマ
トグラフィー法あるいはプロティンＡ−アフィニティー
クロマトグラフィー［たとえば続生化学講座５ 免疫生
化学研究法Ｐ２Ｏ３コにより該切断部分を除去したタン
パク質を用いることができる。As the second binding protein or second antibody, a method using a protease (pepsin, papain, trypsin, plasmin, etc.) for the second binding protein or first antibody [
For example, follow-up biochemistry experiment course 5 immunobiochemistry research methods P89
~ PIOI], and then gel filtration chromatography or protein A-affinity chromatography [for example, Immune Biochemistry Research Method P2O3 Co., Ltd.] can be used to remove the cleaved portion of the protein.
不溶性固体としては、ケイ酸質無機担体［ガラス（ポー
ラス、ツヤ消しガラスなど）、シリカゲル、ベントナイ
トなど］、磁性体、および有機担体（プラスチック、デ
キストラン、口紙など）があげられる。これらのうち、
好ましくは、簡便かつ安定して抗体が結合でき、さらに
、取扱いが容易なガラス（ガラスピーズおよびガラス試
験管）、およびプラスチック（プラスチックチューブお
よびプラスチックトレイ）である。Insoluble solids include silicic acid inorganic carriers [glass (porous, matte glass, etc.), silica gel, bentonite, etc.], magnetic substances, and organic carriers (plastic, dextran, lip paper, etc.). Of these,
Preferable materials are glass (glass beads and glass test tubes) and plastic (plastic tubes and plastic trays), which can be easily and stably bound to antibodies and are also easy to handle.
不溶性固体上に第二結合タンパク質又は第二抗体を結合
させる方法としては、ガラスと抗体を化学的に結合させ
る（シランカップリング剤および必要により架橋剤をも
ちいて）、または物理吸着させる方法［たとえば、米国
特許第４２８０９９２号明細書および同第３６５２７６
１号明細書記載の方法コあるいは、プラスチックに抗体
を物理吸着させる方法［たとえば、イー・エングバール
、ジェー・ジョンソン、ピー・パールマン；バイオキム
、パイオフィス、アクタ（Ｅ、Ｅｎｇｖａｌ Ｉ、Ｊ、
Ｊｏｎｓ−ｓｏｎ、Ｐ、Ｐａｒｌｍａｎｎ；Ｂｉｏｃｈ
ｌｍ、Ｂｊｏｐｙｓ、Ａｃｔａ）、２５１（１９７＋）
４２７〜４３４記載の方法］がある。Methods for binding the second binding protein or the second antibody onto the insoluble solid include chemically bonding the glass and the antibody (using a silane coupling agent and optionally a crosslinking agent), or physical adsorption [e.g. , U.S. Patent Nos. 4,280,992 and 365,276
1, or a method of physically adsorbing antibodies to plastic [for example, E. Engval, J. Johnson, P. Perlman; Biochim, Pioffice, Acta (E., Engval I., J.
Jons-son, P., Parlmann; Bioch.
lm, Bjopys, Acta), 251 (197+)
427-434].
標識抗体又は第三抗体に用いる抗体としては、第一結合
タンパク質又は第一抗体の切断除去部分の全部又は一部
を特異的に認識する抗体［たとえば、第一結合タンパク
質がマウスＩｇＧモノクローナル抗体で切断除去部分が
Ｆｃ部分なら抗マウスＩｇＧＦｃ抗体、あるいは第一結
合タンパク質がマウスＩｇＡモノクローナル抗体で切断
除去部分がＦｃ部分なら抗マウスＩｇＡ Ｆｃ抗体など
コがあげられる。The antibody used for the labeled antibody or the third antibody is an antibody that specifically recognizes all or part of the first binding protein or the cleaved portion of the first antibody [for example, if the first binding protein is cleaved with a mouse IgG monoclonal antibody] Examples include anti-mouse IgG Fc antibody if the removed portion is the Fc portion, or anti-mouse IgA Fc antibody if the first binding protein is a mouse IgA monoclonal antibody and the cut and removed portion is the Fc portion.
標識抗体又は第三抗体に標識する標識物質としては、酵
素（ペルオキシダーゼ、アルカリフォスファターゼ、お
よびβ−ガラクトシダーゼなど）、放射線同位元素（！
Ｈ，１２ＳＪなど）、蛍光物質（フルオレセインイソチ
オシアネート、ローダミンなど）、化学発光物質（イソ
ルミノール誘導体、Ｎ−メチルアクリジウム誘導体など
）などがあげられる。好ましくは、抗体標識が容易で、
かつ高い感度が得られるペルオキシダーゼおよび１２５
Ｉである。Labeling substances for labeling the labeled antibody or third antibody include enzymes (peroxidase, alkaline phosphatase, β-galactosidase, etc.), radioisotopes (!
H, 12SJ, etc.), fluorescent substances (fluorescein isothiocyanate, rhodamine, etc.), chemiluminescent substances (isoluminol derivatives, N-methylacridium derivatives, etc.), and the like. Preferably, antibody labeling is easy;
Peroxidase and 125 that provide high sensitivity and
It is I.
請求項１記載の免疫複合体は、（１）不溶性固体上の第
二結合タンパク質と被測定物質を結合させたのち、洗浄
し、この被測定物質と第一結合タンパク質を結合させた
のち、再び洗浄し、さらにこの第一結合タンパク質と標
識抗体を結合させる方法、（２）第一結合タンパク質、
被測定物質、不溶性固体上の第二結合タンパク質を同時
に結合させたのち、洗浄し、さらにこの第一結合タンパ
ク質と標識抗体を結合させる方法、および、（３）第一
結合タンパク質、被測定物質、不溶性固体上の第二結合
タンパク質、標識抗体を同時に結合させる方法によって
得ることができる。たとえば、（１）第二結合タンパク
質を結合させた不溶性固体、および被測定物質を緩衝液
中でインキュベーション（たとえば、５〜５０℃、５分
〜２日）し結合させたのち、不溶性固体を洗浄する。つ
ぎに第−結合タンパク質を含む緩衝液中に不溶性固体を
移し、インキュベーション（たとえば、５〜５０℃、５
分〜２日）し結合させたのち、再び不溶性固体を洗浄す
る。The immune complex according to claim 1 is prepared by: (1) binding the second binding protein on the insoluble solid to the analyte, washing the analyte, binding the analyte to the first binding protein, and then combining the analyte with the second binding protein on the insoluble solid; a method for washing and further binding this first binding protein to a labeled antibody; (2) a first binding protein;
A method of simultaneously binding an analyte and a second binding protein on an insoluble solid, washing the first binding protein, and further binding a labeled antibody to the first binding protein, and (3) a first binding protein, a analyte, It can be obtained by a method of simultaneously binding a second binding protein and a labeled antibody on an insoluble solid. For example, (1) the insoluble solid to which the second binding protein is bound and the substance to be measured are incubated in a buffer solution (e.g., 5 to 50°C, 5 minutes to 2 days) to bind, and then the insoluble solid is washed. do. The insoluble solid is then transferred into a buffer containing the first binding protein and incubated (e.g., 5-50°C, 50°C).
After binding (from 1 minute to 2 days), the insoluble solids are washed again.
さらに標識抗体を含む緩衝液中に不溶性固体を移し、イ
ンキュベーション（たとえば、５〜５０℃、５分〜２日
）し結合させることにより免疫複合体を得る。（２）第
一結合タンパク質、被測定物質、第二結合タンパク質を
結合させた不溶性固体を緩衝液中でインキュベート（た
とえば、５〜５０℃、５分〜２日）し結合させたのち、
不溶性固体を洗浄する。次に、標識抗体を含む緩衝液中
に不溶性固体を移し、インキュベート（たとえば、５〜
５０℃、５分〜２日）し結合させることにより免疫複合
体を得る。または、（３）第一結合タンパク質、被測定
物質、第二結合タンパク質を結合させた不溶性固体、標
識抗体を緩衝溶液中でインキュベート（たとえば、５〜
５０℃、５分〜２日）し結合させることにより免疫複合
体を得ることができる。Furthermore, the insoluble solid is transferred to a buffer solution containing a labeled antibody, and incubated (eg, 5 to 50°C, 5 minutes to 2 days) to allow binding, thereby obtaining an immune complex. (2) After incubating the insoluble solid to which the first binding protein, analyte, and second binding protein are bound in a buffer solution (e.g., 5 to 50°C, 5 minutes to 2 days) to allow binding,
Wash away insoluble solids. Next, transfer the insoluble solids into a buffer containing the labeled antibody and incubate (e.g.
An immune complex is obtained by binding at 50° C. for 5 minutes to 2 days). Alternatively, (3) incubate the first binding protein, the analyte, the insoluble solid bound to the second binding protein, and the labeled antibody in a buffer solution (for example,
An immune complex can be obtained by binding at 50°C for 5 minutes to 2 days).
免疫複合体の標識物質量の測定は、標識物質に応じて酵
素量を酵素活性の測定により測定する方法、放射線同位
元素量を放射線測定装置を用いて測定する方法、蛍光物
質量を蛍光光度計により測定する方法、化学発光物質量
を酵素を用いた化学発光により測定する方法などにより
行うことができる。たとえば、標識物質にペルオキシダ
ーゼを用いた場合、０−フェニレンジアミンと過酸化水
素溶液、４−アミノアンチピリンと過酸化水素溶液など
の発色基質溶液中で免疫複合体をインキュベート（たと
えば、５〜５０″Ｃ１５分〜２日）した後分光光度計を
用いて発色量を測定することによりペルオキシダーゼ活
性を測定する方法などをあげることができる。The amount of labeled substances in immune complexes can be measured by measuring the amount of enzymes depending on the labeled substance by measuring enzyme activity, by measuring the amount of radioisotopes using a radiation measuring device, and by measuring the amount of fluorescent substances using a fluorometer. The amount of chemiluminescent substance can be measured by chemiluminescence using an enzyme. For example, when peroxidase is used as a labeling agent, the immune complex is incubated in a chromogenic substrate solution such as 0-phenylenediamine and hydrogen peroxide solution, 4-aminoantipyrine and hydrogen peroxide solution (e.g., 5-50"C15 For example, the peroxidase activity can be measured by measuring the amount of color developed using a spectrophotometer after the peroxidase reaction has been carried out (from 1 minute to 2 days).
請求項２記載の免疫複合体は、請求項１記載の第一結合
タンパク質の代わりに第一抗体、被測定物質の代わりに
被測定抗原性物質、第二結合タンパク質の代わりに第二
抗体、標識抗体の代わりに第三抗体を用いることにより
請求項１記載の発明と同様に得ることができる。The immune complex according to claim 2 comprises a first antibody instead of the first binding protein according to claim 1, an antigenic substance to be measured instead of the substance to be measured, a second antibody instead of the second binding protein, and a label. It can be obtained in the same manner as in the invention described in claim 1 by using a third antibody instead of the antibody.
［実施例］
＝１１−
以下、実施例および比較例により、本発明を更に説明す
るが、本発明はこれに限定されるものではない。[Example] =11- The present invention will be further explained below with reference to Examples and Comparative Examples, but the present invention is not limited thereto.
実施例Ｉ ＣＡ１９−９の測定
ａ）抗ＣＡ１９−９モノクローナル抗体の製造ＣＡ１９
−９に対するモノクローナル抗体は、文献［ヒラリー・
コブロスキー ゼノンφステプルスキケネット・ミッチ
ェル、 ミーンハード・ヘリン； ツマチック セルジ
ェネッティックス（Ｈｉｌａｒｙ Ｋｏｐｒｏｗｓｋｌ
、Ｚｅｎｏｎ Ｓｔｅｐｌｅｗｓｋｉ、Ｋｅｎｎｅｔｈ
Ｍｉｔｃｈｅｌｌ、Ｍｅｅｎｈａｒｄ Ｈｅ１ｙｎ
；Ｓｏｍａｔｊｃ Ｃｅ１ｌ Ｇｅｎｅｔｉｃｓ）Ｖｏ
ｌ、５．Ｎｏ［ｉ、１９７９．ｐｐ、９５７−９７２１
に記載の方法に従って製造した。Example I Measurement of CA19-9 a) Production of anti-CA19-9 monoclonal antibody CA19
Monoclonal antibodies against -9 have been used in the literature [Hillary et al.
Kobrowski Zenon φ Stepskikenette Mitchell, Meanhard Helin; Tumatic Cell Genetics (Hilary Koprowskl)
, Zenon Steplewski, Kenneth
Mitchell, Meenhard Helyn
; Somatjc Ce1l Genetics) Vo
l, 5. No [i, 1979. pp, 957-9721
It was manufactured according to the method described in .
簡単には、５Ｗ１１１Ｅｉ細胞（大日本製薬社から入手
可能） ｌＸ１０８個をＢＡＬＢ／ｃマウスの静脈内に
投与し免疫した。 １ケ月後に再び５Ｗ１１１Ｂ細胞１
ｘｌＯ’個を投与し３日後に肺臓を摘出して肺細胞を採
取した。Briefly, 1×108 5W111Ei cells (available from Dainippon Pharmaceutical Co., Ltd.) were intravenously administered to BALB/c mice for immunization. 5W111B cells 1 again after 1 month
Three days after administration of xlO', the lungs were removed and lung cells were collected.
ＲＰＭＩＩＥｉ４０培地にて洗浄した後、肺細胞全量を
２ｘ１０７個のマウスミエローマ細胞（Ｐ３−ＮＳＩ／
ｌ−Ａｇ ４，１）と混ぜ、３７℃の４２．５％ポリエ
チレングリコール１５４０および７．５％ジメチルスル
フオキシドを含むＲＰＭＩＩＥｉ４０培地１ｍｌ中で１
培地１金ｌ中た。１分後にその細胞懸濁物をＲＰＭＩＩ
Ｅｉ４０培地５ｍｌで徐々培地５ヒｌ。それらの細胞を
遠心分離し、洗浄した後、ＨＡＴ培地（ヒポキサンチン
、アミノプテリン、チミジン、ＩＯ％牛脂児血清を含む
ＲＰ旧１６４０培地）を２０１になるように加えて、９
６ウエル マイクロプレートに０．２ｍｌずつ分注して
２週間培養した後、増殖したウェル中の培養上清の抗体
活性を測定した。After washing with RPMIIEi40 medium, the total amount of lung cells was transformed into 2x107 mouse myeloma cells (P3-NSI/
l-Ag 4,1) in 1 ml of RPMIIEi40 medium containing 42.5% polyethylene glycol 1540 and 7.5% dimethyl sulfoxide at 37°C.
The medium was in 1 gold liter. After 1 minute, the cell suspension was transferred to RPMII.
Gradually add 5 ml of Ei40 medium to 5 ml of medium. After centrifuging and washing the cells, add HAT medium (RP old 1640 medium containing hypoxanthine, aminopterin, thymidine, IO% tallow serum) to 201,9
After dispensing 0.2 ml into a 6-well microplate and culturing for 2 weeks, the antibody activity of the culture supernatant in the proliferated wells was measured.
次に、活性の認められたウェルの細胞を限界希釈法を使
用して繰り返してクローン化した。クローン化した細胞
をマウスの腹腔内に投与し腹水腫瘍として成長させた後
、腹水を採取した。この腹水中モノクローナル抗体をア
フィ・ゲル・プロティンＡ ＭＡＰＳキット（バイオラ
ッド社）を用いて精製単離し、以下の検討に用いた。Cells from active wells were then repeatedly cloned using limiting dilution. The cloned cells were intraperitoneally administered to mice to grow as an ascites tumor, and the ascites fluid was collected. This monoclonal antibody in ascites was purified and isolated using Affi-Gel Protein A MAPS kit (Bio-Rad) and used in the following studies.
ｂ）抗ＣＡ１９−９モノクローナル抗体のＦｃ部分の切
断除去
抗ＣＡ１９−９モノクローナル抗体２０Ｂを０．１Ｍ
ＮａＣ１０，１Ｍ酢酸ナトリウム緩衝液（ｐＨ４，５）
で透析した。透析後、０．４ｍｇペプシン（ベーリンガ
ー マンハイム社）を加え、３７℃で６時間インキュベ
ートした。次に、この溶液に０．１Ｍ水酸化ナトリウム
水溶液を加えｐＨを７に合わせたのち、ウルトロゲルＡ
ｃＡ４４ （ニルケイビーフアルマシア社）カラム（１
，５ｃｍｘ９０ｃｍ）でゲルろ過を行いＦｃ部分を含む
分画を除いた。さらに、アフィΦゲルΦプロティンＡ
ＭＡＰＳキットを用いて未消化のモノクローナル抗体を
除去し、モノクローナル抗体Ｆ（ａｂ’）２分画を得た
。b) Cleavage and removal of Fc portion of anti-CA19-9 monoclonal antibody Anti-CA19-9 monoclonal antibody 20B at 0.1 M
NaC10, 1M sodium acetate buffer (pH 4, 5)
Dialyzed with After dialysis, 0.4 mg pepsin (Boehringer Mannheim) was added and incubated at 37°C for 6 hours. Next, 0.1M sodium hydroxide aqueous solution was added to this solution to adjust the pH to 7, and then Ultrogel A
cA44 (Nilkey Beef Almacia) column (1
, 5 cm x 90 cm) to remove the fraction containing the Fc portion. In addition, Affi Φ Gel Φ Protein A
Undigested monoclonal antibody was removed using a MAPS kit to obtain a monoclonal antibody F(ab')2 fraction.
ｃ） Ｆｃ部分を切断除去した抗ＣＡｌ９−９モノク
ローナル抗体結合ガラスピーズの作製
米国特許第６５２７Ｅｉ１号明細書の方法に従い、ガラ
スピーズの表面にＦｃ部分切断除去抗ＣＡ１９−９モノ
クロ一ナル抗体をコーティングした。c) Preparation of anti-CA19-9 monoclonal antibody-bound glass beads with the Fc portion cut and removed According to the method described in US Patent No. 6527Ei1, the surface of glass beads was coated with the anti-CA19-9 monoclonal antibody with the Fc portion cut and removed. .
ｄ）ペルオキシダーゼ標識抗マウスＩｇＧ Ｆｃ抗体の
作製
抗マウスＩｇＧ Ｆｃ抗体（バインディングサイト社）
を文献［ニス・ヨシタケ、エム・イマガヮ、イー拳イシ
カワ、エトール； ジェイ、バイオケム（Ｓ、ＹＯ５Ｈ
ＩＴＡＫＥ 、Ｍ、Ｉ ＩＭＡＧＡＷＡ 、Ｅ 、 ｌ
５ＨＩＫＡＷＡ 、ｅｔ 、ａｌ 、ｉＪ、ＢＩ。d) Preparation of peroxidase-labeled anti-mouse IgG Fc antibody Anti-mouse IgG Fc antibody (Binding Site Co., Ltd.)
References [Nisu Yoshitake, M. Imagawa, Eken Ishikawa, Ettor; Jay, Biochem (S, YO5H
ITAKE, M, I IMAGAWA, E, l
5HIKAWA, et al, iJ, BI.
ｃｈｅｍ、、 Ｖｏｌ、９２（１９８２） １４１３−
＋４２４コに記載の方法にてペルオキシダーゼと結合し
、ペルオキシダーゼ標識抗マウスＩｇＧ Ｆｃ抗体を得
た。この試薬は通常１％牛血清アルブミン含を緩衝液で
１０〜５０００倍に希釈して使用した。chem, Vol. 92 (1982) 1413-
This was combined with peroxidase using the method described in 424, to obtain a peroxidase-labeled anti-mouse IgG Fc antibody. This reagent usually contains 1% bovine serum albumin and was used after being diluted 10 to 5000 times with a buffer.
ｅ） ＣＡ１９−９標準溶液の調整
５Ｗ−１１１８細胞をｌθ％牛脂児血清含有ＲＰＭｌｌ
Ｂ４０培地で培養し、培養土清液を採取した。培養上清
液中ノＣＡ１９−９′ａ度ヲイム／ クロアＣＡｌ９−
９ （富士レヒオ社）を用いて測定し、濃度が３０．Ｅ
ｉＯ，１２０，２４０ｕｎｉｔ／ｍｌなるように１％牛
血清アルブミン含を緩衝液で希釈し標準溶液とした。e) Preparation of CA19-9 standard solution 5W-1118 cells were added to RPMll containing lθ% beef tallow serum.
The cells were cultured in B40 medium, and culture soil liquid was collected. CA19-9'a degrees in culture supernatant/Cloa CAl9-
9 (Fuji Regio), and the concentration was 30. E
A standard solution containing 1% bovine serum albumin was diluted with a buffer solution to give iO, 120, 240 units/ml.
ｆ） ＣＡ１９−９標準溶液の測定
ＣＡ１９−９２４０ｕｎＨ／ｍｌを含む標準溶液５０／
ＩＺＩ、抗ＣＡ１９−９モノクローナル抗体ＩＯμｇ／
ｍｌ含有０．０２Ｍリン酸緩衝液１００μｌおよび１％
牛血清アルブミン含有０．０２Ｍ ＩＪン酸緩衝液３０
０μｌを入れた試験管にＦｃ部分切断除去抗ＣＡｌ９−
９モノクロ一ナル抗体結合ガラスピーズを１個入れイン
キュベーション（３７℃、 １５ 分間）したのち、生
理食塩水にてビーズを洗浄した。次に、ペルオキシダー
ゼ標識抗マウスＩｇＧ Ｆｃ抗体含有０．０２Ｍ リン
酸緩衝液５００μｌ中にビーズを移し、インキュベーシ
ョン（３７℃、１５分間）した。再度、生理食塩水にて
ビーズを洗浄したのち、ビーズを基質溶液（過酸化水素
含有オルト−フェニレンジアミン溶液）５００μｌ中に
移し、インキュベーション（３７’Ｃ，１５分間）した
のち、１．５規定硫酸水溶液３＋ｎ＋を加えて反応を停
止した。この液の４９２ｎｍの吸光度を測定し、ビーズ
に結合した酵素の酵素活性を測定した。f) Measurement of CA19-9 standard solution Standard solution containing 50/ml CA19-9240unH/ml
IZI, anti-CA19-9 monoclonal antibody IOμg/
ml containing 100 μl of 0.02M phosphate buffer and 1%
0.02M IJ acid buffer containing bovine serum albumin 30
Fc partial cleavage removed anti-CAl9-
9 monoclonal antibody-conjugated glass beads were added and incubated (37°C, 15 minutes), and then the beads were washed with physiological saline. Next, the beads were transferred to 500 μl of 0.02 M phosphate buffer containing peroxidase-labeled anti-mouse IgG Fc antibody and incubated (37° C., 15 minutes). After washing the beads again with physiological saline, the beads were transferred to 500 μl of a substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide), incubated (37'C, 15 minutes), and then washed with 1.5 N sulfuric acid. The reaction was stopped by adding aqueous solution 3+n+. The absorbance of this solution at 492 nm was measured to determine the enzyme activity of the enzyme bound to the beads.
同様に、ＣＡ１９−９標準溶液３０．Ｇｏ、１２０ｕｎ
ｉｔ／ｍｌお上び０ブランクの測定も行った。Similarly, CA19-9 standard solution 30. Go, 120un
It/ml and 0 blank measurements were also performed.
第１図に本測定法によるＣＡ１９−９標準溶液の測定結
果を示した。FIG. 1 shows the measurement results of the CA19-9 standard solution using this measurement method.
比較例１ サンドウィッチ法ＥＩＡによるＣＡｌＢ−９
の測定
実施例１と比較するため、抗ＣＡ１９−９モノクローナ
ル抗体を用いたサンドウィッチ法酵素免疫測定法（ＥＩ
Ａ）によりＣＡ１９−９の測定を行った。Comparative Example 1 CAIB-9 by sandwich method EIA
Measurement In order to compare with Example 1, a sandwich enzyme immunoassay (EI) using an anti-CA19-9 monoclonal antibody was performed.
CA19-9 was measured by A).
ａ） 抗ｃＡ１９−９モノクローナル抗体の製造ＩＢ 実施例１．ａ）に準じた。a) Production of anti-cA19-9 monoclonal antibody IB Example 1. According to a).
ｂ）抗ＣＡ１９−９モノクローナル抗体結合ガラスピー
ズの作製
米国特許筒［１５２７［ｉ１号明細書の方法に従い、ガ
ラスピーズの表面に抗ＣＡ１９−９モノクローナル抗体
をコーティングした。b) Preparation of anti-CA19-9 monoclonal antibody-conjugated glass beads The surface of glass beads was coated with an anti-CA19-9 monoclonal antibody according to the method described in US Pat. No. 1,527 [i1].
Ｃ）ペルオキシダーゼ標識抗ＣＡ１９−９モノクローナ
ル抗体の作製
抗ＣＡｌ９−９モノクローナル抗体を文献［ニス・ヨシ
タケ、エム・イマガワ、イー・イシカワ、エトール；
ジェイ、バイオケム（ｓ、ｙｏｓ■ＩＴＡＫＥ、Ｍ、Ｉ
ｒＭＡＧＡＷＡ、Ｅ、ｌ５ＨＩＫＡＷＡ、ｅｔ、ａｌ、
；Ｊ、Ｂｉｏｃｈｅｍ、、 Ｖｏｌ、９２（＋９８２）
１４１３−１４２４１記載の方法にてペルオキシダー
ゼと結合し、ペルオキシダーゼ標識抗ＣＡｌ９−９モノ
クローナル抗体を得た。この試薬は通常１％牛血清アル
ブミン含有緩衝液で１０〜５０００倍に希釈して使用し
た。C) Preparation of peroxidase-labeled anti-CA19-9 monoclonal antibody Anti-CA19-9 monoclonal antibody was prepared from the literature [Yoshitake Nis, M Imagawa, E Ishikawa, Etol;
Jay, Biochem (s, yos ■ ITAKE, M, I
rMAGAWA, E, l5HIKAWA, et al.
;J,Biochem,, Vol, 92(+982)
1413-14241 to obtain a peroxidase-labeled anti-CAl9-9 monoclonal antibody. This reagent was usually diluted 10 to 5000 times with a buffer containing 1% bovine serum albumin.
ｄ） ＣＡ１９−９標準溶液の調整 実施例１．ｅ）に準じた。d) Preparation of CA19-9 standard solution Example 1. According to e).
ｅ） ＣＡ１９−９標準溶液ノ測定
ＣＡ１９−９２４０ｕｎｉｔ／ｍｌを含む標準溶液５０
μｌおよび１％牛血清アルブミン含を０．０２Ｍ ＩＪ
ン酸緩衝液４００μｌを入れた試験管に抗ＣＡ１９−９
モノクローナル抗体結合ガラスピーズを１個入れインキ
ュベーション（３７℃、１５分間）したのち、生理食塩
水にてビーズを洗浄した。次に、ペルオキシダーゼ標識
抗ＣＡ１９−９モノクローナル抗体含有０．０２Ｍリン
酸緩衝液５００μｌ中にビーズを移し、インキュベーシ
ョン（３７℃。e) Measurement of CA19-9 standard solution 50 standard solutions containing CA19-9240 units/ml
0.02M IJ containing μl and 1% bovine serum albumin
Add anti-CA19-9 to a test tube containing 400 μl of acid buffer.
After adding one monoclonal antibody-bound glass bead and incubating it (37°C, 15 minutes), the bead was washed with physiological saline. Next, the beads were transferred to 500 μl of 0.02 M phosphate buffer containing peroxidase-labeled anti-CA19-9 monoclonal antibody and incubated (37° C.).
１５分間）した。再度、生理食塩水にてビーズを洗浄し
たのち、ビーズを基質溶液（過酸化水素台をオルト−フ
ェニレンジアミン溶液） ５００μｌ中に移し、インキ
ュベーション（３７℃、１５分間）したのち、１．５規
定硫酸水溶液３１を加えて反応を停止した。この液の４
９２ｎｉ＋の吸光度を測定し、ビーズに結合した酵素の
酵素活性を測定した。15 minutes). After washing the beads again with physiological saline, the beads were transferred to 500 μl of a substrate solution (hydrogen peroxide solution and ortho-phenylenediamine solution), incubated (37°C, 15 minutes), and then washed with 1.5 N sulfuric acid. Aqueous solution 31 was added to stop the reaction. 4 of this liquid
The absorbance of 92ni+ was measured to determine the enzyme activity of the enzyme bound to the beads.
同様に、ＣＡ１９−９標準溶液３０，６０，１２０ｕｎ
ｉｔ／ｍｌおよび０ブランクの測定も行った。Similarly, CA19-9 standard solution 30, 60, 120un
It/ml and 0 blank measurements were also taken.
第１図に本測定法によるＣＡ１９−９標準溶液の測定結
果を示した。FIG. 1 shows the measurement results of the CA19-9 standard solution using this measurement method.
実施例２
ａ） 抗■Ｂｓモノクローナル抗体の製造■Ｂｓに対
するモノクローナル抗体は文献［ケイ・クニトモ、ニス
・ヤハラ、エイチ・サジ、ティー・ホソイ；ボックス
サンジーニス （Ｋ 、Ｋｕｎｉｔｏｍｏ 。Example 2 a) Production of anti-Bs monoclonal antibodies ■ Monoclonal antibodies against Bs are described in the literature [Kunitomo K, Nis Yahara, H Saji, T Hosoi;
Sangenis (K, Kunitomo.
Ｓ、Ｙａｈａｒａ、Ｈ，５ａｊｊ、Ｔ、Ｈｏ５ｏｉ；Ｖ
ｏｘ Ｓａｎｇｕｉｎｊｓ） Ｖｏｌ。S, Yahara, H, 5ajj, T, Ho5oi; V
ox Sanguinjs) Vol.
４５、Ｎｏ、２．１９８３．ｐｐ、１０４−１１１コに
記載の方法に従って製造した。45, No. 2.1983. It was produced according to the method described in pp. 104-111.
簡単には、ＨＢｓＢｓ抗原５００μミドリ十字社製）を
フロイントの完全アジュバントとともにＢＡＬＢ／ｃマ
ウスの腹腔内に投与し免疫した。１０日後に再びＨＢｓ
抗原１００μｇを静脈内に投与し３日後に肺臓を摘出し
て肺細胞を採取した。ＲＰＭ１１８４０培地にて洗浄し
た後、肺細胞全量を２ｘｌＯ７個のマウスミエローマ細
胞（Ｐ３−ＮＳＩ／ｌ−Ａｇ ４．１）と混ぜ、３７℃
の４２．５％ポリエチレングリコール１５４０および７
．５％ジメチルスルフオキシドを含むＲＰＭ１１６４０
培地１１中で１分間融合させた。１分後にその細胞懸濁
物をＲＰＭｌｌ［ｉ４０培地５１で徐々に希釈した。そ
れらの細胞を遠心分離し、洗浄した後、ＨＡＴ培地（ヒ
ポキサンチン、アミノプテリン、チミジン、ＩＯ％牛脂
児血清を含むＲＰＭＩ＋６４０培地）を２０ｍ ｌにな
るように加えて、９８ウエルマイクロプレートに０．２
ｍｌずつ分注して２週間培養した後、増殖したウェル中
の培養上清の抗体活性を測定した。Briefly, BALB/c mice were immunized by intraperitoneally administering 500 µm of HBsBs antigen (manufactured by Midori Juji) together with complete Freund's adjuvant. HBs again after 10 days
100 μg of antigen was administered intravenously, and 3 days later, the lungs were removed and lung cells were collected. After washing with RPM11840 medium, the total amount of lung cells was mixed with 2xlO7 mouse myeloma cells (P3-NSI/l-Ag 4.1) and incubated at 37°C.
42.5% polyethylene glycol 1540 and 7
．． RPM11640 with 5% dimethyl sulfoxide
The cells were allowed to fuse for 1 minute in medium 11. After 1 minute, the cell suspension was gradually diluted with 51 ml of RPM11 i40 medium. After centrifuging and washing the cells, add HAT medium (RPMI+640 medium containing hypoxanthine, aminopterin, thymidine, and IO% tallow serum) to 20 ml, and add 0.0 mL to a 98-well microplate. 2
After dispensing the cells in ml portions and culturing them for two weeks, the antibody activity of the culture supernatant in the wells in which the cells had grown was measured.
次に、活性の認められたウェルの細胞を限界希釈法を使
用して繰り返してクローン化した。クローン化した細胞
をマウスの腹腔内に投与し腹水腫瘍として成長させた後
、腹水を採取した。この腹水中モノクローナル抗体をア
フィ・ゲル・プロティンＡ ＭＡＰＳキット（バイオラ
ッド社）を用いて精製単離し、以下の検討に用いた。Cells from active wells were then repeatedly cloned using limiting dilution. The cloned cells were intraperitoneally administered to mice to grow as an ascites tumor, and the ascites fluid was collected. This monoclonal antibody in ascites was purified and isolated using Affi-Gel Protein A MAPS kit (Bio-Rad) and used in the following studies.
ｂ）抗ＨＢｓモノクローナル抗体のＦｃ部分の切断除去
抗ＨＢＳモノクローナル抗体２０ｍｇを０．ＩＭ Ｎａ
Ｃｌ−０，１Ｍ酢酸ナトリウム緩衝液（ｐＨ４，５）で
透析した。透析後、０．４１１１ｇヘフシン（バーリン
ガー マンハイム社）を加え、３７℃で６時間インキュ
ベートした。次に、この溶液に０．１Ｍ水酸化ナトリウ
ム水溶液を加えｐＨを７に合わせたのち、ウルトロゲル
ＡｃＡ４４カラム（１，５ｃｍｘ９０ｃｎ）でゲルろ
過を行いＦｅ部分を含む分画を除いた。さらに、アフイ
・ゲル・プロティンＡＭＳＰＳキットを用いて未消化の
モノクローナル抗体を除去し、モノクローナル抗体Ｆ（
ａｂ′）２分画を得た。b) Cleavage and removal of Fc portion of anti-HBs monoclonal antibody 20 mg of anti-HBS monoclonal antibody was added to 0.0 mg of anti-HBs monoclonal antibody. IM Na
Dialysis was performed with Cl-0, 1M sodium acetate buffer (pH 4, 5). After dialysis, 0.4111g hefsin (Burlinger Mannheim) was added and incubated at 37°C for 6 hours. Next, a 0.1 M aqueous sodium hydroxide solution was added to this solution to adjust the pH to 7, and then gel filtration was performed using an Ultrogel AcA44 column (1.5 cm x 90 cm) to remove the fraction containing the Fe portion. Furthermore, undigested monoclonal antibody was removed using Afui Gel Protein AMSPS kit, and monoclonal antibody F (
ab') 2 fractions were obtained.
ｅ） Ｆｃ部分を切断除去した抗ＨＢｓモノクローナ
ル抗体結合ガラスピーズの作製
米国特許第１１ｉ５２７１１ｉ１号明細書の方法に従い
、ガラスピーズの表゛面にＦｃ部分切断除去抗ＨＢｓモ
ノクローナル抗体をコーティングした。e) Preparation of anti-HBs monoclonal antibody-conjugated glass beads with the Fc portion removed by cleavage According to the method described in US Pat. No. 11i52711i1, the surface of glass beads was coated with an anti-HBs monoclonal antibody with the Fc portion removed by cleavage.
ｄ） 、＋２６１標識抗マウスＩｇＧ Ｆｃ抗体の作製
抗マウスＩｇＧ Ｆｃ抗体（バインディングサイト社）
ヲ文献［バーバラ・ビー・ミツシェル、スタンレイ・エ
ム・シギ；セレクテイツド メンツズインセルラーイム
ノロジ−（Ｂａｒｂａｒａ Ｂ、Ｍｉｓｈｅｌｌ ５ｔ
ａｎｌｅｙ Ｍ、Ｓｈｌｇｌ；５ＥＬＥＣＴＥＤ ＭＥ
ＴＢＯＤＳ ＩＮ ＣＥＬＬＵＬＡＲＩＮＮＵＮＯＬＯ
ＧＹ） ＰＰ、３１５−３１６．１９８０コに記載の方
法にて１２６１と結合し １２６１標識抗マウスＩｇＧ
・Ｆｃ抗体を得た。d) Preparation of +261-labeled anti-mouse IgG Fc antibody Anti-mouse IgG Fc antibody (Binding Site Co., Ltd.)
Literature [Barbara B. Michelle, Stanley M. Siggi; Selected articles in cellular immunology (Barbara B. Michelle 5t
anley M, Shlgl;5ELECTED ME
TBODS IN CELLULARINNUNOLO
GY) PP, 315-316.1980 Co., 1261-labeled anti-mouse IgG
・Fc antibody was obtained.
この試薬は通常１％牛血清アルブミン含有緩衝液で１０
〜５０００倍に希釈して使用した。This reagent is usually prepared in a buffer containing 1% bovine serum albumin for 10 min.
It was used after being diluted ~5000 times.
ｅ） ＨＢｓ標準溶液の調整
ミドリ十字社から入手したＨＢｓ抗原（４０Ｍｇ／）く
イアル）を０．０２Ｍりん酸緩衝液で希釈し、濃度が０
．５゜１．２．５，５，１００ｇ／ｍｌとなるようにＨ
Ｂｓ標準溶液を調製した。e) Preparation of HBs standard solution Dilute HBs antigen (40 Mg/) obtained from Midori Juji Co., Ltd. with 0.02 M phosphate buffer to a concentration of 0.
．． 5゜H so that it becomes 1.2.5, 5,100g/ml
A Bs standard solution was prepared.
ｆ） ＨＢｓ標準溶液の測定
■Ｂｓ １００ｇ／ｍｌを含む標準溶液５０．ｃｚｌ、
抗ＨＢｓモノクローナル抗体ｌＯμｇ／ｍ ｌ含有０．
０２Ｍリン酸緩衝液１００μｌおよび１％牛血清アルブ
ミン含有０．０２Ｍ ！Ｊン酸緩衝液３００μｌを入れ
た試験管にＦｃ部分切断除去抗ＨＢｓモノクローナル抗
体結合ガラスピーズを１個入れインキュベーション（３
７℃、１５分間）したのち、生理食塩水にてビーズを洗
浄した。次に １２６１標識抗マウスＩｇＧ Ｆｃ抗体
含有０．０２Ｍ ！Ｊン酸緩衝液５００μｌ中にビーズ
を移し、インキュベーション（３７６Ｃ，１５分間）し
た。再度、生理食塩水にてビーズを洗浄したのち、ビー
ズに結合した＋２Ｊの量をγ線カウンターで測定した。f) Measurement of HBs standard solution ■Standard solution containing 100 g/ml of Bs 50. czl,
Contains 10 μg/ml of anti-HBs monoclonal antibody.
0.02M containing 100μl of 02M phosphate buffer and 1% bovine serum albumin! Place one piece of glass beads conjugated to anti-HBs monoclonal antibody with Fc partial cleavage removed in a test tube containing 300 μl of J acid buffer and incubate (3
7° C. for 15 minutes), and then the beads were washed with physiological saline. Next, 0.02M containing 1261-labeled anti-mouse IgG Fc antibody! The beads were transferred to 500 μl of J acid buffer and incubated (376C, 15 minutes). After washing the beads again with physiological saline, the amount of +2J bound to the beads was measured using a γ-ray counter.
同様に、ＨＢｓ標準溶液０．５，１，２．５，５ｎｇ／
ｍｌおよび０ブランクの測定も行った。Similarly, HBs standard solution 0.5, 1, 2.5, 5ng/
ml and 0 blank measurements were also taken.
第２図に本測定法によるｌｌＢｓ標準溶液の測定結果を
示した。FIG. 2 shows the measurement results of the llBs standard solution using this measurement method.
比較例２
実施例２と比較するため、抗ＨＢｓモノクローナル抗体
を用いたサンドウィッチ法放射線免疫測定法（ＲＩＡ）
によりＨＢｓ抗原の測定を行った。Comparative Example 2 For comparison with Example 2, sandwich radioimmunoassay (RIA) using anti-HBs monoclonal antibody
HBs antigen was measured using the following method.
ａ）抗ＨＢｓモノクローナル抗体の製造実施例２．８）
に準じた。a) Preparation of anti-HBs monoclonal antibody Example 2.8)
According to.
ｂ）抗ＨＢｓモノクローナル抗体結合ガラスピーズの作
製
米国特許第１３５２７８１号明細書の方法に従い、ガラ
スピーズの表面に抗ＨＢｓモノクローナル抗体をコーテ
ィングした。b) Preparation of anti-HBs monoclonal antibody-conjugated glass beads According to the method described in US Pat. No. 1,352,781, the surface of glass beads was coated with an anti-HBs monoclonal antibody.
ｃ） １２Ｊ標識抗ＨＢｓモノクロ一ナル抗体の作製
抗ＨＢｓモノクローナル抗体を文献［バーバラ・ビー・
ミツシェル、スタンレイ・エム・シギ；セレクティッド
メソッズインセルラーイムノロジー（Ｂａｒｂａｒａ
Ｂ、旧５ｈｅｌｌ ５ｔａｎｌｅｙ Ｍ、Ｓｈｌｇｌ
ｉ ＳＥＬＥＣＴＥＤＭＥＴ■ＯＤＳ ＩＮ ＣＥＬＬ
ｔｌＬＡＲＩＭＭＵＮＯＬＯＧＹ） ＰＰ、３１５−３
１Ｅｉ。c) Preparation of 12J-labeled anti-HBs monoclonal antibody Anti-HBs monoclonal antibody was prepared from the literature [Barbara B.
Mitschel, Stanley M. Siggi; Selected Methods in Cellular Immunology (Barbara)
B, old 5hell 5tanley M, Shlgl
i SELECTEDMET■ODS IN CELL
tlLARIMMUNOLOGY) PP, 315-3
1Ei.
１９８０］に記載の方法にて１２５１と結合し １２６
１標識抗ＨＢｓモノクロ一ナル抗体を得た。この試薬は
通常１％牛血清アルブミン含有緩衝液で１０〜５０００
倍に希釈して使用した。126 by combining with 1251 by the method described in [1980].
1-labeled anti-HBs monoclonal antibody was obtained. This reagent is usually used in a buffer containing 1% bovine serum albumin.
It was diluted twice and used.
ｄ） ＨＢｓ標準溶液の調整 実施例２．０）に準じた。d) Preparation of HBs standard solution According to Example 2.0).
ｅ）■Ｂｓ標準溶液の測定
ＨＢｓｌＯｎｇ／ｍｌを含む標準溶液５０μｌおよび１
％牛血清アルブミン含有０．０２Ｍ リン酸緩衝液４０
０μＩを入れた試験管に抗ＨＢｓモノクローナル抗体結
合ガラスピーズを１個入れインキュベーション（３７℃
、１５分間）したのち、生理食塩水にてビーズを洗浄し
た。e) ■Measurement of Bs standard solution 50 μl of standard solution containing HBslOng/ml and 1
0.02M phosphate buffer containing % bovine serum albumin 40
Place one anti-HBs monoclonal antibody-conjugated glass bead in a test tube containing 0μI and incubate (37°C).
, 15 minutes), then the beads were washed with physiological saline.
次に １２６１標識抗ＨＢｓモノクロ一ナル抗体含有０
．０２Ｍリン酸緩衝液５００μｍ中にビーズを移し、イ
ンキュベーション（３７℃、１５分間）した。再度、生
理食塩水にてビーズを洗浄したのち、ビーズに結合した
１！Ｊの量をγ線カウンターで測定した。Next, 0 containing 1261-labeled anti-HBs monoclonal antibody
．． Beads were transferred to 500 μM of 02M phosphate buffer and incubated (37° C., 15 minutes). After washing the beads again with physiological saline, the 1! The amount of J was measured with a gamma ray counter.
同様に、ｔｌＢｓ標準溶液０．５，１，２．５．５，１
０ｎｇ／ｍ＋および０ブランクの測定も行った。Similarly, tlBs standard solution 0.5, 1, 2.5.5, 1
0 ng/m+ and 0 blank measurements were also performed.
第２図に本測定法によるＢＢｓ標準溶液の測定結果を示
した。Figure 2 shows the measurement results of the BBs standard solution using this measurement method.
［発明の効果コ
本発明の測定方法によれば、従来の免疫測定方法に比較
して高感度な測定が可能となる。特に、請求項２記載発
明の測定方法によれば、従来のモノクローナル抗体を用
いた測定方法に比較して高感度な測定が可能となる。従
来のモノクローナル抗体を用いた測定方法は測定精度は
優れているが測定感度が低いという問題点があったが、
本発明の測定方法によれば測定精度を低下させることな
く測定感度を向上させることができる。更に、高感度の
測定方法を検査薬に応用した場合測定感度が向上するた
めに測定時間の大幅な短縮が可能である。[Effects of the Invention] According to the measurement method of the present invention, highly sensitive measurement is possible compared to conventional immunoassay methods. In particular, according to the measuring method of the invention as claimed in claim 2, a highly sensitive measurement is possible compared to the conventional measuring method using a monoclonal antibody. Conventional measurement methods using monoclonal antibodies have excellent measurement accuracy but have the problem of low measurement sensitivity.
According to the measurement method of the present invention, measurement sensitivity can be improved without reducing measurement accuracy. Furthermore, when a highly sensitive measurement method is applied to a test drug, the measurement sensitivity is improved, making it possible to significantly shorten the measurement time.
以上の点から、本発明は、特に高感度の測定が要求され
る血清またはその他の体液中に極微量台まれている腫瘍
関連抗原、ホルモン、ウィルスなどの検出に応用でき、
かつ、これらの検出時間を短縮することができる。From the above points, the present invention can be applied to the detection of tumor-related antigens, hormones, viruses, etc. contained in extremely small amounts in serum or other body fluids, which require particularly highly sensitive measurement.
Moreover, the time required for these detections can be shortened.
第１図は実施例１および比較例１のＣＡ１９−９の測定
検量線、第２図は実施例２および比較例２のＨＢＳ抗原
の測定検量線である。FIG. 1 shows a measurement calibration curve for CA19-9 in Example 1 and Comparative Example 1, and FIG. 2 shows a measurement calibration curve for HBS antigen in Example 2 and Comparative Example 2.
Claims (1)
ク質（第一結合タンパク質）、不溶性固体上の第一結合
タンパク質の一部分を切断除去したタンパク質（第二結
合タンパク質）、および第一結合タンパク質の切断除去
部分の全部又は一部を認識する標識抗体（標識抗体）を
結合させて得た免疫複合体における標識物質量を測定す
ることにより、該被測定物質を測定することを特徴とす
る被測定物質の免疫測定法。 ２、被測定抗原性物質、被測定抗原性物質を認識するモ
ノクローナル抗体（第一抗体）、不溶性固体上の第一抗
体の一部分を切断除去したモノクローナル抗体（第二抗
体）、および第一抗体の切断除去部分の全部又は一部を
認識する標識抗体（第三抗体）を結合させて得た免疫複
合体における標識物質量を測定することにより、該被測
定抗原性物質を測定することを特徴とする抗原性物質の
免疫測定法。[Claims] 1. A substance to be measured, a protein that specifically binds to the substance to be measured (first binding protein), a protein obtained by cutting and removing a portion of the first binding protein on an insoluble solid (second binding protein) and a labeled antibody (labeled antibody) that recognizes all or part of the cleaved portion of the first binding protein, and the amount of the labeled substance in the immune complex obtained is measured, thereby measuring the analyte. An immunoassay method for a substance to be measured, characterized by: 2. An antigenic substance to be measured, a monoclonal antibody (first antibody) that recognizes the antigenic substance to be measured, a monoclonal antibody obtained by cutting and removing a portion of the first antibody on an insoluble solid (second antibody), and the first antibody. The antigenic substance to be measured is measured by measuring the amount of a labeled substance in an immune complex obtained by binding a labeled antibody (third antibody) that recognizes all or part of the cleaved portion. immunoassay for antigenic substances.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26093088A JPH02107967A (en) | 1988-10-17 | 1988-10-17 | Immunoassay |
| JP63290977A JPH0754323B2 (en) | 1988-10-17 | 1988-11-16 | Immunoassays and immunoassay reagents |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26093088A JPH02107967A (en) | 1988-10-17 | 1988-10-17 | Immunoassay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02107967A true JPH02107967A (en) | 1990-04-19 |
Family
ID=17354744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP26093088A Pending JPH02107967A (en) | 1988-10-17 | 1988-10-17 | Immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02107967A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019522223A (en) * | 2016-07-14 | 2019-08-08 | カイヴォゲン オサケ ユキチュア | Diagnosis of lectin-based cancer |
-
1988
- 1988-10-17 JP JP26093088A patent/JPH02107967A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019522223A (en) * | 2016-07-14 | 2019-08-08 | カイヴォゲン オサケ ユキチュア | Diagnosis of lectin-based cancer |
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