JP3575276B2

JP3575276B2 - Method for measuring hepatocyte growth factor activator inhibitor complex

Info

Publication number: JP3575276B2
Application number: JP10438498A
Authority: JP
Inventors: 猛下村
Original assignee: Mitsubishi Chemical Corp
Current assignee: Mitsubishi Chemical Corp
Priority date: 1998-04-15
Filing date: 1998-04-15
Publication date: 2004-10-13
Anticipated expiration: 2018-04-15
Also published as: JPH11295309A

Description

【０００１】
【発明の属する技術分野】
本発明は、２種類の肝細胞増殖因子活性化因子阻害因子（以下HAI-1またはHAI-2と記載する）と肝細胞増殖因子活性化因子との複合体を各因子特異的に測定できることを特徴とするアッセイ方法に関する。
【０００２】
【従来の技術および発明が解決しようとする課題】
肝細胞増殖因子活性化因子（特開平５−１０３６７０、同６−１４１８５９、同６−１５３９４６、および同６−１５３９６６各号公報参照；肝細胞増殖因子（ＨＧＦ）を一本鎖から二本鎖へ変換する活性を有する因子；以下、ＨＧＦアクチベーターもしくはＨＧＦａｃｔｉｖａｔｏｒと記載する）に対して阻害活性を持つ因子は２種類同定されており（ＨＡＩ−１及びＨＡＩ−２）、これら両因子は同じ細胞株によって産生されることが報告されている（特開平９−９５４９７号、特開平９−９５４９８号）。従って、従来のＨＧＦ前駆体とＨＧＦアクチベーター並びにサンプルを用いたＨＧＦ切断活性阻害の測定方法において溶液中の阻害活性を測定した場合、その活性がどちらの阻害因子に起因するのかは明らかにはできなかった。また、未知の阻害因子が存在した場合にもこれら全体の阻害活性を測定することになり、個々の阻害因子の阻害活性値を得ることは不可能であった。さらに、たとえば阻害因子に対する抗体だけを用いて酵素標識イムノアッセイ（ＥＩＡ）やドットアッセイなどで単に物理的量を測定した場合の値は、分解や修飾を受けた阻害因子が存在した場合には、その阻害活性を反映しないことになる。
【０００３】
そこで本発明は、ＨＧＦアクチベーターと各阻害因子の複合体を個別に定量化できる測定方法を提供することを目的としてなされたものである。
【０００４】
【課題を解決するための手段】
本発明者は上記課題を達成すべく鋭意検討した結果、ＨＧＦアクチベーターとその酵素活性を阻害しない抗ＨＧＦアクチベーター抗体、及び阻害因子（HAI-1、HAI-2）とその阻害活性を阻害しない各HAIに対する抗HAI-1抗体または抗HAI-2抗体を用いることで、ＨＧＦアクチベーターと各阻害因子の複合体を個別に定量化できることを見いだした。
【０００５】
すなわち本発明は、肝細胞増殖因子活性化因子（ＨＧＦアクチベーター）と肝細胞増殖因子活性化因子阻害因子−１（ＨＡＩ−１）又は肝細胞増殖因子活性化因子阻害因子−２（ＨＡＩ−２）とで形成された複合体を、抗肝細胞増殖因子活性化因子阻害因子−１抗体（抗ＨＡＩ−１抗体）又は抗肝細胞増殖因子活性化因子阻害因子−２抗体（抗ＨＡＩ−２抗体）と、抗肝細胞増殖因子活性化因子抗体（抗ＨＧＦアクチベーター抗体）を用いて、イムノアッセイにより検出することにより該複合体を形成している肝細胞増殖因子活性化因子阻害因子−１（ＨＡＩ−１）又は肝細胞増殖因子活性化因子阻害因子−２（ＨＡＩ−２）を個別に定量することを特徴とする肝細胞増殖因子活性化因子阻害因子複合体の測定方法である。
【０００７】
また、本発明の好ましい態様によれば、イムノアッセイが酵素標識イムノアッセイである該測定方法；イムノアッセイが蛍光標識イムノアッセイである該測定方法；イムノアッセイがラジオイムノアッセイである該測定方法；イムノアッセイが発光標識イムノアッセイである該測定方法が提供される。
【０００８】
【発明の実施の形態】
以下、本発明につき詳細に説明する。本発明の肝細胞増殖因子活性化因子阻害因子複合体の測定方法は、アッセイ容器中で肝細胞増殖因子活性化因子阻害因子（HAI-1、HAI-2）と該阻害因子に特異的な肝細胞増殖因子活性化因子とで形成された複合体を、それらを各々の抗体でイムノアッセイにより検出するものである。この方法において、測定サンプル中の各阻害因子特異的活性を定量することが可能となる。
【０００９】
本発明に使用される標準品としてのHAI-1（HAI-I）は種々の細胞株の培養上清や遺伝子工学的手法やペプチド合成によって調製することができる。例えば、特開平９−９５４９７号公報に記載の、後記配列表の配列番号２に記載のアミノ酸配列のうち３７５番目のシステインから４２５番目のシステインを保持した形でバクテリア、酵母や動物細胞等に遺伝子組換え技術によって発現生産させることが可能である。また、特開平９−９５４９７号公報に記載の、後記配列表の配列番号２に記載のアミノ酸配列のうち、ＨＧＦアクチベーター阻害活性を担う第一Kunitzドメインである２５０番目のシステインから３００番目のシステイン部分を含む形で一部欠如させたHAI-1改変体なども用いることができる。このようにして発現されたHAI-1は、各種クロマトグラフィー、疎水性、ハイドロキシアパタイト、陰イオン交換、ターゲットプロテアーゼや抗体を用いたアフィニティークロマトグラフィーおよびゲル濾過カラムクロマトグラフィーに供し得ることができる。必要に応じて、逆相カラムクロマトグラフィー等を精製ステップに組み込むこともできる。同様にHAI-2（HAI-II）も特開平９−９５４９８号に記載の方法等により調製可能である。
【００１０】
ＨＧＦアクチベーターは既に公表ずみの方法で組換え体としてまた天然物として得ることができる。前駆体として得られる場合はトロンビン処理することで活性型として得ることができる。
【００１２】
抗ＨＧＦアクチベーター抗体は、ＨＧＦアクチベーターまたはその断片を動物に免疫することでモノクローナル抗体やポリクローナル抗体として得ることができる。同様に各抗阻害因子抗体は、各阻害因子またはその断片を動物に免疫することでモノクローナル抗体やポリクローナル抗体として得ることができる。イムノアッセイとしては、例えば蛍光標識イムノアッセイ（ＦＩＡ）、酵素標識イムノアッセイ（ＥＩＡ）、ラジオイムノアッセイ（ＲＩＡ）、発光イムノアッセイ等が用いられる。この際に使用される標識の種類としては特に限定されないが、蛍光標識としては例えば、FIFC(Fluorecein Isothio cyanate)、AMCA(7-Amino-4-methylcoumarin-3-acetic acid)、Oregon Green 488、Texas Red、Bodipy、Umbelliferone、Lanthanide chelates等が挙げられる。酵素標識としては例えば、Lysozyme、Malate dehydrogenase、Peroxidase、Gulucose oxidase、Alkaline phosphatase、β-Galactosidase、Horseradish peroxidase等が挙げられる。ラジオイムノアッセイのラジオアイソトープとしては例えば、³H、¹⁴C、⁵⁷Co、⁷⁵Se、¹²⁵I、¹³¹I等が挙げられる。発光標識としては例えばLuminol、Isoluminol、Pyrogallol、Protohaemin、ABEI(Aminobutylethyl-n-isoluminol)、AHEI(Aminohexylethyl-n-ethyl-isoluminol)、Acridinium esters、ATP(Adenosine triphosphate)、NAD(Nicotinamide adenine dinucleotide)、Luciferase、Oxidoreductases等が挙げられる。磁気標識としては例えば、ビオチン磁気、ストレプトアビジン磁気等が挙げられる。
【００１３】
阻害因子の測定方法は大きく分けて次の二つの方法で行うことができる。例えば、一つは抗HGF activator抗体をアッセイプレートに吸着させ、非特異的吸着を防ぐ処理を行った後、HGF activatorをプレートに添加し抗体と反応させる。次に抗体に結合した状態のHGF activatorとサンプルを反応させ肝細胞増殖因子活性化因子・肝細胞増殖因子活性化因子阻害因子複合体を形成させる。最後に酵素標識された抗阻害因子抗体を添加し、複合体に反応させる。場合によっては、ＡＢＣ法などの発色増幅などを行い感度を上げることも可能である。これら酵素による基質の発色を標準品での発色と比較することで、各サンプルの阻害活性を定量することが可能となる。もう一つの方法は、まず最初に阻害因子の抗体をプレートに結合させ、非特異的吸着処理後、サンプルをその抗体と反応させる。その後、HGF activatorを添加し、プレートに結合した阻害因子と複合体を形成させる。最後に、抗HGF activator抗体でその複合体を検出して、サンプル中の各肝細胞増殖因子活性化因子阻害因子複合体を個別に定量できる。
【００１４】
【実施例】
以下の実施例により、本発明をより詳細に説明するが、本発明はその趣旨を越えない限り、以下の実施例によって限定されるものではない。
実施例１精製ＨＡＩ−１の測定１
バッファーＡ（１００ｍＭＮａＣｌ，０．１％ｇｅｌａｔｉｎ，０．０５％Ｔｗｅｅｎ２０を含む５０ｍＭＴｒｉｓ−ＨＣｌ（ｐＨ８）バッファー）と洗浄液（１００ｍＭＮａＣｌ，０．０５％Ｔｗｅｅｎ２０を含む５０ｍＭＴｒｉｓ−ＨＣｌ（ｐＨ８）バッファー）を調製する。
【００１５】
EIA用96ウェル・アッセイフ゜レートに抗HGF activator抗体（A23抗体）を10μg/ml濃度で50μl/wellで添加。37℃で数時間インキュヘ゛ーション後、洗浄液でフ゜レートを洗浄し、ハ゛ッファーＡをウェル一杯に添加した。４℃で一昼夜静置後、再度洗浄液で洗浄後、ハ゛ッファーＡに溶解した200ng/ml濃度の34kDa活性型HGF activatorを50μl/well添加し４℃で一昼夜静置した。洗浄液でフ゜レートを洗浄後、ハ゛ッファーＡで精製標準品のHAI-1を100ng/mlから数ng/mlに調製後、50μl/wellを添加し、再度４℃で一昼夜静置した。フ゜レートを洗浄液で洗浄後、ビオチン標識抗HAI-1抗体（C76-18抗体）をハ゛ッファーＡで至適濃度に希釈し50μl/well添加後、３７度で２時間反応させた。再度洗浄ハ゛ッファーで洗浄し、ABCキット（Vectastain Elite ABC kit）の増幅試薬を添加して室温にて１時間反応させた後、Ｏーフェニレンジアミン発色溶液を添加し発色させた。結果を図１に示す。HAI-1濃度依存的な発色が認められた。
実施例２培養上清中のHAI-1の測定培養上清の調製は以下のようにして行った。HLC-1細胞（ヒト肺癌細胞株；Gann (1976) 67, 483-492 Akagi T & Kimoto T）、MKN45（ヒト胃癌細胞株；財団法人がん研究振興財団(Japanese Cancer Reserch Resources Bank)に登録番号JCRB0254として登録、免疫生物研究所から入手）を6 well培養プレートに５％血清含培地で播種した。コンフルエント後培地で洗浄し、再度無血清培地を添加し37度で培養を継続した。数時間後培養上清を回収し、フィルター濾過により細胞除去後、その培養上清中の阻害活性を測定した。
【００１６】
ＥＩＡ用９６ウェル・アッセイプレートに抗ＨＧＦａｃｔｉｖａｔｏｒ抗体（Ａ２３抗体）を１０μｇ／ｍｌ濃度で５０μｌ／ｗｅｌｌで添加。３７℃で数時間インキュベーション後、洗浄液でプレートを洗浄し、バッファーＡをウェル一杯に添加した。４℃で一昼夜静置後、再度洗浄液で洗浄後、バッファーＡに溶解した１μｇ／ｍｌ濃度の活性型ＨＧＦａｃｔｉｖａｔｏｒを５０μｌ／ｗｅｌｌ添加し４℃で一昼夜静置した。洗浄液でプレートを洗浄後、バッファーＡで精製サンプル標準品としてを１００ｎｇ／ｍｌから数ｎｇ／ｍｌに調製後、５０μｌ／ｗｅｌｌを添加、または培養上清を１／２ずつ希釈系列をつくり５０μｌ／ｗｅｌｌを添加し、再度４℃で一昼夜静置した。プレートを洗浄液で洗浄後、ビオチン標識抗ＨＡＩ−１抗体（Ｃ７６−１８抗体）をバッファーＡで至適濃度に希釈し５０μｌ／ｗｅｌｌ添加後、３７度で２時間反応させた。再度洗浄バッファーで洗浄し、ＡＢＣキット（ＶｅｃｔａｓｔａｉｎＥｌｉｔｅＡＢＣｋｉｔ）の増幅試薬を添加して室温にて１時間反応させた後、Ｏーフェニレンジアミン発色溶液を添加し発色させた。各培養上清中のＨＡＩ−１の活性値は標準品の発色との比較により、標準品換算でｎｇ／ｍｌとして表した。その結果図２に示すとおり、培養液中のＨＡＩ−１の活性が測定された。
実施例３精製ＨＡＩ−１の測定２
ＥＩＡ用９６ウェル・アッセイプレートに抗ＨＡＩ−１抗体（Ｃ７６−１８抗体）を１０μｇ／ｍｌ濃度で５０μｌ／ｗｅｌｌで添加。３７℃で数時間インキュベーション後、洗浄液でプレートを洗浄し、バッファーＡをウェル一杯に添加した。洗浄液でプレートを洗浄後、バッファーＡで精製標準品のＨＡＩ−１を５０ｎｇ／ｍｌから１／２希釈系列で数ｎｇ／ｍｌに調製後、５０μｌ／ｗｅｌｌを添加し、４℃で一昼夜静置した。再度洗浄液で洗浄後、バッファーＡに溶解した５００ｎｇ／ｍｌ濃度の３４ｋＤａ活性型ＨＧＦａｃｔｉｖａｔｏｒを５０μｌ／ｗｅｌｌ添加し再度４℃で一昼夜静置した。プレートを洗浄液で洗浄後、ホースラディッシュ標識抗ＨＧＦａｃｔｉｖａｔｏｒ抗体（Ａ２３抗体）をバッファーＡで至適濃度に希釈し５０μｌ／ｗｅｌｌ添加後、室温で１時間反応させた。再度洗浄バッファーで洗浄し、Ｏーフェニレンジアミン発色溶液を添加し発色させた。結果を図３に示す。この系においてもＨＡＩ−１濃度依存的な発色が認められた。
実施例４ＨＡＩ− ２の測定
ＨＡＩ−２遺伝子のトランスフェクトしたＣＨＯ細胞を調製した。その組換えＣＨＯ細胞５％ＦＢＳを含む培地を用いてをＴ２５フラスコに播種した。コンフルエント後、その培養上清を回収し、フィルターで細胞を除去後、アッセイに供した。
【００１７】
ＥＩＡ用９６ウェル・アッセイプレートに抗ＨＡＩ−２抗体（２Ｎ１２抗体）を１０μｇ／ｍｌ濃度で５０μｌ／ｗｅｌｌで添加。３７℃で数時間インキュベーション後、洗浄液でプレートを洗浄し、バッファーＡをウェル一杯に添加した。洗浄液でプレートを洗浄後、バッファーＡで組換えＨＡＩ−２を含む培養上清中を１／２希釈系列で調製後、各ウェルに５０μｌ／ｗｅｌｌを添加し、４℃で一昼夜静置した。再度洗浄液で洗浄後、バッファーＡに溶解した５００ｎｇ／ｍｌ濃度の３４ｋＤａ活性型ＨＧＦａｃｔｉｖａｔｏｒを５０μｌ／ｗｅｌｌ添加し再度４℃で一昼夜静置した。プレートを洗浄液で洗浄後、ホースラディッシュ標識抗ＨＧＦａｃｔｉｖａｔｏｒ抗体（Ａ２３抗体）をバッファーＡで至適濃度に希釈し５０μｌ／ｗｅｌｌ添加後、室温で１時間反応させた。再度洗浄バッファーで洗浄し、Ｏーフェニレンジアミン発色溶液を添加し発色させた。結果を図４に示す。この系においてもＨＡＩ−２濃度に比例する培地希釈依存的な発色が認められた。
【００１８】
【発明の効果】
本発明によれば、肝細胞増殖因子活性化因子と肝細胞増殖因子活性化因子阻害因子の複合体のうち、HAI-1複合体とHAI-2複合体を測り分けることができるので、より詳細にＨＧＦやＨＧＦアクチベーター並びに各阻害因子の機能解析を行うことができるようになり、さらにはこれら因子が関わる疾病等の診断にも応用可能となる。
【図面の簡単な説明】
【図１】本願アッセイ系の一次抗体を抗ＨＧＦａｃｔｉｖａｔｏｒ抗体、二次抗体を酵素標識抗ＨＡＩ−１抗体を用いて精製ＨＡＩ−１の検量線を求めたものである。
【図２】本願アッセイ系の一次抗体を抗ＨＧＦａｃｔｉｖａｔｏｒ抗体、二次抗体を酵素標識抗ＨＡＩ−１抗体を用いて培養上清中のＨＡＩ−１の活性を定量したものである。
【図３】本願アッセイ系の一次抗体を抗ＨＡＩ−１抗体、二次抗体を酵素標識抗ＨＧＦａｃｔｉｖａｔｏｒ抗体を用いて精製ＨＡＩ−１の検量線を求めたものである。
【図４】本願アッセイ系の一次抗体を抗ＨＡＩ−２抗体、二次抗体を酵素標識抗ＨＧＦａｃｔｉｖａｔｏｒ抗体を用いて精製ＨＡＩ−２の検量線を求めたものである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides that a complex of two types of hepatocyte growth factor activator inhibitor (hereinafter referred to as HAI-1 or HAI-2) and a hepatocyte growth factor activator can be specifically measured for each factor. It relates to a characteristic assay method.
[0002]
2. Description of the Related Art
Hepatocyte growth factor activating factor (see JP-A-5-103670, JP-A-6-141589, JP-A-6-153946, and JP-A-6-153966); Hepatocyte growth factor (HGF) is converted from a single chain to a double chain. Two types of factors having inhibitory activity against HGF activator or HGF activator (hereinafter referred to as HGF activator) have been identified (HAI-1 and HAI-2), and these two factors are the same cell. It is reported that it is produced by a strain (JP-A-9-95497, JP-A-9-95498). Therefore, when the inhibitory activity in a solution is measured by a conventional method for measuring HGF cleavage activity inhibition using a HGF precursor, an HGF activator, and a sample, it is not clear which inhibitor is responsible for the activity. Did not. In addition, even when unknown inhibitors exist, the total inhibitory activity of these inhibitors is measured, and it is impossible to obtain the inhibitory activity value of each inhibitor. Furthermore, for example, when a physical quantity is simply measured by an enzyme-labeled immunoassay (EIA) or a dot assay using only an antibody against an inhibitor, the value obtained when a degraded or modified inhibitor is present is It will not reflect the inhibitory activity.
[0003]
Accordingly, an object of the present invention is to provide a measurement method capable of individually quantifying a complex of an HGF activator and each inhibitor.
[0004]
[Means for Solving the Problems]
The present inventor has inhibited the above-mentioned problems result to intensive studies to achieve the, H GF activator and anti-HGF activator antibody does not inhibit the enzymatic activity, and inhibitor (HAI-1, HAI-2 ) and its inhibitory activity By using an anti-HAI-1 antibody or anti-HAI-2 antibody against each HAI, it was found that the complex of the HGF activator and each inhibitor could be individually quantified.
[0005]
That is, the present invention relates to hepatocyte growth factor activator (HGF activator) and hepatocyte growth factor activator inhibitor-1 (HAI-1) or hepatocyte growth factor activator inhibitor-2 (HAI-2). ) And an anti-hepatocyte growth factor activator inhibitor-1 antibody (anti-HAI-1 antibody) or an anti-hepatocyte growth factor activator inhibitor-2 antibody (anti-HAI-2 antibody) ) And an anti-hepatocyte growth factor activator antibody (anti-HGF activator antibody), which is detected by immunoassay to form the hepatocyte growth factor activator inhibitor-1 (HAI) -1) or a method for measuring a hepatocyte growth factor activator inhibitor complex, which is characterized by individually quantifying hepatocyte growth factor activator inhibitor-2 (HAI-2).
[0007]
Also, according to a preferred embodiment of the present invention, the immunoassay is an enzyme-labeled immunoassay the measurement method; immunoassay is a fluorescent label immunoassay the measurement method; in immunoassay luminescent label immunoassay; immunoassay the measurement method is radioimmunoassay Certain such measurement methods are provided.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail. The method for measuring a hepatocyte growth factor activator inhibitor complex according to the present invention comprises the steps of: determining a hepatocyte growth factor activator inhibitor (HAI-1, HAI-2) and a hepatocyte specific to the inhibitor in an assay container; the complex formed by the cell growth factor activator, and detects by immunoassay them in each antibody. In this method, it becomes possible to quantify each inhibitor-specific activity in the measurement sample .
[0009]
HAI-1 (HAI-I) as a standard used in the present invention can be prepared by culture supernatants of various cell lines, genetic engineering techniques or peptide synthesis. For example, the gene is expressed in bacteria, yeast, animal cells, etc. in a form retaining the cysteine at positions 375 to 425 in the amino acid sequence of SEQ ID NO: 2 described in Japanese Patent Application Laid-Open No. 9-95497. Expression can be produced by recombinant techniques. In addition, among the amino acid sequences described in SEQ ID NO: 2 in the sequence listing described later in Japanese Patent Application Laid-Open No. 9-95497, the first Kunitz domain responsible for HGF activator inhibitory activity is the first Kunitz domain from the 250th cysteine to 300th cysteine. A modified HAI-1 or the like partially deleted in a form including a portion can also be used. The HAI-1 thus expressed can be subjected to various types of chromatography, hydrophobicity, hydroxyapatite, anion exchange, affinity chromatography using a target protease or antibody, and gel filtration column chromatography. If necessary, reverse phase column chromatography or the like can be incorporated in the purification step. Similarly, HAI-2 (HAI-II) can be prepared by the method described in JP-A-9-95498 or the like.
[0010]
The HGF activator can be obtained as a recombinant or as a natural product in a method already published. When obtained as a precursor, it can be obtained as an active form by treating with thrombin.
[0012]
The anti- HGF activator antibody can be obtained as a monoclonal antibody or a polyclonal antibody by immunizing an animal with the HGF activator or a fragment thereof. Similarly, each anti-inhibitor antibody can be obtained as a monoclonal antibody or a polyclonal antibody by immunizing an animal with each inhibitor or a fragment thereof. As the immunoassay, for example, a fluorescence-labeled immunoassay (FIA), an enzyme-labeled immunoassay (EIA), a radioimmunoassay (RIA), a luminescence immunoassay and the like are used. The type of label used at this time is not particularly limited, and examples of the fluorescent label include FIFC (Fluorecein Isothio cyanate), AMCA (7-Amino-4-methylcoumarin-3-acetic acid), Oregon Green 488, and Texas. Red, Bodipy, Umbelliferone, Lanthanide chelates and the like. Examples of the enzyme label include Lysozyme, Malate dehydrogenase, Peroxidase, Gulucose oxidase, Alkaline phosphatase, β-Galactosidase, Horseradish peroxidase and the like. The radioisotope of radioimmunoassay for ^{^{^{example, 3 H, 14 C, 57}}} Co, 75 Se, 125 I, 131 I and the like. Examples of luminescent labels include Luminol, Isoluminol, Pyrogallol, Protohaemin, ABEI (Aminobutylethyl-n-isoluminol), AHEI (Aminohexylethyl-n-ethyl-isoluminol), Acridinium esters, ATP (Adenosine triphosphate), NAD (Nicotinamide adenine dinucleotide), and Lucifer. , Oxidoreductases and the like. Examples of the magnetic label include biotin magnetism, streptavidin magnetism, and the like.
[0013]
The method of measuring the inhibitor can be roughly divided into the following two methods. For example, one involves adsorbing an anti-HGF activator antibody to an assay plate and performing a treatment to prevent non-specific adsorption, and then adding HGF activator to the plate to react with the antibody. Next, the HGF activator bound to the antibody is reacted with the sample to form a hepatocyte growth factor activating factor / hepatocyte growth factor activating factor inhibitor complex. Finally, an enzyme-labeled anti-inhibitor antibody is added to react with the complex. In some cases, it is possible to increase the sensitivity by performing color development amplification such as an ABC method. By comparing the color development of the substrate by these enzymes with the color development of the standard product, it becomes possible to quantify the inhibitory activity of each sample. Another method is to first bind the inhibitor antibody to the plate, and after non-specific adsorption treatment, react the sample with the antibody. Thereafter, HGF activator is added to form a complex with the inhibitor bound to the plate. Finally, the complex can be detected with an anti-HGF activator antibody and each hepatocyte growth factor activator inhibitor complex in the sample can be individually quantified.
[0014]
【Example】
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples as long as the gist of the present invention is not exceeded.
Example 1 Measurement 1 of Purified HAI- 1
Buffer A (50 mM Tris-HCl (pH 8) buffer containing 100 mM NaCl, 0.1% gelatin, 0.05% Tween 20) and washing solution (50 mM Tris-HCl (pH 8) containing 100 mM NaCl, 0.05% Tween 20) Buffer).
[0015]
Anti-HGF activator antibody (A23 antibody) was added to EIA 96-well assay plate at a concentration of 10 μg / ml at 50 μl / well. After incubation at 37 ° C. for several hours, the plate was washed with a washing solution, and Buffer A was added to the entire well. After standing at 4 ° C. overnight and then again with a washing solution, 50 μl / well of a 34 kDa activated HGF activator at a concentration of 200 ng / ml dissolved in Buffer A was added, and the mixture was allowed to stand at 4 ° C. overnight. After washing the plate with the washing solution, the purified standard product HAI-1 was adjusted from 100 ng / ml to several ng / ml in Buffer A, 50 μl / well was added, and the mixture was again allowed to stand at 4 ° C. overnight. After washing the plate with a washing solution, a biotin-labeled anti-HAI-1 antibody (C76-18 antibody) was diluted to an optimal concentration with Buffer A, added at 50 μl / well, and reacted at 37 ° C. for 2 hours. After washing again with a washing buffer, an amplification reagent of an ABC kit (Vectastain Elite ABC kit) was added and reacted at room temperature for 1 hour, and then an O-phenylenediamine coloring solution was added to develop a color. The results are shown in FIG. HAI-1 concentration-dependent color development was observed.
Example 2 Measurement of HAI-1 in culture supernatant The culture supernatant was prepared as follows. HLC-1 cells (human lung cancer cell lines; Gann (1976) 67, 483- 492 Akagi T & Kimoto T), MKN45 ( human gastric cancer cell line; registration number to the Foundation for Cancer Research Foundation (Japanese Cancer Reserch Resources Bank) (Registered as JCRB0254, obtained from the Institute for Immunity and Biology) was seeded on a 6-well culture plate in a medium containing 5% serum. After confluence, the cells were washed with a medium, a serum-free medium was added again, and the culture was continued at 37 ° C. After several hours, the culture supernatant was recovered, the cells were removed by filtration with a filter, and the inhibitory activity in the culture supernatant was measured.
[0016]
Anti-HGF activator antibody (A23 antibody) was added to a 96-well assay plate for EIA at a concentration of 10 μg / ml at 50 μl / well. After incubation at 37 ° C. for several hours, the plate was washed with the washing solution, and Buffer A was added to the entire well. After standing at 4 ° C for 24 hours, washing with a washing solution again, an active HGF activator at a concentration of 1 µg / ml dissolved in buffer A was added at 50 µl / well, and the mixture was allowed to stand at 4 ° C overnight. After washing the plate with the washing solution, prepare a purified sample standard from 100 ng / ml to several ng / ml with buffer A, then add 50 μl / well, or make a dilution series of the culture supernatant in ずつ to 50 μl / well. Was added and left again at 4 ° C. overnight. After the plate was washed with a washing solution, a biotin-labeled anti-HAI-1 antibody (C76-18 antibody) was diluted to an optimal concentration with buffer A, added at 50 μl / well, and reacted at 37 ° C. for 2 hours. After washing with a washing buffer again, an amplification reagent of an ABC kit (Vectastain Elite ABC kit) was added and reacted at room temperature for 1 hour. Then, an O-phenylenediamine coloring solution was added to develop color. The activity value of HAI-1 in each culture supernatant was expressed as ng / ml in terms of standard by comparing with the color development of the standard. As a result, as shown in FIG. 2, the activity of HAI-1 in the culture solution was measured.
Example 3 Measurement 2 of Purified HAI-1
Anti-HAI-1 antibody (C76-18 antibody) was added at a concentration of 10 μg / ml at 50 μl / well to a 96-well assay plate for EIA. After incubation at 37 ° C. for several hours, the plate was washed with the washing solution, and Buffer A was added to the entire well. After washing the plate with a washing solution, HAI-1 as a purified standard product was prepared from 50 ng / ml to several ng / ml in a 1/2 dilution series with buffer A, and 50 μl / well was added, followed by standing at 4 ° C. overnight. . After washing again with the washing solution, 50 μl / well of a 34 kDa activated HGF activator at a concentration of 500 ng / ml dissolved in buffer A was added, and the mixture was again allowed to stand at 4 ° C. overnight. After the plate was washed with a washing solution, a horseradish-labeled anti-HGF activator antibody (A23 antibody) was diluted to an optimal concentration with buffer A, added at 50 μl / well, and reacted at room temperature for 1 hour. After washing with a washing buffer again, an O-phenylenediamine coloring solution was added to develop a color. The results are shown in FIG. Also in this system, color development dependent on HAI-1 concentration was observed.
CHO cells transfected with the measurement HAI-2 gene of Example 4 HAI- 2 was prepared. Using a medium containing 5% FBS of the recombinant CHO cells, T25 flasks were inoculated. After confluence, the culture supernatant was collected, the cells were removed with a filter, and then used for the assay.
[0017]
Anti-HAI-2 antibody (2N12 antibody) was added at a concentration of 10 μg / ml to a 96-well assay plate for EIA at 50 μl / well. After incubation at 37 ° C. for several hours, the plate was washed with the washing solution, and Buffer A was added to the entire well. After washing the plate with the washing solution, the culture supernatant containing the recombinant HAI-2 was prepared in a Ａ dilution series with the buffer A, and 50 μl / well was added to each well, followed by standing at 4 ° C. overnight. After washing again with the washing solution, 50 μl / well of a 34 kDa activated HGF activator at a concentration of 500 ng / ml dissolved in buffer A was added, and the mixture was again allowed to stand at 4 ° C. overnight. After the plate was washed with a washing solution, a horseradish-labeled anti-HGF activator antibody (A23 antibody) was diluted to an optimal concentration with buffer A, added at 50 μl / well, and reacted at room temperature for 1 hour. After washing with a washing buffer again, an O-phenylenediamine coloring solution was added to develop a color. FIG. 4 shows the results. Also in this system, color development dependent on the dilution of the culture medium, which is proportional to the HAI-2 concentration, was observed.
[0018]
【The invention's effect】
According to the present invention, among the complexes of hepatocyte growth factor activator and hepatocyte growth factor activator inhibitor, the HAI-1 complex and the HAI-2 complex can be separately measured, so In addition, functional analysis of HGF, HGF activator and each inhibitory factor can be performed, and further, it can be applied to diagnosis of diseases and the like related to these factors.
[Brief description of the drawings]
FIG. 1 shows a calibration curve of purified HAI-1 obtained by using an anti-HGF activator antibody as a primary antibody and an enzyme-labeled anti-HAI-1 antibody as a secondary antibody in the assay system of the present invention.
FIG. 2 shows the results of quantifying the activity of HAI-1 in a culture supernatant using an anti-HGF activator antibody as a primary antibody and an enzyme-labeled anti-HAI-1 antibody as a secondary antibody in the assay system of the present invention.
FIG. 3 shows a calibration curve of purified HAI-1 obtained by using an anti-HAI-1 antibody as a primary antibody and an enzyme-labeled anti-HGF activator antibody as a secondary antibody in the assay system of the present invention.
FIG. 4 shows a calibration curve of purified HAI-2 obtained using an anti-HAI-2 antibody as a primary antibody and an enzyme-labeled anti-HGF activator antibody as a secondary antibody.

Claims

Hepatocyte growth factor activator (HGF activator) and hepatocyte growth factor activator inhibitor-1 ( HAI-1) or hepatocyte growth factor activator inhibitor-2 (HAI-2) and the complex, anti-hepatocyte growth factor activator inhibitor-1 antibody (anti-HAI-1 antibody) or Kokimo cell growth factor activator inhibitor 2 antibody (anti-HAI-2 antibody), anti-liver A hepatocyte growth factor activator inhibitor-1 (HAI-1) or a hepatocyte which forms the complex by detecting by immunoassay using a cell growth factor activator antibody (anti-HGF activator antibody) A method for measuring a hepatocyte growth factor activator inhibitor complex, which comprises individually quantifying cell growth factor activator inhibitor-2 (HAI-2) .

Immunoassay, measurement methods according to claim 1 is an enzyme-labeled immunoassay.

Immunoassay, measurement methods according to claim 1 is a fluorescent label immunoassay.

Immunoassay, measurement methods according to claim 1 which is radioimmunoassay.

Immunoassay, measurement methods according to claim 1 is a luminescent label immunoassay.