JP6485854B2 - Anti-mutant protein S antibody, diagnostic composition containing the antibody, method for detecting mutant human protein S, and kit containing the antibody - Google Patents
Anti-mutant protein S antibody, diagnostic composition containing the antibody, method for detecting mutant human protein S, and kit containing the antibody Download PDFInfo
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- JP6485854B2 JP6485854B2 JP2014194080A JP2014194080A JP6485854B2 JP 6485854 B2 JP6485854 B2 JP 6485854B2 JP 2014194080 A JP2014194080 A JP 2014194080A JP 2014194080 A JP2014194080 A JP 2014194080A JP 6485854 B2 JP6485854 B2 JP 6485854B2
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Description
本発明は、特定のアミノ酸変異を有する変異型ヒトプロテインＳに特異的な抗体、該抗体を含む免疫学的診断用組成物、該抗体を用いて変異型ヒトプロテインＳを検出する方法、ならびに、該抗体を含む検出用キットおよび診断用キットに関する。 The present invention relates to an antibody specific to mutant human protein S having a specific amino acid mutation, an immunological diagnostic composition containing the antibody, a method for detecting mutant human protein S using the antibody, and The present invention relates to a detection kit and a diagnostic kit containing the antibody.
血栓症、例えば静脈血栓症等は、後天的な危険因子や環境因子の他に、単一遺伝子性かつ常染色体優性であって、様々な浸透率を有する多数の遺伝子変異または遺伝子多型によって、そのリスクが増大することがわかってきた。かかる遺伝子変異または遺伝子多型（まとめて「遺伝的欠陥」ともいう）の例としては、凝固促進タンパク質（フィブリノーゲン、プロトロンビン等）、抗凝固タンパク質（後述）、その他の血栓症関連タンパク質（アンジオテンシンＩ転換酵素等）をコードする遺伝子における遺伝的欠陥が挙げられる。
特に抗凝固タンパク質を代表するプロテインＣ、プロテインＳおよびアンチトロンビンＩＩＩの遺伝的欠陥は、静脈血栓症患者において高い割合で存在し、血栓症の素因の６０％超は遺伝的要因である（非特許文献１、特許文献５）。
Thrombosis, such as venous thrombosis, is due to numerous genetic mutations or polymorphisms that are monogenic and autosomal dominant, with various penetrances, in addition to acquired risk factors and environmental factors. It has been found that the risk increases. Examples of such gene mutations or gene polymorphisms (collectively referred to as “genetic defects”) include procoagulant proteins (fibrinogen, prothrombin, etc.), anticoagulant proteins (described later), and other thrombosis-related proteins (angiotensin I conversion). A genetic defect in a gene encoding an enzyme, etc.).
In particular, genetic defects such as protein C, protein S and antithrombin III, which are representative of anticoagulant proteins, are present in a high proportion in patients with venous thrombosis, and more than 60% of the predisposition to thrombosis is a genetic factor (non-patented Document 1, Patent Document 5).
プロテインＳは、生体内の血液凝固系の制御機構において中心的に機能する血漿タンパク質である。血漿中に分泌された成熟型プロテインＳのタンパク質部分は６３５アミノ酸残基で構成され、分子内に１７個のジスルフィド結合を含むマルチドメイン型一本鎖ポリペプチドである。プロテインＳは、血液凝固第ＶＩＩ因子、第ＩＸ因子、プロトロンビン、プロテインＣ等と同様、ビタミンＫ依存性タンパク質ファミリーに分類される。天然型プロテインＳはその分子内に４種類のドメイン構造を有しており、アミノ末端側から、γ−カルボキシルグルタミン酸（Ｇｌａ）ドメイン、トロンビン感受性領域、４つの連続した上皮細胞成長因子（ＥＧＦ）様ドメインおよび性ホルモン結合グロブリン（ＳＨＢＧ）様ドメインが存在する（特許文献６）。主に血液中に存在するプロテインＳは、活性化プロテインＣの補欠因子（補助因子）であり、活性化プロテインＣの活性を上昇させることができ、血液中での活性化プロテインＣの働きに欠かせないものである。活性化プロテインＣは、ヒトにおける血液凝固を促進する活性化血液凝固第Ｖ因子（第Ｖａ因子）および活性化血液凝固第ＶＩＩＩ因子（第ＶＩＩＩａ因子）を分解することにより、血液凝固反応を抑制する役割を担った因子である。プロテインＳは、Ｃ４ｂ結合タンパク質（補体第４因子ｂ結合タンパク質；Ｃ４ｂｐ）と１対１で特異的に結合し、複合体を形成する。すなわち、Ｃ４ｂ結合タンパク質は、プロテインＳのリガンドとなる。通常、健常者の血液中（血漿中）のプロテインＳは、その約６０％が「プロテインＳ−Ｃ４ｂｐ複合体」（すなわち結合型）であり、その約４０％は「遊離状態のプロテインＳ」（すなわち遊離型）である。 Protein S is a plasma protein that functions centrally in the control mechanism of the blood coagulation system in the living body. The protein portion of mature protein S secreted into plasma is a multidomain single-chain polypeptide composed of 635 amino acid residues and containing 17 disulfide bonds in the molecule. Protein S is classified into the vitamin K-dependent protein family, as is blood coagulation factor VII, factor IX, prothrombin, protein C and the like. Natural protein S has four types of domain structures in its molecule. From the amino-terminal side, γ-carboxylglutamic acid (Gla) domain, thrombin sensitive region, four consecutive epidermal growth factor (EGF) -like There are domains and sex hormone binding globulin (SHBG) -like domains (Patent Document 6). Protein S present mainly in blood is a prosthetic factor (cofactor) of activated protein C, which can increase the activity of activated protein C and is lacking in the function of activated protein C in blood. It is not allowed. Activated protein C suppresses blood coagulation by degrading activated blood coagulation factor V (factor Va) and activated blood coagulation factor VIII (factor VIIIa) that promote blood coagulation in humans It is a factor that played a role. Protein S specifically binds one-to-one with a C4b binding protein (complement factor 4b binding protein; C4bp) to form a complex. That is, the C4b binding protein becomes a ligand for protein S. Usually, about 60% of protein S in the blood (plasma) of healthy individuals is “protein S-C4bp complex” (ie, conjugated), and about 40% is “free protein S” ( That is, free type).
血栓症発症リスクを増大させるプロテインＳの遺伝的欠陥は、その量的欠損および質的異常に大別される。
プロテインＳの量的欠損を検出するには、ヒトプロテインＳを特異的に認識するモノクローナル抗体およびＣ４ｂｐとプロテインＳとの複合体を特異的に認識するモノクローナル抗体（特許文献１および６）、Ｃ４ｂ結合タンパク質とプロテインＳとの結合を阻害する抗Ｃ４ｂｐ抗体（特許文献２）、遊離型とＣ４ｂｐ結合型とのヒトプロテインＳを２価金属イオンの存在下においてのみ認識する、いわゆる立体構造特異的モノクローナル抗体（特許文献３）、遊離型プロテインＳに特異的なモノクローナル抗体（特許文献４）、プロテインＳのＣ４ｂｐとの結合部位以外の部位に特異的に結合するモノクローナル抗体（特許文献７および８ならびに非特許文献２）等の抗体を使用して、血中の結合型／遊離型プロテインＳ濃度の測定を行う。
Protein S genetic defects that increase the risk of developing thrombosis are broadly divided into quantitative deficiencies and qualitative abnormalities.
In order to detect quantitative deficiency of protein S, a monoclonal antibody that specifically recognizes human protein S, a monoclonal antibody that specifically recognizes a complex of C4bp and protein S (Patent Documents 1 and 6), and C4b binding Anti-C4bp antibody that inhibits binding between protein and protein S (Patent Document 2), so-called three-dimensional structure-specific monoclonal antibody that recognizes human protein S of free type and C4bp binding type only in the presence of divalent metal ions (Patent document 3), monoclonal antibody specific to free protein S (patent document 4), monoclonal antibody that specifically binds to a site other than the binding site of protein S to C4bp (patent documents 7 and 8 and non-patent document) Measure the concentration of bound / free protein S in the blood using antibodies such as literature 2)
他方、プロテインＳの質的異常については、試料中の総プロテインＳのタンパク質量および試料中の総プロテインＳの活性値を別々に測定し、この測定により得た総プロテインＳのタンパク質量と総プロテインＳの活性値とを、比活性を求める等により比較することによって、プロテインＳ徳島（Ｋ１５５Ｅ変異）を検出することができた（特許文献７および非特許文献２）。
また、市販のキット（STA試薬シリーズ プロテインＳ（クロット）、ロシュ・ダイアグノスティックス（株））等を使用する活性化プロテインＣの抗凝固活性を促進するコファクター活性測定または遺伝子検査によってもプロテインＳの質的異常を検出することができる。
On the other hand, for the qualitative abnormality of protein S, the protein amount of total protein S in the sample and the activity value of total protein S in the sample were measured separately, and the protein amount of total protein S and total protein obtained by this measurement were measured separately. Protein S Tokushima (K155E mutation) could be detected by comparing the activity value of S with specific activity or the like (Patent Document 7 and Non-Patent Document 2).
Proteins can also be measured by cofactor activity measurement or genetic testing that promotes the anticoagulant activity of activated protein C using commercially available kits (STA reagent series Protein S (Clot), Roche Diagnostics Co., Ltd.), etc. A qualitative abnormality of S can be detected.
例えば日本人（主にモンゴロイド人種）である静脈血栓塞栓症患者１７３名において、プロテインＳ遺伝子とともにアンチトロンビン遺伝子およびプロテインＣ遺伝子を検査したところ、５５名（約３２％）から３９種のアミノ酸変異を伴う変異が同定された。かかるアミノ酸変異には、例えば、本発明者らが同定したプロテインＳ遺伝子のＫ１９６Ｅ変異（非特許文献３）の他に、プロテインＣのＫ１９３del変異や同Ｖ３３９Ｍ変異等が含まれていた。プロテインＳのＫ１９６Ｅ変異のヘテロ接合体について、活性化部分トロンボプラスチン時間（ＡＰＴＴ）を指標にしてプロテインＳのコファクター活性を測定した結果、変異型プロテインＳのコファクター活性値は平均１６％の低下を示したが、変異型プロテインＳのコファクター活性値が大きくばらつき、健常者由来のプロテインＳのコファクター活性値と大きく重複することから、プロテインＳのコファクター活性を測定するだけでは当該変異の有無を判定することは困難であった（非特許文献３）。 For example, in 173 patients with venous thromboembolism who are Japanese (mainly Mongoloid race), anti-thrombin gene and protein C gene were tested together with protein S gene, and 39 amino acid mutations from 55 (about 32%) A mutation was identified. Such amino acid mutations included, for example, the K193del mutation of protein C and the V339M mutation in addition to the K196E mutation (Non-patent Document 3) of the protein S gene identified by the present inventors. As a result of measuring the cofactor activity of protein S using the activated partial thromboplastin time (APTT) as an index for the heterozygote of the protein S K196E mutation, the cofactor activity value of mutant protein S decreased by an average of 16%. As shown, since the cofactor activity value of mutant protein S varies widely and overlaps with the cofactor activity value of protein S derived from healthy subjects, the presence or absence of the mutation is only measured by measuring the cofactor activity of protein S. It was difficult to determine (Non-patent Document 3).
そこで、本発明は、前記の問題を解決する、Ｋ１９６Ｅ変異を有する変異型プロテインＳを正確かつ簡便に検出する手法を提供することを目的とする。 Accordingly, an object of the present invention is to provide a method for accurately and simply detecting a mutant protein S having a K196E mutation, which solves the above-mentioned problems.
本発明者らは、前記課題を解決すべく鋭意研究を重ねる中で、いわゆる抗ペプチド抗体によるプロテインＳのＫ１９６Ｅ変異の検出に着眼し、そのための抗原として、１９６番目のグルタミン酸を含む所定の１１アミノ酸を選択し、かかる抗原をもとに、野生型ヒトプロテインＳに対するよりも、Ｋ１９６Ｅ変異の変異型プロテインＳに対して、特異的に結合する抗体の作製に成功し、本発明を完成するに至った。 The present inventors focused on the detection of the K196E mutation of protein S using a so-called anti-peptide antibody in earnest research to solve the above-mentioned problems, and the predetermined 11 amino acids containing the 196th glutamic acid as an antigen for that purpose Based on such antigen, the inventors succeeded in producing an antibody that specifically binds to mutant protein S of K196E mutation rather than to wild type human protein S, and completed the present invention. It was.
すなわち、本発明は、抗原として、ＣＫＮＧＦＶＭＬＳＮＫ（配列番号３）に対するよりも、ＣＫＮＧＦＶＭＬＳＮＥ（配列番号２）に対して有意に結合する抗体またはその抗原結合断片に関する。
ＣＫＮＧＦＶＭＬＳＮＫ（配列番号３）は、好ましくは、配列番号１で表される野生型ヒトプロテインＳのアミノ酸配列に含まれる。
本発明の抗体またはその断片は、好ましくは、少なくともＫ１９６Ｅの変異を有する変異型ヒトプロテインＳに特異的である。
本発明の抗体またはその断片は、好ましくは、モノクローナル抗体である。
That is, the present invention relates to an antibody or antigen-binding fragment thereof that significantly binds to CKNGFVMLSNE (SEQ ID NO: 2) as an antigen rather than to CKNGFVMLSNK (SEQ ID NO: 3).
CKNGFVMLSNK (SEQ ID NO: 3) is preferably included in the amino acid sequence of wild-type human protein S represented by SEQ ID NO: 1.
The antibody or fragment thereof of the present invention is preferably specific for mutant human protein S having at least a K196E mutation.
The antibody or fragment thereof of the present invention is preferably a monoclonal antibody.
また、本発明は、本発明の抗体またはその断片を含む、免疫学的診断用組成物に関する。
本発明の免疫学的診断用組成物は、好ましくは血栓塞栓症、例えば静脈血栓塞栓症を診断するためのものである。
The present invention also relates to an immunological diagnostic composition comprising the antibody of the present invention or a fragment thereof.
The immunodiagnostic composition of the present invention is preferably for diagnosing thromboembolism such as venous thromboembolism.
さらに、本発明は、配列番号１で示されるアミノ酸配列の１９６番目のリジン（Ｋ）がグルタミン酸（Ｅ）に変異した非同義変異を少なくとも有する変異型ヒトプロテインＳを試料から検出する方法であって、前記試料を、本発明の抗体またはその断片と接触させる工程を含む、前記方法に関する。
本発明の検出方法は、変異型ヒトプロテインＳを、免疫比濁法、ラテックス凝集法、イムノクロマト法および固相免疫法からなる群から選択される少なくとも１つの免疫学的測定法により検出する工程をさらに含むことが好ましい。
Furthermore, the present invention is a method for detecting from a sample mutant human protein S having at least a non-synonymous mutation in which the 196th lysine (K) of the amino acid sequence represented by SEQ ID NO: 1 is mutated to glutamic acid (E). And said method comprising the step of contacting said sample with an antibody or fragment thereof of the invention.
The detection method of the present invention comprises a step of detecting mutant human protein S by at least one immunoassay selected from the group consisting of immunoturbidimetry, latex agglutination, immunochromatography, and solid phase immunization. Furthermore, it is preferable to include.
さらにまた、本発明は、本発明の抗体またはその断片を含む、配列番号１で示されるアミノ酸配列の１９６番目のリジン（Ｋ）がグルタミン酸（Ｅ）に変異した非同義変異を少なくとも有する変異型ヒトプロテインＳを検出するための検出用キット、および本発明の免疫学的診断用組成物を含む、該変異型ヒトプロテインＳを検出するための診断用キットに関する。 Furthermore, the present invention provides a mutant human having at least a non-synonymous mutation in which the 196th lysine (K) of the amino acid sequence represented by SEQ ID NO: 1 is mutated to glutamic acid (E), comprising the antibody of the present invention or a fragment thereof. The present invention relates to a detection kit for detecting protein S, and a diagnostic kit for detecting mutant human protein S, which comprises the immunological diagnostic composition of the present invention.
本発明の抗体またはその抗原結合断片は、野生型ヒトプロテインＳに比してＫ１９６Ｅ変異に対する特異性が高いため、被験者に由来する例えば血液試料等から、当該変異型ヒトプロテインＳを、正確かつ簡便に検出することができる。
また、プロテインＳのＫ１９６Ｅ変異の検出には、活性化部分トロンボプラスチン時間（ＡＰＴＴ）を指標にするプロテインＳのコファクター活性の測定が困難なため（非特許文献３）、例えば、ＧｅｎｅＣｈｉｐｓ、Ｔａｑｍａｎ対立遺伝子識別法、パイロシーケンス法等の遺伝子検査を行うことも考えられるが、本発明にかかる手法によれば、プロテインＳのＫ１９６Ｅ変異をはるかに低コストで、正確かつ簡便に検出することができる。
本発明によって、プロテインＳの活性が低下し、血栓症のリスクが高まる例えば妊婦等に対し、血栓症のリスク判断のための診断薬として用いることが可能になる。
Since the antibody of the present invention or the antigen-binding fragment thereof has a higher specificity for the K196E mutation than wild-type human protein S, the mutant human protein S can be accurately and simply obtained from, for example, a blood sample derived from a subject. Can be detected.
In addition, the detection of the K196E mutation in protein S is difficult because it is difficult to measure the cofactor activity of protein S using the activated partial thromboplastin time (APTT) as an index (Non-patent Document 3). For example, GeneChips, Taqman alleles Although it is conceivable to perform genetic tests such as an identification method and a pyrosequencing method, according to the method of the present invention, the K196E mutation of protein S can be detected accurately and simply at a much lower cost.
The present invention can be used as a diagnostic agent for determining the risk of thrombosis, for example, for pregnant women whose protein S activity is reduced and the risk of thrombosis is increased.
以下、本発明について、本発明の好適な実施態様に基づき、詳細に説明する。
本発明における「プロテインＳ」（ＰＳ）またはＰＲＯＳ１タンパク質は、ヒト由来のあらゆるＰＳ遺伝子、ｃＤＮＡもしくはＲＮＡの産物、またはＰＳタンパク質が含まれ、例えば配列番号１で表されるアミノ酸配列やGenBankアクセッション番号AAH15801に開示されたアミノ酸配列等からなり、本明細書中「ヒトプロテインＳ」、または単に「プロテインＳ」とも記す。野生型のヒトプロテインＳの代表的なアミノ酸配列を配列番号１に示すが、本発明においては、配列番号１のアミノ酸配列の１９６番目に相当するアミノ酸がグルタミン酸（Ｅ）に置換されたもののみを「変異型ヒトプロテインＳ」と定義する。この変異を有さなければ、これ以外のアミノ酸１個または複数個の欠失、置換および／または付加の変異を有していても、「野生型ヒトプロテインＳ」と定義する。この野生型ヒトプロテインＳのアミノ酸配列は、配列番号１のアミノ酸配列と、好ましくは６０％以上、より好ましくは７０％以上、さらに好ましくは８０％以上、特に好ましくは９０％以上の配列同一性を有する。
Hereinafter, the present invention will be described in detail based on preferred embodiments of the present invention.
“Protein S” (PS) or PROS1 protein in the present invention includes any human-derived PS gene, cDNA or RNA product, or PS protein. For example, the amino acid sequence represented by SEQ ID NO: 1 or GenBank accession number It consists of the amino acid sequence disclosed in AAH15801, etc., and is also referred to as “human protein S” or simply “protein S” in this specification. A typical amino acid sequence of wild-type human protein S is shown in SEQ ID NO: 1. In the present invention, only the amino acid corresponding to the 196th amino acid sequence of SEQ ID NO: 1 is substituted with glutamic acid (E). It is defined as “mutant human protein S”. If it does not have this mutation, it is defined as “wild type human protein S” even if it has one or more other amino acid deletion, substitution and / or addition mutations. The amino acid sequence of the wild type human protein S preferably has a sequence identity of 60% or more, more preferably 70% or more, still more preferably 80% or more, and particularly preferably 90% or more with the amino acid sequence of SEQ ID NO: 1. Have.
＜抗体またはその断片＞
本発明は、一態様において、抗原として、ＣＫＮＧＦＶＭＬＳＮＫ（配列番号３）に対するよりも、ＣＫＮＧＦＶＭＬＳＮＥ（配列番号２）に対して、例えばＥＬＩＳＡにより測定した抗体の結合定数等を指標として、有意に結合する抗体またはその抗原結合断片を提供するものである。すなわち、本発明の抗体またはその抗原結合断片（単に「その断片」とも記す）は、配列番号２のアミノ酸配列を有するペプチドには特異的であって、かつ配列番号３のアミノ酸配列を有するペプチドには有意に結合しないともいえ、好ましくは配列番号２のペプチドと配列番号３のペプチドとに対し交差反応するものではない。
<Antibodies or fragments thereof>
In one aspect, the present invention provides an antibody that binds significantly to CKNGGFVMLSNE (SEQ ID NO: 2) as an antigen, for example, using an antibody binding constant measured by ELISA as an index, rather than CKNGGFVMLSNK (SEQ ID NO: 3). Alternatively, an antigen-binding fragment thereof is provided. That is, the antibody of the present invention or an antigen-binding fragment thereof (also simply referred to as “fragment thereof”) is specific for the peptide having the amino acid sequence of SEQ ID NO: 2 and is the peptide having the amino acid sequence of SEQ ID NO: 3. May not bind significantly, but preferably does not cross-react with the peptide of SEQ ID NO: 2 and the peptide of SEQ ID NO: 3.
本発明における「（抗体の）抗原結合断片」または単に「（抗体の）断片」とは、ＣＫＮＧＦＶＭＬＳＮＥ（配列番号２）のペプチドに対して有意に結合する抗体と同程度に該ペプチドに結合することができる抗体の断片であれば特に限定されるものではないが、かかる断片としては、例えば、Ｆａｂ断片、Ｆ（ａｂ’）２断片、Ｆ（ａｂ’）断片、二重特異性ｓｃＦｖ断片、Ｆｄ断片およびＦａｂ発現ライブラリーにより産生された断片、ペプチドアプタマーから選択されるその抗体の断片等が挙げられる。
本発明の抗体は、その由来として、特に限定されるものではなく、例えば、哺乳動物（マウス、ウサギ、ラット、ヒツジ、ヤギ、ウマ、ヒト等）または鳥類（ニワトリ、ウズラ、キジ、ダチョウ、アヒル等）などが挙げられる。
The “(antibody) antigen-binding fragment” or simply “(antibody) fragment” in the present invention binds to the peptide to the same extent as an antibody that significantly binds to the peptide of CKNGGFVMLSNE (SEQ ID NO: 2). Examples of such fragments include, for example, Fab fragments, F (ab ′) 2 fragments, F (ab ′) fragments, bispecific scFv fragments, Fd And fragments produced by the Fab expression library, fragments of the antibody selected from peptide aptamers, and the like.
The antibody of the present invention is not particularly limited in its origin, and for example, mammals (mouse, rabbit, rat, sheep, goat, horse, human, etc.) or birds (chicken, quail, pheasant, ostrich, duck) Etc.).
本発明において、ＣＫＮＧＦＶＭＬＳＮＫ（配列番号３）は、好ましくは、配列番号１で表される野生型ヒトプロテインＳのアミノ酸配列に含まれる（１８６〜１９６番目の配列に対応）。それと同時に、ＣＫＮＧＦＶＭＬＳＮＥ（配列番号２）は、配列番号１で表される野生型ヒトプロテインＳのアミノ酸配列において、１９６番目のリジン残基（Ｋ）がグルタミン酸残基（Ｅ）に変異した場合の１８６〜１９６番目の配列に対応する。 In the present invention, CKNGGFVMLSNK (SEQ ID NO: 3) is preferably included in the amino acid sequence of wild-type human protein S represented by SEQ ID NO: 1 (corresponding to the 186th to 196th sequences). At the same time, CKNGGFVMLSNE (SEQ ID NO: 2) is 186 when the 196th lysine residue (K) is mutated to a glutamic acid residue (E) in the amino acid sequence of wild-type human protein S represented by SEQ ID NO: 1. Corresponds to the 196th sequence.
本発明の抗体またはその断片は、好ましくは、少なくともＫ１９６Ｅの変異を有する変異型ヒトプロテインＳ（以下、単に「変異型プロテインＳ」とも記す）に特異的である、すなわち、配列番号１で表されるアミノ酸配列を有する野生型ヒトプロテインＳに対するよりも、変異型プロテインＳに対して、有意に結合する。
ヒトプロテインＳは、野生型ヒトプロテインＳのアミノ酸配列において４３５〜４６８番目の領域でＣ４ｂｐと結合するので（特許文献４）、本発明の抗体またはその断片は、遊離型のプロテインＳのみならず、プロテインＳ−Ｃ４ｂｐ複合体における結合型のプロテインＳにも結合することができる。
The antibody of the present invention or a fragment thereof is preferably specific for mutant human protein S having a mutation of at least K196E (hereinafter also simply referred to as “mutant protein S”), ie, represented by SEQ ID NO: 1. It binds significantly to mutant protein S rather than to wild type human protein S having a specific amino acid sequence.
Since human protein S binds to C4bp in the region from 435 to 468 in the amino acid sequence of wild-type human protein S (Patent Document 4), the antibody of the present invention or a fragment thereof is not limited to free protein S, It can also bind to bound protein S in the protein S-C4bp complex.
本発明において「少なくともＫ１９６Ｅの変異を有する変異型ヒトプロテインＳ」とは、配列番号１で表されるアミノ酸配列において、１９６番目のリジン残基（Ｋ）がグルタミン酸残基（Ｅ）に置換している変異の他に、ミスセンス変異、ノンセンス変異、フレームシフト変異、スプライス部位変異およびアミノ酸欠損変異からなる群から選択される非同義変異を含むものであってもよいが、ＣＫＮＧＦＶＭＬＳＮＥ（配列番号２）のアミノ酸配列は保存されている。 In the present invention, “mutant human protein S having at least a K196E mutation” means that the 196th lysine residue (K) is replaced with a glutamic acid residue (E) in the amino acid sequence represented by SEQ ID NO: 1. In addition to the mutation, it may include a non-synonymous mutation selected from the group consisting of a missense mutation, a nonsense mutation, a frameshift mutation, a splice site mutation, and an amino acid deletion mutation, but CKNGFVMLSNE (SEQ ID NO: 2) The amino acid sequence is conserved.
本発明の抗体またはその断片は、好ましくは、モノクローナル抗体である。ＣＫＮＧＦＶＭＬＳＮＫ（配列番号３）に対するよりも、ＣＫＮＧＦＶＭＬＳＮＥ（配列番号２）に対して有意に結合するモノクローナル抗体が複数種存在する場合、各モノクローナル抗体同士を混合した組成物（いわゆるポリクローナル抗体）であってもよい。
本発明の抗体の作製方法としては、例えば、ＣＫＮＧＦＶＭＬＳＮＥ（配列番号２）からなるペプチドを免疫原（抗原）として用いてマウス、好ましくは胚中心Ｂ細胞に核内タンパク質ＧＡＮＰを高発現するトランスジェニックマウス（ＧＡＮＰ(登録商標)マウス）等の前記哺乳動物または前記鳥類に免疫し、この動物の脾臓細胞をマウスミエローマ細胞と融合して得られたハイブリドーマが産生する抗体をスクリーニングするとともに、特定の抗体を産生するハイブリドーマのクローニングを行う従来の方法等が挙げられる。具体例としての詳細は本実施例を参照。
The antibody or fragment thereof of the present invention is preferably a monoclonal antibody. When there are multiple types of monoclonal antibodies that bind significantly to CKNGGFVMLSNE (SEQ ID NO: 2) rather than to CKNGFVMLSNK (SEQ ID NO: 3), even a composition (so-called polyclonal antibody) in which the monoclonal antibodies are mixed together Good.
As a method for producing the antibody of the present invention, for example, a mouse comprising a peptide comprising CKNGGFVMLSNE (SEQ ID NO: 2) as an immunogen (antigen), preferably a transgenic mouse that highly expresses nuclear protein GANP in germinal center B cells. (GANP (registered trademark) mouse) and other mammals or birds are immunized, and antibodies produced by hybridomas obtained by fusing spleen cells of the animals with mouse myeloma cells are screened, and specific antibodies are The conventional method etc. which clone the hybridoma to produce are mentioned. See this example for details as a specific example.
本発明の抗体のような、いわゆる抗ペプチド抗体を作製するのに際し、従来の方法においては、特定のタンパク質の中から特定のアミノ酸配列を選択して免疫原（合成ペプチド）として用いるが、通常、免疫原である合成ペプチドの中にシステイン残基が含まれないように選択することが知られている。システイン残基同士は、タンパク質の三次構造（立体構造）を保持するためジスルフィド結合を形成することがあり、システイン残基を含む選択ペプチドは、タンパク質の立体構造内部に埋もれやすく、その立体障害のため、抗ペプチド抗体が選択ペプチドを介してタンパク質に結合しづらい。
ただし、合成ペプチドを免疫原とするこのような場合、そのままでは免疫原として認識されづらいため、その合成ペプチドのＮ末端またはＣ末端にシステインをあえて加え、そのシステインを介して、キーホールリンペットヘモシアニン（ＫＬＨ）等の抗原性刺激のあるキャリアタンパク質と合成ペプチドとを結合させることがある。
In producing a so-called anti-peptide antibody such as the antibody of the present invention, in a conventional method, a specific amino acid sequence is selected from a specific protein and used as an immunogen (synthetic peptide). It is known to select a synthetic peptide that is an immunogen so that it does not contain a cysteine residue. Cysteine residues may form a disulfide bond because they retain the tertiary structure (steric structure) of the protein, and select peptides containing cysteine residues are likely to be buried within the three-dimensional structure of the protein. The anti-peptide antibody is difficult to bind to the protein via the selected peptide.
However, in such a case where a synthetic peptide is used as an immunogen, it is difficult to recognize it as an immunogen as it is. Therefore, a cysteine is added to the N-terminal or C-terminal of the synthetic peptide, and keyhole limpet hemocyanin is added via the cysteine. A carrier protein having an antigenic stimulus such as (KLH) may be bound to a synthetic peptide.
本発明において使用するＣＫＮＧＦＶＭＬＳＮＥ（配列番号２）の先頭のシステイン残基（Ｃ）は、キャリアタンパク質との結合を目的として加えたものではなく、野生型ヒトプロテインＳのアミノ酸配列（配列番号１）に含まれるもの（１８６番目のアミノ酸）であり、このシステイン残基は、配列番号１に含まれる他のシステイン残基（１９９番目のアミノ酸）とジスルフィド結合を形成することが確認されている。
当業者は通常、免疫抗原としての合成ペプチド内に、ジスルフィド結合に寄与するシステイン残基を含まないように選択するが、本発明は、あえてシステイン残基を含む配列をエピトープとする抗体を作製することで、抗原として認識されやすい抗体を作製することに成功した。
The leading cysteine residue (C) of CKNGGFVMLSNE (SEQ ID NO: 2) used in the present invention is not added for the purpose of binding to a carrier protein, but is added to the amino acid sequence of wild-type human protein S (SEQ ID NO: 1). It is confirmed that this cysteine residue forms a disulfide bond with another cysteine residue (199th amino acid) included in SEQ ID NO: 1.
Although those skilled in the art usually choose not to include a cysteine residue contributing to disulfide bonds in a synthetic peptide as an immunizing antigen, the present invention dares to create an antibody having a sequence containing a cysteine residue as an epitope. As a result, we succeeded in producing an antibody that is easily recognized as an antigen.
＜免疫学的診断用組成物＞
また、本発明は、別の一態様において、本発明の抗体またはその断片を含む、免疫学的診断用組成物を提供するものであり、該組成物は、好ましくは血栓塞栓症、より好ましくは静脈血栓塞栓症、例えば、エコノミークラス症候群やロングフライト血栓症とも呼ばれる肺血栓塞栓症および深部静脈血栓症等の遺伝的素因があるか否かを判定するために使用するものである。
本発明の免疫学的診断用組成物には、本発明の抗体および／またはその断片の他に、例えば、緩衝液等を含んでもよい。
また、本発明の免疫学的診断用組成物の調製方法としては、例えば、本発明の抗体またはその断片の製造方法に準じ、本発明の抗体またはその断片が失活しなければ、従来公知の任意の方法を用いることができる。
<Composition for immunological diagnosis>
In another aspect, the present invention provides an immunological diagnostic composition comprising the antibody of the present invention or a fragment thereof, and the composition is preferably thromboembolism, more preferably It is used to determine whether or not there is a genetic predisposition such as venous thromboembolism, for example, pulmonary thromboembolism and deep vein thrombosis, also called economy class syndrome or long flight thrombosis.
The immunodiagnostic composition of the present invention may contain, for example, a buffer solution in addition to the antibody of the present invention and / or a fragment thereof.
The immunodiagnostic composition of the present invention can be prepared by, for example, a conventionally known method according to the method of producing the antibody of the present invention or a fragment thereof, unless the antibody of the present invention or a fragment thereof is inactivated. Any method can be used.
＜検出方法＞
さらに、本発明は、他の一態様において、配列番号１で示されるアミノ酸配列の１９６番目のリジン（Ｋ）がグルタミン酸（Ｅ）に変異した非同義変異を少なくとも有する変異型ヒトプロテインＳを試料から検出する方法を提供するものであって、該方法は、
前記試料を、本発明の抗体またはその断片と接触させる工程
を含み、好ましくは、
変異型ヒトプロテインＳを、従来公知の免疫比濁法（ＴＩＡ）、ラテックス凝集（ＬＰＩＡ）法、イムノクロマト法（ＩＣ）および固相免疫法からなる群から選択される少なくとも１つの免疫学的測定法により検出する工程
をさらに含むものである。
<Detection method>
Furthermore, in another aspect, the present invention provides a mutant human protein S having at least a non-synonymous mutation in which the 196th lysine (K) of the amino acid sequence represented by SEQ ID NO: 1 is mutated to glutamic acid (E). Providing a method of detecting, comprising:
Contacting the sample with an antibody of the invention or a fragment thereof, preferably,
Mutant human protein S is obtained by using at least one immunoassay selected from the group consisting of conventionally known immunoturbidimetric (TIA), latex agglutination (LPIA), immunochromatography (IC) and solid phase immunization. It further includes a step of detecting by.
本発明の検出方法における「試料」とは、プロテインＳが存在する可能性があり、かつプロテインＳの存在の有無、または含有量（濃度）の測定を行おうとする対象を指し、かかる試料としては、例えば、ヒト由来の血液、血清、血漿、唾液、汗、尿、涙、髄液、羊水、腹水等の体液、または、肝臓、心臓、脳、骨、毛髪、皮膚、爪、筋肉、神経組織等の臓器、組織もしくは細胞等の抽出液など、プロテインＳが含まれる可能性のあるものが挙げられる。
ＥＬＩＳＡの原理を利用する固相免疫法としては、抗体への標識物質の種類により、例えば、放射性同位元素を標識するＲＩＡ、ユーロピウム等の蛍光発光物質を標識する蛍光免疫測定法（ＦＩＡ）、ルミノール等の化学発光物質を標識する化学発光免疫測定法（ＣＬＩＡ）、ペルオキシダーゼ等の酵素を標識する酵素免疫測定法（ＥＩＡ）等が挙げられる。
The “sample” in the detection method of the present invention refers to an object in which protein S may be present, and the presence or absence of protein S, or the content (concentration) of which is to be measured. For example, human blood, serum, plasma, saliva, sweat, urine, tears, cerebrospinal fluid, amniotic fluid, ascites fluid, or liver, heart, brain, bone, hair, skin, nail, muscle, nerve tissue Examples include extracts that may contain protein S, such as extracts of organs, tissues, or cells.
Solid-phase immunization utilizing the principle of ELISA includes, for example, RIA for labeling radioisotopes, fluorescent immunoassay for labeling fluorescent substances such as europium (FIA), luminol, depending on the type of labeling substance for antibodies. And chemiluminescent immunoassay (CLIA) for labeling chemiluminescent substances such as enzyme immunoassay (EIA) for labeling enzymes such as peroxidase.
＜キット＞
さらにまた、本発明は、さらに他の一態様において、本発明の抗体および／またはその断片ならびに免疫学的診断用組成物を夫々含む、配列番号１で示されるアミノ酸配列の１９６番目のリジン（Ｋ）がグルタミン酸（Ｅ）に変異した非同義変異を少なくとも有する変異型ヒトプロテインＳを検出するための検出用キットおよび診断用キットを提供するものである。
これらのキットには、例えば、緩衝液、抗体への標識物質、二次抗体、シグナルを検出するための検出用試薬、またはキットの使用説明書等がさらに含まれていてもよい。
<Kit>
Furthermore, the present invention, in yet another aspect, comprises the antibody of the present invention and / or a fragment thereof and an immunological diagnostic composition, respectively, at the 196th lysine (K) of the amino acid sequence represented by SEQ ID NO: 1. ) Provides a detection kit and a diagnostic kit for detecting mutant human protein S having at least a non-synonymous mutation mutated to glutamic acid (E).
These kits may further contain, for example, a buffer, a labeling substance for the antibody, a secondary antibody, a detection reagent for detecting a signal, or instructions for using the kit.
以下、実施例によって本発明をさらに詳細に説明するが、本発明はこれらによって限定されるものではない。
［実施例１］抗体の作製
配列番号２で示されるＣＫＮＧＦＶＭＬＳＮＥのペプチドを免疫原として免疫したマウスから得られたマウス血清に含まれる抗体の希釈系列と、固相化抗原として夫々、配列番号２のペプチド、配列番号３のペプチド、ＫＬＨを結合した配列番号２のペプチドおよびＫＬＨのみとの抗体抗原反応をＥＬＩＳＡにより確認した。その結果を図１に示す。なお、二次抗体としてＨＲＰ標識抗マウスＩｇＧ（γ）抗体を、ＨＲＰの基質としてオルトフェニレンジアミンを用いた。
次に、以下の常法に従って、前記の免疫したマウスからハイブリドーマを作製し、配列番号３のＣＫＮＧＦＶＭＬＳＮＫペプチドに対するより、配列番号２のペプチドに対して有意に結合するか否かを指標に、所望のモノクローナル抗ペプチド抗体のスクリーニングおよび抗体産生細胞のクローニングを行った。最終的に本発明では３種類のモノクローナル抗体（４Ｂ１、１５Ｃ８、１６Ｅ３）を得ることに成功した。
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these.
[Example 1] Preparation of antibody A dilution series of an antibody contained in mouse serum obtained from a mouse immunized with the CKNGFVMLSNE peptide represented by SEQ ID NO: 2 and an immobilized antigen of SEQ ID NO: 2 respectively. The antibody antigen reaction with the peptide, the peptide of SEQ ID NO: 3, the peptide of SEQ ID NO: 2 conjugated with KLH, and KLH alone was confirmed by ELISA. The result is shown in FIG. An HRP-labeled anti-mouse IgG (γ) antibody was used as the secondary antibody, and orthophenylenediamine was used as the HRP substrate.
Next, according to the following conventional method, a hybridoma is prepared from the immunized mouse, and the desired whether or not it binds significantly to the peptide of SEQ ID NO: 2 rather than to the CKNGGFVMLSNK peptide of SEQ ID NO: 3 Monoclonal anti-peptide antibody screening and antibody-producing cells were cloned. Finally, in the present invention, three types of monoclonal antibodies (4B1, 15C8, 16E3) were successfully obtained.
＜常法＞
モノクローナル抗体を産生するハイブリドーマは、ケーラーとミルシュタインの方法（Koehler & Milstein, Nature, 256, 495-497 (1975)）に準じて得ることができる。すなわち、配列番号２の合成ペプチドのシステイン残基にＫＬＨを結合させたものを用いてマウスを免疫した後、このマウスの脾臓細胞をマウスミエローマ細胞と融合させる。得られたハイブリドーマをマイクロタイタープレートにまき込んで、抗体のスクリーニングと抗体産生細胞のクローニングを行い、このハイブリドーマからモノクローナル抗体を採取した。
採取したハイブリドーマは、通常の培地で継代培養することができ、また液体窒素中で半永久的に保存することができる。所望のモノクローナル抗体は、インビトロにおける培養法により大量調製することができる。インビトロ培養法は、ハイブリドーマを適当な血清培地若しくは無血清培地中で培養することにより実施でき、所望のモノクローナル抗体は培地中に産生される。この培養法によれば、比較的高純度の所望抗体を培養上清として得ることができる。
本実験においては、前記「マウス」として、ＧＡＮＰ(登録商標)マウスを使用した。
<Regular method>
A hybridoma producing a monoclonal antibody can be obtained according to the method of Koehler and Milstein (Koehler & Milstein, Nature, 256, 495-497 (1975)). That is, after immunizing a mouse with a cysteine residue of the synthetic peptide of SEQ ID NO: 2 bound to KLH, the mouse spleen cell is fused with mouse myeloma cell. The obtained hybridoma was placed in a microtiter plate, antibody screening and antibody-producing cell cloning were performed, and a monoclonal antibody was collected from this hybridoma.
The collected hybridoma can be subcultured in a normal medium and can be stored semipermanently in liquid nitrogen. Desired monoclonal antibodies can be prepared in large quantities by in vitro culture methods. The in vitro culture method can be performed by culturing the hybridoma in an appropriate serum medium or serum-free medium, and the desired monoclonal antibody is produced in the medium. According to this culture method, a desired antibody having a relatively high purity can be obtained as a culture supernatant.
In this experiment, a GANP (registered trademark) mouse was used as the “mouse”.
なお、得られたハイブリドーマ４Ｂ１、１５Ｃ８および１６Ｅ３は、２０１４年７月３１日付で、千葉県木更津市かずさ鎌足２−５−８（〒２９２−０８１８）、独立行政法人製品評価技術基盤機構 特許微生物寄託センター（ＮＰＭＤ）に、受領番号NITE AP-01906、NITE AP-01907、およびNITE AP-01908として夫々受領された。 The obtained hybridomas 4B1, 15C8 and 16E3 were dated July 31, 2014, 2-5-8, Kazusa Kamashizu, Kisarazu City, Chiba Prefecture, Japan Received at the Deposit Center (NPMD) as receipt numbers NITE AP-01906, NITE AP-01907, and NITE AP-01908, respectively.
［実施例２］プロテインＳ（42-676）と抗体とのＥＬＩＳＡ測定
固相化抗原として、配列番号１で表される野生型ヒトプロテインＳのアミノ酸配列における４２〜６７６番目の配列からなる遺伝子組み換え体のヒトプロテインＳ（０．８ｍｇ／ｍｌ）と、配列番号１で表される野生型ヒトプロテインＳのアミノ酸配列において１９６番目のリジン残基（Ｋ）がグルタミン酸残基（Ｅ）に変異した場合の４２〜６７６番目の配列からなる遺伝子組み換え体のヒトプロテインＳ（１．０ｍｇ／ｍｌ）の夫々に対し、実施例１で得られた３種のモノクローナル抗体（４Ｂ１、１５Ｃ８、１６Ｅ３）との抗体抗原反応をＥＬＩＳＡにより確認した。
[Example 2] ELISA measurement of protein S (42-676) and antibody Genetic recombination consisting of the 42-676th sequence in the amino acid sequence of wild-type human protein S represented by SEQ ID NO: 1 as a solid-phased antigen When the 196th lysine residue (K) is mutated to a glutamic acid residue (E) in the amino acid sequence of human protein S (0.8 mg / ml) and wild type human protein S represented by SEQ ID NO: 1 Antibodies against the three types of monoclonal antibodies (4B1, 15C8, 16E3) obtained in Example 1 against human recombinant protein S (1.0 mg / ml) comprising the 42nd to 676th sequences of Antigen reaction was confirmed by ELISA.
具体的な手順としては、ＰＢＳ（ナカライテスク社、リン酸緩衝生理食塩水（ｐＨ７．４）製品番号27575−31）で希釈した遺伝子組み換え体のヒトプロテインＳを５０μｌ／ウェル添加し、室温で１時間静置した。ＥＬＩＳＡのサンプルプレートとして、ＮＵＮＣイムノモジュール（フレーム付）マキシソープ（製品番号467466）を使用した。
洗浄バッファー（ＰＢＳ−０．０５％Ｔｗｅｅｎ２０）にてウェルを３回洗浄後、ブロッキングバッファー（０．５％ゼラチンＰＢＳ−０．０５％Ｔｗｅｅｎ２０）を２００μｌ／ウェル添加し、４℃で終夜(約１６時間)静置した。
洗浄バッファーにてウェルを３回洗浄後、抗体希釈液（０．０５％ゼラチンＰＢＳ−０．０５％Ｔｗｅｅｎ２０）で希釈した抗ヒトプロテインＳ−Ｋ１９６Ｅモノクローナル抗体（２μｇ／ｍｌ）を５０μｌ／ウェル添加し、室温で１時間静置した。
As a specific procedure, 50 μl / well of recombinant human protein S diluted with PBS (Nacalai Tesque, phosphate buffered saline (pH 7.4), product number 27575 − 31) was added at room temperature. And left for 1 hour. As an ELISA sample plate, NUNC immuno module (with frame) maxi soap (product number 467466) was used.
After the wells were washed three times with a washing buffer (PBS-0.05% Tween 20), blocking buffer (0.5% gelatin PBS-0.05% Tween 20) was added at 200 μl / well, and overnight at 4 ° C. (about 16 (Time).
After washing the wells three times with the washing buffer, 50 μl / well of anti-human protein S-K196E monoclonal antibody (2 μg / ml) diluted with an antibody diluent (0.05% gelatin PBS-0.05% Tween 20) was added. And left at room temperature for 1 hour.
洗浄バッファーにてウェルを３回洗浄後、抗体希釈液で２５００倍に希釈したＨＲＰ標識抗マウスＩｇＧ（γ）抗体（KPL社(Kirkegaard & Perry Laboratories, Inc.)）を５０μｌ／ウェル添加し、室温で１時間静置した。
洗浄バッファーにてウェルを３回洗浄後、ＴＭＢ（KPL社、SureBlue Reserve TMB Microwell Peroxidase Substrate）を１００μｌ／ウェル添加し、室温で１０分間静置した。反応停止液（１ＮのＨＣｌ）を１００μｌ／ウェル添加し、Thermo Labsystems Multiskan Ascent（サーモバイオアナリシスジャパン社）を使用して吸光度を４５０／６５０ｎｍで夫々の試料について３回測定した。その結果を図２と表１に示す。
After washing the wells three times with a washing buffer, 50 μl / well of HRP-labeled anti-mouse IgG (γ) antibody (KPL (Kirkegaard & Perry Laboratories, Inc.)) diluted 2500-fold with an antibody diluent was added, and room temperature was added. And left for 1 hour.
After the wells were washed three times with a washing buffer, 100 μl / well of TMB (KPL, SureBlue Reserve TMB Microwell Peroxidase Substrate) was added and allowed to stand at room temperature for 10 minutes. A stop solution (1N HCl) was added at 100 μl / well, and the absorbance was measured three times for each sample at 450/650 nm using Thermo Labsystems Multiskan Ascent (Thermo Bioanalysis Japan). The results are shown in FIG.
なお、「プロテインＳ（42-676）」は、配列番号１で表される野生型ヒトプロテインＳのアミノ酸配列における４２〜６７６番目の配列からなる遺伝子組み換え体のヒトプロテインＳを指し、さらにプロテインＳ（42-676）の１９６番目のアミノ酸がリジン残基のものを「野生型」とし、グルタミン酸残基のものを「変異型」とする。
また、プロテインＳ（42-676）は、当該分野において公知の方法に従い調製した。
“Protein S (42-676)” refers to human protein S which is a gene recombinant consisting of sequences 42 to 676 in the amino acid sequence of wild-type human protein S represented by SEQ ID NO: 1. The amino acid at position 196 of (42-676) is defined as “wild type” when the lysine residue, and “mutant” when the glutamic acid residue is present.
Protein S (42-676) was prepared according to a method known in the art.
［実施例３］プロテインＳ（117-283）と抗体とのＥＬＩＳＡ測定
実施例２において、固相化抗原として、野生型プロテインＳ（42-676）の代わりに、配列番号１で表される野生型ヒトプロテインＳのアミノ酸配列における１１７〜２８３番目の配列からなる遺伝子組み換え体の野生型ヒトプロテインＳ（９ｍｇ／ｍｌ）を、変異型プロテインＳ（42-676）の代わりに、配列番号１で表されるヒトプロテインＳのアミノ酸配列において１９６番目のリジン残基（Ｋ）がグルタミン酸残基（Ｅ）に変異した場合の１１７〜２８３番目の配列からなる遺伝子組み換え体のヒトプロテインＳ（５．３ｍｇ／ｍｌ）を用いた以外は、実施例２と同様にしてＥＬＩＳＡ測定を行った。その結果を図３と表２に示す。
[Example 3] ELISA measurement of protein S (117-283) and antibody In Example 2, instead of wild type protein S (42-676), the wild type protein represented by SEQ ID NO: 1 was used as the immobilized antigen. Recombinant wild-type human protein S (9 mg / ml) consisting of the 117th to 283rd sequences in the amino acid sequence of type human protein S is represented by SEQ ID NO: 1 instead of mutant type protein S (42-676). Human protein S (5.3 mg / 5.3 mg) comprising the sequence 117 to 283 when the 196th lysine residue (K) is mutated to the glutamic acid residue (E) in the amino acid sequence of human protein S ELISA measurement was performed in the same manner as in Example 2, except that ml) was used. The results are shown in FIG.
なお、「プロテインＳ（117-283）」は、配列番号１で表される野生型ヒトプロテインＳのアミノ酸配列における１１７〜２８３番目の配列からなる遺伝子組み換え体のヒトプロテインＳを指し、４つの連続した上皮細胞成長因子（ＥＧＦ）様ドメインに対応する。また、プロテインＳ（117-283）の１９６番目のアミノ酸がリジン残基のものを「野生型」とし、グルタミン酸残基のものを「変異型」とする。
また、プロテインＳ（117-283）は、当該分野において公知の方法に従い調製した。
“Protein S (117-283)” refers to human protein S which is a genetic recombinant consisting of the 117th to 283rd sequences in the amino acid sequence of wild-type human protein S represented by SEQ ID NO: 1. Correspond to an epidermal growth factor (EGF) -like domain. In addition, the 196th amino acid of protein S (117-283) has a lysine residue as “wild type”, and a glutamic acid residue has “mutant type”.
Protein S (117-283) was prepared according to a method known in the art.
［実施例４］ウェスタンブロット解析
通常の方法によりウェスタンブロットを行い、実施例２および実施例３の夫々において調製した遺伝子組み換え体のヒトプロテインＳと実施例１で得られたモノクローナル抗体（１５Ｃ８）との結合を検出した。
分子量マーカーとして、プレシジョンＰｌｕｓプロテイン２色スタンダード（BIO-RAD社、製品番号161-0374）を使用した。ゲルとして、スーパーセップエース１０−２０％ゲル（Wako社、製品番号198-15041）を使用した。メンブレンとして、Immun-Blot PVDFメンブレン（BIO-RAD社）を使用した。ブロッキングには５％スキムミルクを使用した。二次抗体として、ａｎｔｉ−ｍｏｕｓｅ−ＩｇＧ−ＨＲＰ（KPL社、製品番号074-1806）を使用し、一次抗体および二次抗体夫々の反応の際は室温で１時間静置した。発色には、immobilon western Chemiluminescent HRP Substrate（ミリポア社）を使用した。画像解析には、LAS-3000を使用した。得られた結果を図４に示す。
[Example 4] Western blot analysis Western blotting was performed by a conventional method, and the recombinant human protein S prepared in each of Examples 2 and 3 and the monoclonal antibody (15C8) obtained in Example 1 were used. Binding was detected.
As a molecular weight marker, Precision Plus Protein 2 color standard (BIO-RAD, product number 161-0374) was used. As the gel, Super Sep Ace 10 − 20% gel (Wako, product number 198-15041) was used. As the membrane, Immun-Blot PVDF membrane (BIO-RAD) was used. 5% skim milk was used for blocking. Anti-mouse-IgG-HRP (KPL, product number 074-1806) was used as a secondary antibody, and the primary antibody and the secondary antibody were allowed to stand at room temperature for 1 hour. For color development, immobilon western Chemiluminescent HRP Substrate (Millipore) was used. LAS-3000 was used for image analysis. The obtained results are shown in FIG.
［実施例５］ヒト血漿中プロテインＳと抗体とのＥＬＩＳＡ測定
野生型プロテインＳおよび変異型プロテインＳを夫々含むヒト血漿サンプルをウェルに添加し、室温で静置した。
コーティングバッファー（５０ｍＭ 炭酸／炭酸水素バッファー、ｐＨ９．６）を、０．１５９ｇのＮａ２ＣＯ３および０．２９３ｇのＮａＨＣＯ３を１００ｍｌの水に溶解させて調製した。このコーティングバッファーを用いて希釈した１０μｇ／ｍｌのPolyclonal Rabbit Anti-Human Protein S（DAKO社、製品番号A0348）を１００μｌ／ウェル添加し、４℃で終夜(約１６時間)静置した。洗浄バッファー（ＴＢＳ−０．１％Ｔｗｅｅｎ２０）にて３回洗浄後、ブロッキングバッファー（１％ＢＳＡ−ＴＢＳ）を２００μｌ／ウェル添加し、室温で２時間静置した。さらに洗浄バッファーにて３回洗浄後、試料希釈液（ＴＢＳ）で希釈した試料（ヒト血漿１／２０）を１００μｌ／ウェル添加し、室温で２時間静置した。洗浄バッファーにて３回洗浄後、ＨＲＰ標識抗ヒトプロテインＳ−Ｋ１９６Ｅモノクローナル抗体（１５Ｃ８）を抗体希釈液（１％ＢＳＡ−ＴＢＳ−０．１％Ｔｗｅｅｎ２０）で５μｇ／ｍｌに希釈し、１００μｌ／ウェル添加し、室温で１時間静置した。洗浄バッファーにて５回洗浄後、ＴＭＢを１００μｌ／ウェル添加し、室温で１０分間静置した。反応停止液（１Ｎ ＨＣｌ）を１００μｌ／ウェル添加し、吸光度４５０／６５０ｎｍで測定した。その結果を図５と表３に示す。なお、ＨＲＰはPeroxidase Labeling Kit NH2（Dojindo社）を用いて標識した。また、１０×ＴＢＳ（ｐＨ７．４）は６．０６ｇのＴｒｉｓおよび５．８ｇのＮａＣｌを１００ｍｌの水に溶解させて調製した。
[Example 5] ELISA measurement of protein S and antibody in human plasma Human plasma samples each containing wild-type protein S and mutant protein S were added to wells and allowed to stand at room temperature.
A coating buffer (50 mM carbonate / bicarbonate buffer, pH 9.6) was prepared by dissolving 0.159 g Na 2 CO 3 and 0.293 g NaHCO 3 in 100 ml water. 100 μl / well of 10 μg / ml Polyclonal Rabbit Anti-Human Protein S (DAKO, product No. A0348) diluted with this coating buffer was added and allowed to stand at 4 ° C. overnight (about 16 hours). After washing 3 times with a washing buffer (TBS-0.1% Tween 20), 200 μl / well of blocking buffer (1% BSA-TBS) was added and allowed to stand at room temperature for 2 hours. Further, after washing three times with a washing buffer, 100 μl / well of a sample (human plasma 1/20) diluted with a sample diluent (TBS) was added and allowed to stand at room temperature for 2 hours. After washing three times with the washing buffer, the HRP-labeled anti-human protein S-K196E monoclonal antibody (15C8) was diluted to 5 μg / ml with an antibody diluent (1% BSA-TBS-0.1% Tween 20), and 100 μl / well. Add and let stand at room temperature for 1 hour. After washing 5 times with the washing buffer, 100 μl / well of TMB was added and allowed to stand at room temperature for 10 minutes. The reaction stop solution (1N HCl) was added at 100 μl / well, and the absorbance was measured at 450/650 nm. The results are shown in FIG. HRP was labeled using Peroxidase Labeling Kit NH2 (Dojindo). 10 × TBS (pH 7.4) was prepared by dissolving 6.06 g of Tris and 5.8 g of NaCl in 100 ml of water.
＜考察＞
実施例２および３のＥＬＩＳＡの結果から、実施例１で得られた３種類のモノクローナル抗体（４Ｂ１、１５Ｃ８、１６Ｅ３）のいずれも、Ｋ１９６Ｅの変異を有するヒトプロテインＳの遺伝子組み換え体と有意に結合することがわかった。
実施例４のウェスタンブロットの結果（図４）から、実施例１で得られたモノクローナル抗体１５Ｃ８は、ヒトプロテインＳの全長（42-676）およびＥＧＦ様ドメイン（117-283）のいずれにも同じように結合することがわかった。また、同結果から、モノクローナル抗体１５Ｃ８が、Ｋ１９６Ｅ変異を有するヒトプロテインＳのＥＧＦ様ドメイン（117-283）を標的とすることも確認することができた。
図４において、ヒトプロテインＳの全長（42-676）およびＥＧＦ様ドメイン（117-283）の各バンドのシグナル強弱が、ＳＤＳ−ＰＡＧＥに供した各タンパク質のモル数に差があること（ただし、ＳＤＳ−ＰＡＧＥにロードした各タンパク質の重量は等しい）、分子量の大きいタンパク質ほどブロッティング効率が低くなる傾向にあること等が原因であることを本発明者らは突きとめている。
<Discussion>
From the ELISA results of Examples 2 and 3, any of the three monoclonal antibodies (4B1, 15C8, 16E3) obtained in Example 1 significantly bound to the recombinant protein of human protein S having the K196E mutation. I found out that
From the Western blot result of FIG. 4 (FIG. 4), the monoclonal antibody 15C8 obtained in Example 1 is the same for both the full length of human protein S (42-676) and the EGF-like domain (117-283). It was found that they combined. The results also confirmed that monoclonal antibody 15C8 targets the EGF-like domain (117-283) of human protein S having the K196E mutation.
In FIG. 4, the signal intensity of each band of the full length of human protein S (42-676) and EGF-like domain (117-283) is different in the number of moles of each protein subjected to SDS-PAGE (however, The present inventors have found that the cause is that blotting efficiency tends to be lower as the protein having a higher molecular weight is equal to the weight of each protein loaded on SDS-PAGE.
本発明に係るアミノ酸配列（配列番号２）のＮ末端のシステイン残基は、ヒトプロテインＳのアミノ酸配列（配列番号１）中のＥＧＦ様ドメイン（117-283）に含まれる１８６番目のシステイン残基に相当するものであり、このシステイン残基は、配列番号１に含まれるＥＧＦ様ドメイン中の１９９番目のシステイン残基とジスルフィド結合を形成することが確認されている。当業者は通常、免疫抗原としての合成ペプチド内に、抗原性の低下をもたらすジスルフィド結合に寄与するシステイン残基を含まないように選択するものである。しかしながら、本発明では、敢えてシステイン残基を含む配列、すなわちＥＧＦ様ドメイン中の別のシステイン残基との間でジスルフィド結合を形成するシステイン残基を含む配列をエピトープとして選択したことで、驚くべきことに、ジスルフィド結合を含むネイティブコンフォメーション変異体を認識する抗体を分離することに成功した。 The cysteine residue at the N-terminal of the amino acid sequence (SEQ ID NO: 2) according to the present invention is the 186th cysteine residue contained in the EGF-like domain (117-283) in the amino acid sequence of human protein S (SEQ ID NO: 1). It has been confirmed that this cysteine residue forms a disulfide bond with the 199th cysteine residue in the EGF-like domain contained in SEQ ID NO: 1. Those skilled in the art usually choose not to include cysteine residues contributing to disulfide bonds that lead to reduced antigenicity in synthetic peptides as immunizing antigens. However, in the present invention, it is surprising that a sequence containing a cysteine residue, that is, a sequence containing a cysteine residue that forms a disulfide bond with another cysteine residue in the EGF-like domain was selected as an epitope. In particular, we have succeeded in isolating antibodies that recognize native conformational mutants containing disulfide bonds.
また、実施例５のヒト血漿中のプロテインＳのＥＬＩＳＡの結果（表３、図５）から、実施例１で得られたモノクローナル抗体１５Ｃ８が、ヒト血漿中においてＫ１９６Ｅの変異を有するヒトプロテインＳと有意に結合することがわかった。したがって、本発明は、血液等の生体試料中であってもヒトプロテインＳを検出することが可能であることから、血液検査等の実用に耐え得ることが示された。
さらに、プロテインＳの変異を検出する従来法（特許文献７および非特許文献２）は、試料中の総プロテインＳのタンパク質量および試料中の総プロテインＳの活性値を別々に測定し、比活性を求めること等を特徴とするが、本発明においてはプロテインＳの変異を検出するためにプロテインＳの活性値の測定は不要であることから、簡便にプロテインＳの変異を検出することが可能である。
Further, from the results of ELISA for protein S in human plasma of Example 5 (Table 3, FIG. 5), the monoclonal antibody 15C8 obtained in Example 1 was found to be human protein S having a K196E mutation in human plasma. It was found to bind significantly. Therefore, the present invention can detect human protein S even in a biological sample such as blood, and thus has been shown to be able to withstand practical use such as blood tests.
Furthermore, the conventional methods for detecting protein S mutations (Patent Document 7 and Non-Patent Document 2) separately measure the protein amount of total protein S in a sample and the activity value of total protein S in the sample, and determine the specific activity. However, in the present invention, it is not necessary to measure the protein S activity value in order to detect the protein S mutation, so that it is possible to easily detect the protein S mutation. is there.
本発明は、血栓症患者、例えば静脈血栓塞栓症患者の治療方針に遺伝性の素因情報を提供することができる。それによって、血栓症等の疾患の予防、診断および治療等に役立てることができる。 The present invention can provide hereditary predisposition information to treatment strategies for thrombotic patients, for example, venous thromboembolism patients. Thereby, it can be used for prevention, diagnosis and treatment of diseases such as thrombosis.
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| US5028527A (en) * | 1988-02-22 | 1991-07-02 | Applied Bio Technology | Monoclonal antibodies against activated ras proteins with amino acid mutations at position 13 of the protein |
| DE102005026163A1 (en) * | 2004-08-24 | 2006-03-02 | Dade Behring Marburg Gmbh | Antibodies directed against the Marburg I polymorphism of Factor VII activating protease (FSAP), their production and use |
| DK2622348T3 (en) * | 2010-09-30 | 2015-12-14 | Deutsches Krebsforsch | Means and methods for diagnosing cancer using an antibody which specifically binds to braf v600e |
| JP5839256B2 (en) * | 2011-03-14 | 2016-01-06 | 学校法人九州文化学園 | Reagent used in detection method of total protein S abnormality |
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