JPH03200066A - Method for measuring activated human protein c - Google Patents
Method for measuring activated human protein cInfo
- Publication number
- JPH03200066A JPH03200066A JP33810589A JP33810589A JPH03200066A JP H03200066 A JPH03200066 A JP H03200066A JP 33810589 A JP33810589 A JP 33810589A JP 33810589 A JP33810589 A JP 33810589A JP H03200066 A JPH03200066 A JP H03200066A
- Authority
- JP
- Japan
- Prior art keywords
- human protein
- antibody
- inhibitor
- protein
- 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
- 229940100689 human protein c Drugs 0.000 title claims abstract description 37
- 101500025568 Homo sapiens Saposin-D Proteins 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 28
- 101001053641 Homo sapiens Plasma serine protease inhibitor Proteins 0.000 claims abstract description 15
- 102000049532 human SERPINA5 Human genes 0.000 claims abstract description 15
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- 230000008105 immune reaction Effects 0.000 claims abstract description 5
- 239000007795 chemical reaction product Substances 0.000 claims description 5
- 101000823116 Homo sapiens Alpha-1-antitrypsin Proteins 0.000 claims description 4
- 102000051631 human SERPINA1 Human genes 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 abstract description 11
- 239000008280 blood Substances 0.000 abstract description 11
- 101710081722 Antitrypsin Proteins 0.000 abstract description 10
- 230000001475 anti-trypsic effect Effects 0.000 abstract description 10
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- 239000000243 solution Substances 0.000 description 27
- 101800004937 Protein C Proteins 0.000 description 17
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- 229960000856 protein c Drugs 0.000 description 17
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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、活性化ヒトプロティンＣに特異的なモノクロ
ーナル抗体、ヒトプロティンＣインヒビターに特異的な
モノクローナル抗体および、ヒトα１アンチトリプシン
に特異的なモノクローナル抗体を利用した活性化ヒトプ
ロティンＣの測定方法に関するものである。Detailed Description of the Invention (Field of Industrial Application) The present invention provides a monoclonal antibody specific for activated human protein C, a monoclonal antibody specific for human protein C inhibitor, and a monoclonal antibody specific for human α1 antitrypsin. This invention relates to a method for measuring activated human protein C using a monoclonal antibody.
（従来の技術）
血液凝固の制御反応の重要なものとして、ブロテアーゼ
による凝固因子の分解反応がある。この反応の主たるも
のは、プロティンＣによるものである。(Prior Art) An important reaction for controlling blood coagulation is the decomposition reaction of coagulation factors by protease. This reaction is mainly caused by protein C.
プロティンＣはＧｌａ含有凝固因子（ビタミンに依存性
因子）の一つであり、循環血液中では２本鎖の前駆体と
して存在する。プロティンＣはトロンビンにより高分子
鎖のアミノ末端から１２個のアミノ酸が遊離して活性化
プロティンＣとなる。これが、凝固系のＶａ因子と■ａ
囚子を分解して失活させる。また、活性化プロティンＣ
はブラスミノーゲンアクチベーターインヒビターの活性
を抑制して線溶の凡退を起こす作用もある。Protein C is one of the Gla-containing coagulation factors (vitamin-dependent factors) and exists as a two-chain precursor in circulating blood. Protein C becomes activated protein C by liberating 12 amino acids from the amino terminus of the polymer chain by thrombin. This is the Va factor of the coagulation system and ■a
Decompose and inactivate the prisoner. In addition, activated protein C
also has the effect of suppressing the activity of blasminogen activator inhibitor and causing regression of fibrinolysis.
ヒトプロティンＣインヒビターは、血液凝固制御因子の
活性化プロティンＣの生理的阻害因子の１つである。精
製されたプロティンＣインヒビターは、試験管内では、
Ｘａ因子、トロンビン、ＸＩａ因子、血漿カリクレイン
、さらに線溶系プロテアーゼの組織プラスミノーゲンア
クチベーターや尿由来ブラスミノーゲンアクチベーター
（ウロキナーゼ）を阻害する。しかし、通常プロティン
Ｃがほとんど活性化されない状態での血液凝固過程を試
験管内で行うと、プロティンＣインヒビター活性の低下
はほとんど見られない。また、生体内でのプロティンＣ
インヒビターの濃度の変動はプロティンＣ濃度の変動と
良く一致することも知られている。これらのことより、
プロティンＣインヒビターは生体内ではプロティンＣと
最も良く反応するものと考えられる。Human protein C inhibitor is one of the physiological inhibitors of activated protein C, a blood coagulation regulator. Purified protein C inhibitor in vitro:
It inhibits factor Xa, thrombin, factor XIa, plasma kallikrein, as well as fibrinolytic protease tissue plasminogen activator and urine-derived plasminogen activator (urokinase). However, when the blood coagulation process is carried out in vitro in a state in which protein C is normally hardly activated, little decrease in protein C inhibitor activity is observed. In addition, protein C in vivo
It is also known that variations in inhibitor concentration closely match variations in protein C concentration. Than these things,
Protein C inhibitors are thought to react best with protein C in vivo.
また、プロティンＣに対する別のインヒビターとして、
α、アンチトリプシンが作用することが報告されている
（Ｍ、Ｊ、Ｈｅｅｂ＆Ｊ、Ｈ。In addition, as another inhibitor of protein C,
α, antitrypsin has been reported to act (M, J, Heeb & J, H.
Ｇｒｉ ｆ ｆ ｉｎ、Ｊ、Ｂｉｏｌ、Ｃｈｅｍ。Gri f f in, J, Biol, Chem.
２６３．１１６１３．（１９８８））。263.11613. (1988)).
以上のことより、活性化プロティンＣをそのインヒビタ
ーとの複合体として測定することにより、血液凝固阻止
活性を検出することになる。また、前血栓状態とされる
糖尿病や、ＤＩＣの病理診断が可能になる。プロティン
Ｃの血中濃度は、健常人では約２〜８ ｍｇ／Ｉである
。From the above, blood coagulation inhibitory activity can be detected by measuring activated protein C as a complex with its inhibitor. Furthermore, pathological diagnosis of diabetes mellitus, which is considered to be a pre-thrombotic state, and DIC becomes possible. The blood concentration of protein C is approximately 2 to 8 mg/I in healthy people.
その測定方法としては、プロティンＣに対スるモノクロ
ーナル抗体を用いたＥＩＡが現在行われているが、活性
化されたプロティンＣの測定としては厳格な意味で向い
ていない。As a measurement method, EIA using a monoclonal antibody against protein C is currently being performed, but it is not suitable for measuring activated protein C in a strict sense.
（発明が解決しようとする課題）
本発明の目的は、従来の方法よりも正確に、短時間に、
高感度に活性化ヒトプロティンＣを免疫学的に測定する
方法を提供することにある。(Problems to be Solved by the Invention) The purpose of the present invention is to
The object of the present invention is to provide a method for immunologically measuring activated human protein C with high sensitivity.
（課題を解決するための手段）
本発明者らは上記課題に関し鋭意検討した結果、本発明
に到達した。(Means for Solving the Problems) The present inventors have made extensive studies regarding the above problems, and as a result, have arrived at the present invention.
すなわち本発明は、試料中の活性化ヒトプロティンＣを
測定する方法において、
（ａ） ・活性化ヒトプロティンＣを特異的に認識す
る、固相に固定化されたモノクローナル抗体（１）、
・試料、
・ヒトプロテインＣインヒビターを特異的に認識すると
ころの標識されたまたは標識されていないモノクローナ
ル抗体（２）
・および、ヒトα、アンチトリプシンを特異的に認識す
るところの標識されたまたは標識されていないモノクロ
ーナル抗体（３）を接触させ
（ｂ） （ａ）で標識されていないモノクローナル抗
体（２） 、 （３）を使用した場合には（ａ）で生じ
る免疫反応生成物、またはモノクローナル抗体（２）お
よび（３）を特異的に認識する標識された抗体を接触さ
せ、
（ｅ）遊離の又は固定化された標識化抗体の標識を、直
接的または間接的に検出して免疫反応生成物を定量する
ことを特徴とする活性化ヒトプロティンＣの測定方法で
ある。That is, the present invention provides a method for measuring activated human protein C in a sample, comprising: (a) - a monoclonal antibody (1) immobilized on a solid phase that specifically recognizes activated human protein C; - a sample.・A labeled or unlabeled monoclonal antibody that specifically recognizes human protein C inhibitor (2) ・A labeled or unlabeled monoclonal antibody that specifically recognizes human α, antitrypsin (b) When monoclonal antibodies (2) and (3) not labeled in (a) are used, the immunoreaction products generated in (a), or the monoclonal antibodies (2) ) and (3), and (e) directly or indirectly detect the label of the free or immobilized labeled antibody to detect the immune reaction product. This is a method for measuring activated human protein C, which is characterized by quantifying activated human protein C.
本発明のヒトプロティンＣインヒビター測定法は、ヒト
プロティンＣに特異的なモノクローナル抗体、ヒトプロ
ティンＣインヒビターに特異的なモノクローナル抗体お
よび、ヒトα、アンチトリプシンに特異的なモノクロー
ナル抗体を利用した血液中のヒトプロティンＣの免疫学
的測定法にあり、以下その詳細について説明する。The human protein C inhibitor measurement method of the present invention utilizes a monoclonal antibody specific to human protein C, a monoclonal antibody specific to human protein C inhibitor, and a monoclonal antibody specific to human α and antitrypsin. This method is an immunoassay method for human protein C, and the details thereof will be explained below.
本発明方法において、用いられるモノクローナル抗体は
、調製自体公知である方法（Ｇ。In the method of the present invention, the monoclonal antibodies used can be prepared by methods known per se (G.
Ｋｏｈｌｅｒ＆Ｃ，Ｍｉ １ｓｔｅｉｎ。Kohler & C, Mi 1stein.
Ｎａｔｕｒｅ、２５６，４９５．（１９７５））に準じ
て製造することができる。Nature, 256,495. (1975)).
以上の方法により、ヒトプロティンＣ１ヒトプロテイン
Ｃインヒビター又はヒトα、アンチトリプシンを特異的
に認識する複数種のモノクローナル抗体を得た。それぞ
れの抗体の結合定数は、］０７〜ＩＯＩＩＭ−１の範囲
内であった。これらのモノクローナル抗体を使って、ヒ
トプロティンＣを定量的に測定できるサンドイツチ法に
よる免疫学的測定方法が可能となった。By the above method, multiple types of monoclonal antibodies that specifically recognize human protein C1 human protein C inhibitor, human α, and antitrypsin were obtained. The binding constants of each antibody were within the range of ]07 to IOIIM-1. Using these monoclonal antibodies, it has become possible to perform an immunoassay method based on the Sand-Deutsch method that allows quantitative measurement of human protein C.
本発明方法に用いられる抗活性化ヒトプロティンＣ抗体
を固相に固定化する方法は、公知の方法を採用でき、固
相としては例えば、ポリスチレン、ポリエチレン、ポリ
塩化ビニル、ポリカーボネート、セファロース粒子、ラ
テックス、アガロース、セルロース、ポリメタアクリレ
ートなどが使用される。A known method can be used to immobilize the anti-activated human protein C antibody used in the method of the present invention on a solid phase. Examples of the solid phase include polystyrene, polyethylene, polyvinyl chloride, polycarbonate, Sepharose particles, and latex. , agarose, cellulose, polymethacrylate, etc. are used.
また抗ヒトプロティンＣインヒビター抗体および抗ヒト
α、アンチトリプシン抗体を標識する場合、標識化の方
法とその検出方法もなんら限定されるものでなく、公知
の方法により標識化および検出することができる。抗ヒ
トプロティンＣインヒビター抗体および抗ヒトα１アン
チトリプシン抗体が標識されていないものを用いる場合
には、これらの抗体又は免疫反応生成物を特異的に認識
する標識された抗体を用いる。Furthermore, when an anti-human protein C inhibitor antibody and an anti-human α, antitrypsin antibody are labeled, the method of labeling and the method of detection thereof are not limited at all, and the labeling and detection can be performed by known methods. When using unlabeled anti-human protein C inhibitor antibodies and anti-human α1 antitrypsin antibodies, labeled antibodies that specifically recognize these antibodies or immune reaction products are used.
標識として間接的に検出されるもの、例えば酵素を用い
る場合、標識物質としては例えば、ペルオキシダーゼ、
β−Ｄ−ガラクトシダーゼ、アルカリホスファターゼ、
ウレアーゼ、カタラーゼ、β−グルクロニダーゼなどの
酵素が使われる。また直接的に検出される標識、例えば
放射性物質としては、Ｊ ＋２５１．または１３１工
等が、蛍光物質を使用する方法としては、例えば、フル
オレスカミン、フルオレラセンチオシアネート、テトラ
ローダミンイソチオシアネート等が常法によりモノクロ
ーナル抗体に結合される。しかしながら、標識物質は上
記物質に何ら限定されるべきものではない。When using something that can be detected indirectly as a label, such as an enzyme, examples of the labeling substance include peroxidase,
β-D-galactosidase, alkaline phosphatase,
Enzymes such as urease, catalase, and β-glucuronidase are used. Also, examples of labels that can be detected directly, such as radioactive substances, include J+251. As a method using a fluorescent substance, for example, fluorescamine, fluorella centiocyanate, tetrarhodamine isothiocyanate, etc. are bound to a monoclonal antibody by a conventional method. However, the labeling substance should not be limited to the above-mentioned substances.
測定に使用される試薬は、上記物質以外にも、基質、溶
解剤、緩衝剤、洗浄剤、反応停止剤等の公知の試薬が用
いられる。In addition to the above-mentioned substances, known reagents used in the measurement include substrates, solubilizers, buffers, detergents, reaction terminators, and the like.
試料、抗体の添加順序には特に限定はない。There are no particular limitations on the order in which samples and antibodies are added.
最終的に生成した免疫反応生成物、すなわち固定化され
た標識化抗体の標識又は遊離の抗体の標識を検出し、定
量すればよい。The finally produced immunoreaction product, that is, the immobilized labeled antibody label or the free antibody label, may be detected and quantified.
（作用）
活性化ヒトプロティンＣは、試料中に共存するヒトプロ
ティンＣインヒビターまたはヒトα１アンチトリプシン
と複合体を形成している。従ってこの複合体を定量する
ことで、活性化ヒトプロティンＣを間接的に定量するこ
とができる。ヒトプロティンＣインヒビターは、ヒトプ
ロティンＣへの結合力は強いが、例えば血中での存在量
が少く、一方、ヒトα、アンチトリプシンはヒトプロテ
ィンＣへの結合力は弱いが、血中では多量に存在する。(Effect) Activated human protein C forms a complex with human protein C inhibitor or human α1 antitrypsin coexisting in the sample. Therefore, by quantifying this complex, activated human protein C can be indirectly quantified. Human protein C inhibitor has a strong binding force to human protein C, but is present in small amounts in the blood, while human α and antitrypsin have a weak binding force to human protein C, but are present in large amounts in the blood. exists in
そこで、活性化ヒトプロティンＣ−ヒトプロティンＣイ
ンヒビター複合体及び活性化ヒトプロティンＣ−ヒトα
１アンチトリプシン複合体の両方を測定することで、よ
り厳密に活性化ヒトプロティンＣを測定することができ
る。従って本発明方法では、この複合体を抗活性化プロ
ティンＣ抗体及び抗ヒトプロティンＣインヒビター抗体
、又は抗活性化ヒトプロティンＣ抗体及び抗ヒトα１ア
ンチトリプシン抗体でサンドイッチし、測定するのであ
る。Therefore, activated human protein C-human protein C inhibitor complex and activated human protein C-human α
By measuring both antitrypsin and antitrypsin complexes, activated human protein C can be measured more precisely. Therefore, in the method of the present invention, this complex is sandwiched between an anti-activated protein C antibody and an anti-human protein C inhibitor antibody, or an anti-activated human protein C antibody and an anti-human α1 antitrypsin antibody, and then measured.
尚、試料中にヒトプロティンＣインヒビターとα、アン
チトリプシンが存在しない場合は、外部から添加して複
合体を形成させてから本発明法により測定すべきである
。しかし、試料がヒト血液であれば、両者ともその中に
含まれているので、外部から添加する必要はなく、そう
いったことからも試料はヒト血液であることが好ましい
。If human protein C inhibitor, α, and antitrypsin are not present in the sample, they should be added externally to form a complex and then measured using the method of the present invention. However, if the sample is human blood, since both are contained therein, there is no need to add them from the outside, and for this reason as well, it is preferable that the sample is human blood.
（発明の効果）
以上の説明から明らかなように本発明によれば、（１）
血液中の活性化ヒトプロティンＣ濃度は、１〜２００ｎ
ｇ／ｍｌの範囲内で測定することができ、
（２）従来法に比べて極めて簡便な操作で短時間に、か
つより厳密な意味での活性化ヒトプロティンＣを感度よ
く多数の検体の測定が可能である。(Effects of the Invention) As is clear from the above explanation, according to the present invention, (1)
Activated human protein C concentration in blood is 1-200n
(2) Compared to conventional methods, activated human protein C can be measured in a short time with extremely simple operations, and in a more precise sense, a large number of samples can be measured with high sensitivity. is possible.
（実施例）
以下に本発明の詳細な実施例を説明する。しかし、本発
明はこれら実施例にのみに限定されるものではない。(Example) Detailed examples of the present invention will be described below. However, the present invention is not limited to these examples.
（モノクローナル抗体の調製）
（Ａ）抗原感作動物細胞の調製
ヒトプロティンＣ１ヒトプロテインＣインヒビター、又
はヒトα１アンチトリプシンを抗原としてＢ ａ １
ｂ ／ ｃマウス（♀）をそれぞれ免疫した。(Preparation of monoclonal antibodies) (A) Preparation of antigen-sensitized animal cells B a 1 using human protein C1 human protein C inhibitor or human α1 antitrypsin as an antigen.
b/c mice (♀) were each immunized.
免疫は、マウスの腹腔にフロイントの完全アジュバント
と抗原１００μｇ／匹とを乳化させた血液１００μｌを
投与した。２週間後に追加免疫として抗原１００μｇ／
匹をフロイントの不完全アジュバントと乳化させたちの
１００μｌをマウス腹腔に投与した。１週間後最終免疫
として抗原１００μｇ／匹をリン酸ｔ１！衝化生理食塩
水（０，８５％ＮａＣｌ含有０．０１％リン酸緩衝液、
ｐＨ７，２：以下ＰＢＳ）に溶解したちの１００μｌを
腹腔内に投与した。３日後この処置マウスの肺臓を無菌
的に取出した。１５％子牛脂児血清（以下１５％ＦＣＳ
と省略する）を含むＤＭＥＭＩ Ｏｍ １を注射器で吸
い取り２７ゲージの注射針をつけた。肺臓を水冷してお
いたデイツシュに入れ、注射針で数か新式をあけた。注
射針を差し込み還流し肺臓細胞をデイツシュに流出させ
た。流出液をナイロンメツシュで濾過し遠心チューブに
入れ、１０００ ｒ ｐｍで１０分間遠心分離して上澄
をすてた。細胞ペレット中の赤血球を０．１５Ｍ塩化ア
ンモニウム溶液（１ｍＭエチレンジアミン４酢酸−２ナ
トリウム塩（以下ＥＤＴＡと省略する）を含む０．０１
Ｍ炭酸緩衝液、ｐＨ７，２）で溶血させ遠心分離し、さ
らに細胞ペレットをＤＭＥＭで２回同様に遠心洗浄して
肺細胞とした。For immunization, 100 μl of blood emulsified with Freund's complete adjuvant and 100 μg of antigen/mouse was administered into the abdominal cavity of the mouse. Two weeks later, a booster dose of 100 μg of antigen/
The mice were emulsified with incomplete Freund's adjuvant and 100 μl of the emulsified mixture was intraperitoneally administered to the mice. One week later, as final immunization, 100 μg/mouse of antigen was given to phosphate t1! Enhanced physiological saline (0.01% phosphate buffer containing 0.85% NaCl,
100 μl of the solution dissolved in pH 7.2 (hereinafter referred to as PBS) was administered intraperitoneally. Three days later, the lungs of the treated mice were aseptically removed. 15% calf fat serum (hereinafter referred to as 15%FCS)
DMEMI Om 1 containing (abbreviated as ) was aspirated with a syringe and a 27-gauge needle was attached. I put the lungs into a water-cooled date bag and injected them with a few needles. A syringe needle was inserted and reflux was performed to cause the lung cells to flow out into the tissue. The effluent was filtered through a nylon mesh, placed in a centrifuge tube, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The red blood cells in the cell pellet were dissolved in a 0.15M ammonium chloride solution (0.01% containing 1mM ethylenediaminetetraacetic acid-disodium salt (hereinafter abbreviated as EDTA)).
The cells were hemolyzed with M carbonate buffer, pH 7.2) and centrifuged, and the cell pellet was centrifugally washed twice with DMEM to obtain lung cells.
（Ｂ）骨髄腫細胞の調製
骨髄腫細胞としてはＢａ１ｂ／ｃマウス由来の８−アザ
グアニン耐性株として、ＳＰ２１０−Ａｇ１４（以下Ｓ
Ｐ ２１０と省略する）を使用した。(B) Preparation of myeloma cells SP210-Ag14 (hereinafter S
P210) was used.
細胞融合を行う１週間前まで２０μｇ／ｍｌの８−アザ
グアニン、１５％ＦＣ３を含むＤＭＥＭで培養し、その
後細胞融合日まで１５％ＦＣＳを含むＤＭＥＭを使用し
た。細胞融合直前に、５Ｐ２１０は無菌的にＤＭＥＭで
１１０００ｒｐで１０分間遠心洗浄を２回繰り返し調製
した。The cells were cultured in DMEM containing 20 μg/ml 8-azaguanine and 15% FC3 until one week before cell fusion, and then DMEM containing 15% FCS was used until the day of cell fusion. Immediately before cell fusion, 5P210 was aseptically prepared in DMEM by repeating centrifugal washing twice at 11,000 rpm for 10 minutes.
（Ｃ）細胞融合
上記（Ａ）項で調製した肺臓細胞と上記（Ｂ）項で調製
した骨髄腫細胞を５＝１の割合で混合遠心（１０００ｒ
ｐｍ、１０分）し細胞ペレットを集めた。遠心チューブ
を軽くたたいて細胞ペレットを壁面にうずく広げた。そ
の中に３７℃に暖めておいた５０％ＰＥＧ （ＭＥＲＫ
社製ポリエチレングリコール４０００）を含むＤＭＥＭ
溶液０．５ｍｌを遠心チューブを回しながら少しずつ滴
下した。１分間ゆっくりと遠心チューブを回転させ混合
した後、３０秒に１ｍｌの割合で遠心チューブを回転し
ながら３７℃に加温しておいたＤＭＥＭを１０回加えた
。つぎにＦｅ２を２ｍｌゆっくりと入れ、１１０００ｒ
ｐ、１０分間遠心した。細胞ペレットを１５％ＦＣＳと
ｌＸｌ０−’Ｍヒポキサンチン、４Ｘ１０−’Ｍアミノ
プテリン、１．６Ｘ１０−５Ｍチミジンを含むＤＭＥＭ
（以下ＨＡＴ培地と省略する）で２回遠心洗浄（１０
００ｒｐｍ、１０分間）した。この培養液を９６ウエル
ブレー） （Ｆａ ｌ ｃｏｎ＃３０４２）に５Ｘ１０
’細胞個／ウェルになるように２００μＩずつ分注した
。３日目ごとにＨＡＴ培地を１００μｌ／ウエル交換し
た。３週間後からは、ｌＸｌ０−’Ｍヒポキサンチン、
１．６Ｘ１０−’Ｍチミジンと１５％ＦＣＳを含むＤＭ
ＥＭ （以下ＨＴ培地と省略する）を培地交換に用いた
。(C) Cell fusion The lung cells prepared in section (A) above and the myeloma cells prepared in section (B) above were mixed at a ratio of 5=1 and centrifuged (1000r
pm, 10 min) and collected the cell pellet. The cell pellet was spread on the wall by tapping the centrifuge tube. 50% PEG (MERK
DMEM containing polyethylene glycol 4000)
0.5 ml of the solution was dropped little by little while rotating the centrifuge tube. After mixing by slowly rotating the centrifuge tube for 1 minute, DMEM that had been heated to 37° C. was added 10 times while rotating the centrifuge tube at a rate of 1 ml every 30 seconds. Next, slowly add 2ml of Fe2 and add 11000r
p, centrifuged for 10 minutes. The cell pellet was transferred to DMEM containing 15% FCS and 1X10-'M hypoxanthine, 4X10-'M aminopterin, 1.6X10-5M thymidine.
(hereinafter abbreviated as HAT medium) twice by centrifugal washing (10
00 rpm for 10 minutes). Transfer this culture solution to a 96-well plate (Falcon #3042) at 5×10
'200 μl was dispensed at a rate of 1 cell/well. The HAT medium was replaced with 100 μl/well every third day. After 3 weeks, lXl0-'M hypoxanthine,
DM with 1.6X10-'M thymidine and 15% FCS
EM (hereinafter abbreviated as HT medium) was used for medium exchange.
（Ｄ）ハイブリドーマの選択
９６ウエルプレートに細胞コロニーが認められるｌＯ０
日目後から固相酵素免疫測定法を行い、培養上清に特異
的抗体が存在するかどうか調べた。(D) Selection of hybridomas Cell colonies observed in 96-well plate lO0
After a day, solid-phase enzyme immunoassay was performed to examine the presence of specific antibodies in the culture supernatant.
９６ウエルプレート平底（インターメッド社製）に、各
抗原２μｇ ／ ｍ １を５０μｌ／ウェル分注し、３
７℃で１．５時間静置する。ウェルに残っている溶液を
除去し、ＰＢＳに０．０４％ツイーン（ｔｗｅｅｎ）−
２０を含んだ溶液（以下ＰＢＳ−Ｔ）で３回洗浄した後
、０．１％ウシ血清アルブミン（以下ＢＳＡ）を溶解し
たＰＢＳ−Ｔ溶液３００μｌを各ウェルに加えて、３７
℃で１．５時間ブロッキング処理した。つぎに各ウェル
に上記培養上清を１００μｍずつ分注し３７℃で１．５
時間静置した。これらのウェルをＰＢＳＴ溶液で３回洗
浄した後、ペルオキシダーゼ標識ラビット抗マウスＩｇ
Ｇ抗体（ジャクソン社製）４０００倍希釈を５０μｍ／
ウェルずつ分注し、３７℃で１，５時間静置した。ＰＢ
Ｓ−Ｔ溶液で３回洗浄したのち、基質溶液（１，２％
２，２−アジノジ＝（３−エチルベンズチアゾリン硫酸
）−ジアンモニウム塩（ＡＢＴＳ）及び０．０１％過酸
化水素（Ｈ２０□）を含有する０、１Ｍクエン酸緩衝液
（ｐＨ５，１））を各ウェルに１００μｍ添加した。３
０分間室温で放置し、２００ｍＭシュウ酸溶液を１００
μｌを加えて酵素反応を停止させた。４１５ｎｍでの吸
光度を測定し、酵素活性が認められたウェルに特異的モ
ノクローナル抗体を産生ずるハイブリドーマが存在する
ことがわかる。以上のようにして、抗体価の強い抗体産
生ハイブリドーマを取得した。Dispense 50 μl/well of 2 μg/ml of each antigen into a 96-well plate with a flat bottom (manufactured by Intermed), and
Leave at 7°C for 1.5 hours. Remove remaining solution in the wells and add 0.04% tween-
After washing three times with a solution containing 20 (hereinafter referred to as PBS-T), 300 μl of a PBS-T solution containing 0.1% bovine serum albumin (hereinafter referred to as BSA) was added to each well.
Blocking treatment was performed at ℃ for 1.5 hours. Next, 100 μm of the above culture supernatant was dispensed into each well and heated to 37°C for 1.5 μm.
Let it stand for a while. After washing these wells three times with PBST solution, peroxidase-labeled rabbit anti-mouse Ig
G antibody (manufactured by Jackson) 4000 times diluted at 50μm/
The solution was dispensed into each well and allowed to stand at 37°C for 1.5 hours. P.B.
After washing three times with S-T solution, substrate solution (1,2%
0,1M citrate buffer (pH 5,1) containing 2,2-azinodi=(3-ethylbenzthiazoline sulfate)-diammonium salt (ABTS) and 0.01% hydrogen peroxide (H20□)). 100 μm was added to each well. 3
Leave at room temperature for 0 minutes, then add 200mM oxalic acid solution to 100%
The enzymatic reaction was stopped by adding μl. The absorbance at 415 nm was measured, and it was found that hybridomas producing specific monoclonal antibodies were present in wells in which enzyme activity was observed. As described above, an antibody-producing hybridoma with a strong antibody titer was obtained.
（Ｅ）コンデショニングメデウムの調製２６ゲージの注
射針をつけた注射器に１０ｍ１の冷蔵しておいた０、３
４Ｍサッカロース溶液を吸い取った。Ｂａ１ｂ／ｃマウ
ス（♂）をを椎脱臼させ、無菌的に腹腔内に上記溶液を
注入した。(E) Preparation of conditioning medium In a syringe fitted with a 26 gauge needle, 10 ml of refrigerated 0.3
The 4M sucrose solution was blotted. A Ba1b/c mouse (male) was subjected to vertebrae dislocation, and the above solution was injected intraperitoneally in a sterile manner.
注入後５分以内に左側腹部に１８ゲージの注射針をつけ
水冷しておいた注射器にて腹腔内溶液を回収した。氷冷
しておいた遠心チューブに上記回収液を流し込み、１１
０００ｒｐで５分間遠心分離した。遠心後上清を廃棄し
、細胞ベレットに］５％ＦＣ５−ＤＭＥＭを加え攪拌し
プッシュに入れた。３７℃、５％炭酸ガス濃度、９５％
湿度で一晩培養した。培養上清を集め、０．２２μｍの
メンブレンフィルターで濾過し、これをコンデショニン
グメデウムとした。Within 5 minutes after injection, the intraperitoneal solution was collected using a water-cooled syringe with an 18-gauge needle attached to the left flank. Pour the recovered solution into an ice-cooled centrifuge tube, and proceed to step 11.
Centrifuged at 000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 5% FC5-DMEM was added to the cell pellet, stirred, and placed in a push tube. 37℃, 5% carbon dioxide concentration, 95%
Cultured overnight in humidity. The culture supernatant was collected, filtered through a 0.22 μm membrane filter, and used as a conditioning medium.
（Ｆ）クローニング
抗体産生を認めるハイブリドーマについて限界希釈法を
用いて単一クローンにした。上記（Ｅ）項で作製したコ
ンデショニングメデウムを１ｍｌ含む１（ＡＴ培地２０
ｍ１を用意した。クローニングしたいハイブリドーマ細
胞を各ウェルに１個になるように上記培養液中に調整し
、２００μｌ／ウエルずつ９６ウエルプレート（Ｆａｌ
ｃｏｎ＃３０４２）に分注した。培養１０日目前後から
細胞コロニーが認められるウェルについて、上記（Ｄ）
項に記載した固相酵素免疫測定法に準じて抗体産生ハイ
ブリドーマを選択し、さらに再度クローニングを繰り返
し単一／１イブリドーマを樹立した。最終的に２７クロ
ーンの／Ｘイブリドーマを確立した。(F) Cloning Hybridomas capable of producing antibodies were made into a single clone using the limiting dilution method. 1 containing 1 ml of the conditioning medium prepared in (E) above (AT medium 20
m1 was prepared. Adjust the hybridoma cells to be cloned into the above culture medium so that there is one cell in each well, and add 200 μl/well to a 96-well plate (Fal
con#3042). For wells where cell colonies are observed from around the 10th day of culture, see (D) above.
Antibody-producing hybridomas were selected according to the solid-phase enzyme immunoassay method described in Section 1, and cloning was repeated again to establish single hybridomas. Finally, 27 clones of /X hybridomas were established.
（Ｇ）抗体の精製
Ｂａ１ｂ／ｃマウス（♂）６〜１０週令の腹腔にブリス
タン（２，６，１０，１４−テトラメチルペンタデカン
）を０．５ｍｌ／匹投与した。２週間後上記（Ｆ）項で
得られた各特異的抗体産生ハイプリドーマ株をマウス腹
腔内に各クローンについて２Ｘ１０６細胞個／匹移植し
た。１０日目前後に生成した腹水を、１８ゲージの注射
針を腹腔に差し込み、１／２０量の０．２Ｍ−ＥＤＴＡ
をいれた遠心チューブに滴下させた。遠心チューブを４
００Ｏｒｐｍで１０分間遠心し、上清を集めた。採取し
た上清を５０％硫酸アンモニウム沈殿分画法にしたがっ
て粗精製し、０．０５％アジ化ナトリウムを含むＰＢＳ
溶液に透析後、イオン交換クロマトグラフィー、ゲル濾
過をおこない精製した。メルカプトエタノール還元下で
の１２％５ＤＳ−ポリアクリルアミド電気泳動で１本の
重鎮と１本の軽鎖の２本のバンドになったことで抗体の
純度を確認した。(G) Purification of antibody Bristane (2,6,10,14-tetramethylpentadecane) was administered into the abdominal cavity of 6-10 week old Ba1b/c mice (female) in an amount of 0.5 ml/mouse. Two weeks later, each specific antibody-producing hybridoma line obtained in the above (F) was intraperitoneally transplanted into mice at 2×10 6 cells/mouse for each clone. Ascites generated around the 10th day was injected with 1/20 volume of 0.2M-EDTA by inserting an 18-gauge needle into the abdominal cavity.
was dropped into a centrifuge tube containing 4 centrifuge tubes
Centrifugation was performed at 00 rpm for 10 minutes, and the supernatant was collected. The collected supernatant was crudely purified according to a 50% ammonium sulfate precipitation fractionation method, and added to PBS containing 0.05% sodium azide.
After dialysis against a solution, it was purified by ion exchange chromatography and gel filtration. The purity of the antibody was confirmed by the formation of two bands, one heavy chain and one light chain, in 12% 5DS-polyacrylamide electrophoresis under mercaptoethanol reduction.
（酵素標識法によるヒトプロティンＣの測定）（Ａ）抗
ヒトプロティンＣ抗体の固定化未処理マイクロタイター
プレート（９６ウエル・ヌンクプレート、インターメツ
ド社製）の各ウェルにＯ，１Ｍ炭酸ナトリウム緩衝液（
ｐＨ９，６）に溶解した３μｇ ／ ｍ １のマウス由
来の抗ヒトプロティンＣ抗体の溶液２００μ工を加えて
、４℃−夜インキユベートした。次に、各ウェルの溶液
を除去し、ＰＢＳ−Ｔで３回洗浄した後、０．１％ＢＳ
Ａを溶解したＰＢＳ−Ｔ溶液３００μｌを各ウェルに加
えて、４℃でブロッキング処理しそのまま保存した。(Measurement of human protein C by enzyme labeling method) (A) Immobilization of anti-human protein C antibody In each well of an untreated microtiter plate (96-well Nunc plate, manufactured by Intermed), O, 1M sodium carbonate buffer (
200 μg of a solution of 3 μg/ml mouse-derived anti-human protein C antibody dissolved in pH 9.6) was added and incubated overnight at 4°C. Next, the solution in each well was removed and washed three times with PBS-T, followed by 0.1% BS.
300 μl of a PBS-T solution in which A was dissolved was added to each well, subjected to blocking treatment at 4° C., and stored as is.
（Ｂ）西洋ワサビペルオキシダーゼ（以下ＨＲＰ）標識
抗体の調製：０．３Ｍ重炭酸ナトリウム緩衝液（ｐＨ８
，１）に溶解したＨＲＰ溶液（５ｍｇ／ ｍ １ ）に
１％ １−フルオロ−２，４−ジニトロベンゼンのエタ
ノール溶液０．１ｍｌを加え、室温にて１時間反応させ
た。その溶液に０．０６Ｍ過ヨウ素酸ナトリウム１．０
ｍｌを添加し３０分反応させた。未反応の過ヨウ素酸ナ
トリウムを０．１６Ｍのエチレングリコール１．Ｏｍｌ
を加えて除去した後、０．０１Ｍ炭酸ナトリウム緩衝液
（ｐＨ９，５）で透析した。次に、マウス由来抗ヒトプ
ロティンＣインヒビター・モノクローナル抗体５ｍｇを
加えて５〜６時間反応させた。水素化ホウ素ナトリウム
５ｍｇを添加して４℃中で一夜放置した。この後、未反
応の水素化ホウ素ナトリウムを除去するため、０．８５
％塩化ナトリウムを含む１０ｍＭリン酸ナトリウム緩衝
液（ｐＨ７，１）に対して４℃で一夜攪拌しながら透析
した。上記反応物をＴＳＫ−ゲルＧ−３０００５Ｗ （
東ソー株式会社製、商品名）を用いて高速液体クロマト
グラフィーにて精製し、ＨＲＰｅＡ２抗体とした。同様
にして、抗ヒトα１アンチトリプシン抗体の標識抗体も
同様に調製した。(B) Preparation of horseradish peroxidase (HRP) labeled antibody: 0.3M sodium bicarbonate buffer (pH 8
, 1) was added with 0.1 ml of 1% 1-fluoro-2,4-dinitrobenzene in ethanol solution (5 mg/m 1 ), and reacted at room temperature for 1 hour. Add 1.0 0.06M sodium periodate to the solution.
ml was added and reacted for 30 minutes. Unreacted sodium periodate was dissolved in 0.16M ethylene glycol 1. Oml
was added and removed, and then dialyzed against 0.01M sodium carbonate buffer (pH 9.5). Next, 5 mg of mouse-derived anti-human protein C inhibitor monoclonal antibody was added and reacted for 5 to 6 hours. 5 mg of sodium borohydride was added and left at 4°C overnight. After this, in order to remove unreacted sodium borohydride, 0.85
Dialysis was performed against 10 mM sodium phosphate buffer (pH 7.1) containing % sodium chloride at 4° C. overnight with stirring. The above reaction product was mixed with TSK-Gel G-30005W (
It was purified by high performance liquid chromatography using Tosoh Corporation (trade name) to obtain an HRPeA2 antibody. Similarly, a labeled antibody of anti-human α1 antitrypsin antibody was prepared in the same manner.
（Ｃ）血液中のヒトプロティンＣの定量本実施例中の（
Ａ）で記述した方法で作製したマイクロタイタープレー
トを室温にもどし、ＰＢＳ−Ｔ溶液で洗浄した後、既知
量の活性化ヒトプロティンＣ・インヒビター複合体を含
む標準血液を各ウェルにそれぞれ２０μｍ加えた。つぎ
に本実施例（Ｂ）で得た２つのＨＲＰ標識抗体をＰＢＳ
−Ｔ溶液で希釈し、各ウェルに１００μｌずつ添加した
。そのまま室温で３時間インキュベートした後、溶液を
除去しＰＢＳ−Ｔ溶液で３回洗浄した。それに、１．２
％ ＡＢＴＳ及び０６０１％過酸化水素（Ｈ２０□）を
含有する０、１Ｍクエン酸緩衝液（ｐＨ４，１）から成
る基質溶液を各ウェルに２００μｍ添加し、室温で３０
分間酵素反応させた後、２００ｍＭシュウ酸溶液を１０
０μｌ加えて酵素反応を停止させた。上記マイクロタイ
タープレートを各ウェルについて、波長４１５ｎｍ、対
照波長４９２ｎｍの吸光強度を自動マイクロタイタープ
レートリーダー（東ソー株式会社製、ＭＰＲ−Ａ４、商
品名）で測定した。(C) Quantification of human protein C in blood (
The microtiter plate prepared by the method described in A) was brought to room temperature, washed with PBS-T solution, and 20 μm of standard blood containing a known amount of activated human protein C inhibitor complex was added to each well. . Next, the two HRP-labeled antibodies obtained in Example (B) were added to PBS.
-T solution and added 100 μl to each well. After incubating at room temperature for 3 hours, the solution was removed and the plate was washed three times with PBS-T solution. Besides, 1.2
200 μm of substrate solution consisting of 0.1 M citrate buffer (pH 4.1) containing 0.6% ABTS and 1% hydrogen peroxide (H20□) was added to each well and incubated for 30 min at room temperature.
After enzymatic reaction for 1 minute, 200mM oxalic acid solution was added to
The enzyme reaction was stopped by adding 0 μl. For each well of the microtiter plate, the absorption intensity at a wavelength of 415 nm and a reference wavelength of 492 nm was measured using an automatic microtiter plate reader (manufactured by Tosoh Corporation, MPR-A4, trade name).
結果を表１に示す。The results are shown in Table 1.
表１
表１から明らかなように、試料中のヒトプロティンＣは
１〜２００ｎｇ／ｍｌの範囲で定量できることが確認さ
れた。Table 1 As is clear from Table 1, it was confirmed that human protein C in the sample could be quantified in the range of 1 to 200 ng/ml.
Claims (1)
て、 （ａ）・活性化ヒトプロテインＣを特異的に認識する、
固相に固定化されたモノクローナル抗体（１）、 ・試料、 ・ヒトプロテインＣインヒビターを特異的に認識すると
ころの標識されたまたは標識さ れていないモノクローナル抗体（２） ・および、ヒトα＿１アンチトリプシンを特異的に認識
するところの標識されたまたは標識されていないモノク
ローナル抗体（３） を接触させ （ｂ）（ａ）で標識されていないモノクローナル抗体（
２）、（３）を使用した場合には（ａ）で生じる免疫反
応生成物、またはモノクローナル抗体（２）および（３
）を特異的に認識する標識された抗体を接触させ、 （ｃ）遊離の又は固定化された標識化抗体の標識を、直
接的または間接的に検出して免疫反応生成物を定量する ことを特徴とする活性化ヒトプロテインＣの測定方法。[Claims] A method for measuring activated human protein C in a sample, comprising: (a) specifically recognizing activated human protein C;
A monoclonal antibody (1) immobilized on a solid phase, - a sample, - a labeled or unlabeled monoclonal antibody that specifically recognizes human protein C inhibitor (2), and human α_1 antitrypsin. Contact with a labeled or unlabeled monoclonal antibody (3) that specifically recognizes (b) (a).
When using 2) and (3), the immune reaction product generated in (a) or the monoclonal antibodies (2) and (3)
) and (c) directly or indirectly detecting the label of the free or immobilized labeled antibody to quantify the immune reaction product. Characteristic method for measuring activated human protein C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33810589A JPH03200066A (en) | 1989-12-28 | 1989-12-28 | Method for measuring activated human protein c |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33810589A JPH03200066A (en) | 1989-12-28 | 1989-12-28 | Method for measuring activated human protein c |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03200066A true JPH03200066A (en) | 1991-09-02 |
Family
ID=18314961
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33810589A Pending JPH03200066A (en) | 1989-12-28 | 1989-12-28 | Method for measuring activated human protein c |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03200066A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002029015A1 (en) | 2000-10-02 | 2002-04-11 | Oklahoma Medical Research Foundation | Assay for rapid detection of human activated protein c and highly specific monoclonal antibody therefor |
| WO2004009641A1 (en) * | 2002-07-22 | 2004-01-29 | Chugai Seiyaku Kabushiki Kaisha | aPC NON-NEUTRALIZING ANTIBODY |
-
1989
- 1989-12-28 JP JP33810589A patent/JPH03200066A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002029015A1 (en) | 2000-10-02 | 2002-04-11 | Oklahoma Medical Research Foundation | Assay for rapid detection of human activated protein c and highly specific monoclonal antibody therefor |
| US6989241B2 (en) | 2000-10-02 | 2006-01-24 | Oklahoma Medical Research Foundation | Assay for rapid detection of human activated protein C and highly specific monoclonal antibody therefor |
| WO2004009641A1 (en) * | 2002-07-22 | 2004-01-29 | Chugai Seiyaku Kabushiki Kaisha | aPC NON-NEUTRALIZING ANTIBODY |
| US7517965B2 (en) | 2002-07-22 | 2009-04-14 | Chugai Seiyaku Kabushiki Kaisha | Non-neutralizing anti-aPC antibodies |
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