JP5280916B2 - Anti-rat osteocalcin monoclonal antibody - Google Patents
Anti-rat osteocalcin monoclonal antibody Download PDFInfo
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- JP5280916B2 JP5280916B2 JP2009083899A JP2009083899A JP5280916B2 JP 5280916 B2 JP5280916 B2 JP 5280916B2 JP 2009083899 A JP2009083899 A JP 2009083899A JP 2009083899 A JP2009083899 A JP 2009083899A JP 5280916 B2 JP5280916 B2 JP 5280916B2
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- osteocalcin
- monoclonal antibody
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Description
本発明は、ラットオステオカルシンに対して特異性を有する抗オステオカルシンモノクローナル抗体及びその使用方法に関する。 The present invention relates to an anti-osteocalcin monoclonal antibody having specificity for rat osteocalcin and a method for using the same.
オステオカルシン（以下ＯＣと略す）はビタミンＫ依存性のＧｌａ残基を有するカルシウム結合タンパク質であり、骨芽細胞により合成される。一般に骨の代謝が亢進している骨疾患において血中レベルの上昇が認められ、骨代謝の生化学的な指標となるといわれている。近年、閉経後骨粗鬆症、ミエローマ、ベージェット病、慢性腎不全、副甲状腺機能亢進症等の診断に有用な指標であることが示されてきた。この目的のため、ポリクローナル抗体を用いたラジオイムノアッセイ法（例えば非特許文献１）が開発され、ヒト血中のＯＣ濃度を測定することが可能となった。また、モノクローナル抗体を用いるエンザイムイムアッセイ方法も本発明者らにより先に提案されている（例えば特許文献１）。 Osteocalcin (hereinafter abbreviated as OC) is a calcium-binding protein having a vitamin K-dependent Gla residue and is synthesized by osteoblasts. In general, an increase in blood level is observed in a bone disease in which bone metabolism is promoted, and it is said to be a biochemical index of bone metabolism. In recent years, it has been shown to be a useful index for diagnosis of postmenopausal osteoporosis, myeloma, Paget disease, chronic renal failure, hyperparathyroidism, and the like. For this purpose, a radioimmunoassay method using a polyclonal antibody (for example, Non-Patent Document 1) has been developed, and it has become possible to measure the OC concentration in human blood. In addition, an enzyme enzyme assay method using a monoclonal antibody has been previously proposed by the present inventors (for example, Patent Document 1).
骨芽細胞は、骨髄細胞中に存在する間葉系幹細胞から分化する。骨芽細胞の分化過程をｉｎ ｖｉｔｒｏで再現し、モニタリングすることは、前記疾病の病態モデルの解明ためにも有用であり、病態モデルとしてラットやマウスが多く用いられている。 Osteoblasts differentiate from mesenchymal stem cells present in bone marrow cells. Reproducing and monitoring the differentiation process of osteoblasts in vitro is also useful for elucidating the pathological model of the disease, and rats and mice are often used as the pathological model.
しかしながら動物細胞の培養時にはウシ血清が通常使用されているため、骨髄細胞の分化マーカーであるＯＣ測定時においては、ウシ血清中に存在するウシＯＣの存在が問題となる。
そこで、操作が簡便で、かつ測定精度、特異性、再現性に優れたＯＣ分別測定手段の開発が望まれていた。
However, since bovine serum is usually used when culturing animal cells, the presence of bovine OC present in bovine serum becomes a problem when measuring OC, which is a differentiation marker for bone marrow cells.
Thus, it has been desired to develop an OC fraction measurement means that is simple in operation and excellent in measurement accuracy, specificity, and reproducibility.
本発明者らは鋭意努力を重ね、従来入手されていなかったラット及び／又はマウスＯＣを特異的に認識する抗ＯＣモノクローナル抗体の創成に成功し、当該モノクローナル抗体を使用することにより、従来の課題が解決されること、当該モノクローナル抗体を使用すれば、培養細胞中のラット及び／又はマウスＯＣのモニタリングにも有用であることを見出し本発明を完成させた。 The present inventors have made extensive efforts and succeeded in creating an anti-OC monoclonal antibody that specifically recognizes rat and / or mouse OC, which has not been obtained in the past. Thus, the present inventors have found that the use of the monoclonal antibody is useful for monitoring rat and / or mouse OC in cultured cells.
本発明の第１の発明は、配列表の配列番号１で表されるラットオステオカルシンのＣ末端部位と反応し、配列表の配列番号２で表されるウシオステオカルシンのＣ末端部位と反応しないことを特徴とする抗オステオカルシンモノクローナル抗体に関する。本発明の第１の発明は、抗オステオカルシンモノクローナル抗体が、ハイブリドーマＦＥＲＭ Ｐ−２１４９６より産生され得るモノクローナル抗体ＲａｔＯＣ−Ｃ ９−１２Ｈが含まれる。 1st invention of this invention reacts with the C terminal part of the rat osteocalcin represented by sequence number 1 of a sequence table, and does not react with the C terminal part of the bovine osteocalcin represented by sequence number 2 of a sequence table. It relates to a characteristic anti-osteocalcin monoclonal antibody. The first invention of the present invention includes the monoclonal antibody RatOC-C 9-12H in which the anti-osteocalcin monoclonal antibody can be produced from the hybridoma FERM P-21396.
本発明の第２の発明は、被験試料中のオステオカルシンを測定する試薬において、本発明の第１の発明の抗オステオカルシンモノクローナル抗体を含むオステオカルシン測定試薬に関する。本発明の第２の発明は、さらに下記（Ａ）〜（Ｃ）より選択される少なくとも１つのモノクローナル抗体を含む請求項３記載のオステオカルシン測定試薬が含まれる；
（Ａ）γ−カルボキシグルタミン酸残基を有するオステオカルシンと反応し、γ−カルボキシグルタミン酸残基を脱炭酸してグルタミン酸残基としたオステオカルシンとは反応しない抗オステオカルシンモノクローナル抗体；
（Ｂ）オステオカルシンの中央部位と反応する抗オステオカルシンモノクローナル抗体；
（Ｃ）オステオカルシンのＮ末端部位と反応する抗オステオカルシンモノクローナル抗体。
The second invention of the present invention relates to a reagent for measuring osteocalcin comprising the anti-osteocalcin monoclonal antibody of the first invention of the present invention in a reagent for measuring osteocalcin in a test sample. The second invention of the present invention includes the osteocalcin measuring reagent according to claim 3, further comprising at least one monoclonal antibody selected from the following (A) to (C);
(A) an anti-osteocalcin monoclonal antibody that reacts with osteocalcin having a γ-carboxyglutamic acid residue and does not react with osteocalcin obtained by decarboxylating the γ-carboxyglutamic acid residue into a glutamic acid residue;
(B) an anti-osteocalcin monoclonal antibody that reacts with the central site of osteocalcin;
(C) An anti-osteocalcin monoclonal antibody that reacts with the N-terminal site of osteocalcin.
本発明の第３の発明は、本発明の第１の発明の抗オステオカルシンモノクローナル抗体又は本発明の第２の発明のオステオカルシン測定試薬を使用する被検試料中のオステオカルシンの測定方法に関する。本発明の第３の発明は、ウシ血清を含有する被検試料中のラットオステオカルシンを測定するオステオカルシンの測定方法、当該被験試料が、生体由来試料又は培養細胞由来試料から選ばれる測定方法を含む。 The third invention of the present invention relates to a method for measuring osteocalcin in a test sample using the anti-osteocalcin monoclonal antibody of the first invention of the present invention or the osteocalcin measuring reagent of the second invention of the present invention. The third invention of the present invention includes a method for measuring osteocalcin for measuring rat osteocalcin in a test sample containing bovine serum, and a method for measuring the test sample selected from a biological sample or a cultured cell-derived sample.
本発明の第４の発明は、本発明の第１の発明の抗オステオカルシンモノクローナル抗体を産生する、寄託番号ＦＥＲＭ Ｐ−２１４９６で表されるハイブリドーマ細胞に関する。 The fourth invention of the present invention relates to a hybridoma cell represented by the deposit number FERM P-21396, which produces the anti-osteocalcin monoclonal antibody of the first invention of the present invention.
本発明により、操作が簡便で、測定精度が高く、特異性、再現性にも優れた抗ＯＣモノクローナル抗体が安定して提供される。
本発明の試薬は、サンプル中のＯＣの絶対量の変化を調べることができ、マウスやラット、それらの培養細胞を使用する骨代謝のモニタリング手法として用いることができる。さらに、本発明の抗体は、ウシのＯＣを認識しないため、ウシ血清が混在する可能性のあるサンプルであっても、特異的かつ正確にオステオカルシンを測定することができ、ことに顕著な効果を有する。
The present invention stably provides an anti-OC monoclonal antibody that is easy to operate, has high measurement accuracy, and is excellent in specificity and reproducibility.
The reagent of the present invention can examine changes in the absolute amount of OC in a sample, and can be used as a method for monitoring bone metabolism using mice, rats, or cultured cells thereof. Furthermore, since the antibody of the present invention does not recognize bovine OC, it can measure osteocalcin specifically and accurately even in samples that may contain bovine serum, and has a remarkable effect. Have.
本発明で「オステオカルシン」（ＯＣ）とはビタミンＫ依存性のＧｌａ残基を有するカルシウム結合タンパク質であって、γ−カルボキシグルタミン酸（Ｇｌａ）残基を有する天然のＧｌａ型ＯＣ、γ−カルボキシグルタミン酸を脱炭酸してグルタミン酸（Ｇｌｕ）残基としたＧｌｕ型ＯＣが含まれる。また、ＯＣの断片、特に血清中に含まれるＮ末端が欠損したＯＣの断片も含まれる。 In the present invention, “osteocalcin” (OC) is a calcium binding protein having a vitamin K-dependent Gla residue, and is a natural Gla-type OC having γ-carboxyglutamic acid (Gla) residue, γ-carboxyglutamic acid. A Glu-type OC that is decarboxylated to a glutamic acid (Glu) residue is included. Also included are OC fragments, particularly OC fragments lacking the N-terminus contained in serum.
本発明で「モノクローナル抗体」とは、単一クローンの抗体生産細胞が分泌する抗体を意味し、単クローン（性）抗体ともいう。特定の抗原決定基を認識する抗体であり、アミノ酸配列の一次構造が均一である。本発明のモノクローナル抗体は、細胞融合法により調製されるハイブリドーマが産生する抗体に加えて、抗体産生細胞のｍＲＮＡ等を用いて遺伝子工学的に作製された抗体も含まれる。 In the present invention, the “monoclonal antibody” means an antibody secreted by a single clone antibody-producing cell, and is also referred to as a monoclonal (sex) antibody. It is an antibody that recognizes a specific antigenic determinant and has a uniform primary structure of amino acid sequence. The monoclonal antibody of the present invention includes antibodies produced by genetic engineering using mRNA or the like of antibody-producing cells, in addition to antibodies produced by hybridomas prepared by a cell fusion method.
本発明で、「配列表の配列番号１で表されるラットＯＣのＣ末端部位と反応し、配列表の配列番号２で表されるウシＯＣのＣ末端部位と反応しない」とは、本発明のモノクローナル抗体が、前記配列表の配列番号２で表されるウシＯＣのＣ末端部位と免疫反応を強く起こさないことを意味する。特に限定はされないが、例えば、本発明のモノクローナル抗体を用いて、配列表の配列番号２で表される配列を含むＯＣを測定した場合、その測定値は、当該ＯＣと同濃度の配列表の配列番号１で表される配列を含むＯＣを測定した場合の測定値に比べて極めて低値、又は実質的に測定値として意味の無い数値であり、１％以下、好ましくは０．１％以下、特に好ましくは０．０１％以下である。最も好ましい態様において、配列表の配列番号２で表される配列を含むＯＣの測定値は検出限界以下である。 In the present invention, “reacts with the C-terminal site of rat OC represented by SEQ ID NO: 1 in the sequence listing and does not react with the C-terminal site of bovine OC represented by SEQ ID NO: 2 of the sequence listing”. This means that the monoclonal antibody does not cause an immune reaction strongly with the C-terminal part of bovine OC represented by SEQ ID NO: 2 in the sequence listing. Although not particularly limited, for example, when the OC containing the sequence represented by SEQ ID NO: 2 in the Sequence Listing is measured using the monoclonal antibody of the present invention, the measured value is the same as that in the Sequence Listing at the same concentration as the OC. Compared to the measured value when the OC containing the sequence represented by SEQ ID NO: 1 is measured, it is a value that is extremely low or substantially meaningless as a measured value, and is 1% or less, preferably 0.1% or less Particularly preferably, it is 0.01% or less. In the most preferred embodiment, the measured value of OC containing the sequence represented by SEQ ID NO: 2 in the sequence listing is below the detection limit.
（１）本発明の抗ＯＣモノクローナル抗体
すなわち本発明を概説すれば、本発明は被験試料中のＯＣを測定する試薬において、配列表の配列番号１で表されるラットＯＣのＣ末端部位と反応し、配列表の配列番号２で表されるウシＯＣのＣ末端部位と反応しないモノクローナル抗体に関するものである。すなわち、本発明のモノクローナル抗体は、ラットＯＣと反応し、ウシＯＣとは反応しない抗体である。本発明の抗体はＯＣのＣ末端ペプチドを認識するため、Ｎ末端を欠損するＯＣであっても測定することができ、この点でも有用である。
(1) Anti-OC monoclonal antibody of the present invention In summary, the present invention is a reagent for measuring OC in a test sample and reacts with the C-terminal site of rat OC represented by SEQ ID NO: 1 in the sequence listing. In addition, the present invention relates to a monoclonal antibody that does not react with the C-terminal site of bovine OC represented by SEQ ID NO: 2 in the sequence listing. That is, the monoclonal antibody of the present invention is an antibody that reacts with rat OC but does not react with bovine OC. Since the antibody of the present invention recognizes the C-terminal peptide of OC, even an OC lacking the N-terminus can be measured, which is also useful in this respect.
本発明の抗体は、ウシ血清を含む試料であっても、ウシ血清に混在するウシＯＣの影響を受けることなくラットＯＣを特異的に測定することができる。したがって、ウシの血液、血清、血漿などのウシＯＣを含む培地で培養したラット培養細胞の抽出物や培養上清中のラットＯＣを特異的かつ高感度に測定することができる。例えば、間葉系幹細胞などの前駆細胞から骨芽細胞への分化過程において、ＯＣをマーカーとしてモニタリングする際に、ウシ血清を含む培地を使用しても細胞の分化を正確かつ高感度にモニターすることができる。 The antibody of the present invention can specifically measure rat OC without being affected by bovine OC mixed with bovine serum even in a sample containing bovine serum. Accordingly, the extract of cultured rat cells cultured in a medium containing bovine OC such as bovine blood, serum, and plasma, and rat OC in the culture supernatant can be specifically and highly sensitively measured. For example, in the process of differentiation from progenitor cells such as mesenchymal stem cells to osteoblasts, cell differentiation is accurately and highly sensitively monitored using a medium containing bovine serum when monitoring using OC as a marker. be able to.
本発明の別の態様として、本発明の抗体は配列表の配列番号１で表されるラットＯＣのＣ末端部位及び配列番号４で表わされるマウスＯＣのＣ末端部位と反応し、配列表の配列番号２で表されるウシＯＣのＣ末端部位と反応しないモノクローナル抗体である。したがって、本発明の抗体はウシ血清を含む試料であっても、ウシ血清に混在するウシＯＣの影響を受けることなくマウスＯＣを特異的に測定することができる。すなわち、ウシの血液、血清、血漿などのウシＯＣを含む培地で培養したマウス培養細胞の抽出物や培養上清中のマウスＯＣを特異的かつ高感度に測定することができる。 In another embodiment of the present invention, the antibody of the present invention reacts with the C-terminal site of rat OC represented by SEQ ID NO: 1 in the sequence listing and the C-terminal site of mouse OC represented by SEQ ID NO: 4, and the sequences of the sequence listing It is a monoclonal antibody that does not react with the C-terminal part of bovine OC represented by No. 2. Therefore, even if the antibody of the present invention is a sample containing bovine serum, mouse OC can be specifically measured without being affected by bovine OC mixed in bovine serum. That is, the extract of cultured mouse cells cultured in a medium containing bovine OC such as bovine blood, serum, and plasma, and mouse OC in the culture supernatant can be specifically and highly sensitively measured.
また、本発明の抗体にペプシン、パパイン等のタンパク質分解酵素を作用させ、抗体のＦｃ部分を除去して得られる、Ｆ（ａｂ’）２、Ｆａｂ’、Ｆａｂ等のフラグメントも本発明で使用する抗体に含まれる。 In addition, fragments of F (ab ′) 2 , Fab ′, Fab and the like obtained by allowing a protease such as pepsin and papain to act on the antibody of the present invention to remove the Fc part of the antibody are also used in the present invention. Included in antibodies.
さらに、得られたモノクローナル抗体を基に、遺伝子工学的に製造される組換え抗体や、定常領域を他の抗体の定常領域に置換したキメラ抗体であっても良い。このような抗体は、二重特異性抗体（二価抗体）、ｓｃＦｖ、Ｆａｂ３、Ｄｉａｂｏｄｙ、Ｔｒｉａｂｏｄｙ、Ｔｅｔｒａｂｏｄｙ、Ｍｉｎｉｂｏｄｙ、Ｂｉｓ−ｓｃＦｖ、（ｓｃＦｖ）２−Ｆｃ、ｉｎｔａｃｔ−ＩｇＧが例示され、Ｈｏｌｌｉｇｅｒら、Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ、第２３巻、第９号、ｐ．１１２６−３６（２００５）に詳述される。 Furthermore, it may be a recombinant antibody produced by genetic engineering based on the obtained monoclonal antibody, or a chimeric antibody in which the constant region is replaced with the constant region of another antibody. Examples of such antibodies include bispecific antibodies (bivalent antibodies), scFv, Fab 3 , Diabody, Triabody, Tetabobody, Minibody, Bis-scFv, (scFv) 2 -Fc, intact-IgG, and Holliger et al. , Nature Biotechnology, Vol. 23, No. 9, p. 1126-36 (2005).
本発明のモノクローナル抗体は、いわゆる細胞融合法によって作製されたハイブリドーマを使用して製造される。前記ハイブリドーマは、抗体産生細胞の集団と骨髄腫細胞と融合ハイブリドーマを形成させ、該ハイブリドーマをクローン化し、配列番号１で表わされる配列を含むＯＣを認識する抗体を産生するクローンを取捨選択し、さらに本発明の目的に好適なクローンを選別することによって樹立される。
抗体産生細胞には、配列番号１で表わされる配列を含むＯＣ又はその一部によって免疫された動物の脾細胞、リンパ節細胞、Ｂリンパ球等が利用できる。免疫させる動物としては、マウス、ウマ、ヤギ、ウサギ等が挙げられる。免疫原として用いる前記ＯＣ又はその一部を天然から得る場合は、血清画分あるいは血漿画分から塩析、透析やアフィニティークロマトグラフィー、イオン交換クロマトグラフィー、疎水クロマトグラフィー等各種クロマトグラフィーを用いて精製することが可能である。また、ＯＣを産生する骨髄細胞等、適当な細胞を培養し、その培養上清から精製することも可能である。また、人工的にＯＣ又はその一部のポリペプチド又はペプチドを合成して調製することができる。かくして得られたＯＣ又はその一部を、例えばＫＬＨ（Ｋｅｙｈｏｌｅ Ｌｉｍｐｅｔ Ｈｅｍｏｃｙａｎｉｎ）に代表されるキャリアタンパクと結合後、又はＰＶＰ（ポリビニルピロリドン）と混合後、フロイントのアジュバントと混合し、動物の免疫に使用する。又はＯＣ又はその一部を直接フロイントのアジュバントと混合し、動物の免疫に使用する。免疫は動物の皮下、筋肉内あるいは腹腔内に１回に２０〜２００μｇの抗原−アジュバント混合物を、２〜３週間に１回、３〜７回投与することにより行われる。最終免疫より約３〜５日後、免疫動物から抗体産生細胞を分取する。
The monoclonal antibody of the present invention is produced using a hybridoma produced by a so-called cell fusion method. The hybridoma forms a fusion hybridoma with a population of antibody-producing cells and myeloma cells, clones the hybridoma, selects clones that produce an antibody recognizing OC containing the sequence represented by SEQ ID NO: 1, and It is established by selecting clones suitable for the purposes of the present invention.
As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like of animals immunized with OC containing the sequence represented by SEQ ID NO: 1 or a part thereof can be used. Examples of animals to be immunized include mice, horses, goats, rabbits and the like. When the OC or a part thereof used as an immunogen is obtained from nature, it is purified from the serum fraction or plasma fraction using various chromatographic methods such as salting out, dialysis, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, etc. It is possible. It is also possible to culture appropriate cells such as bone marrow cells that produce OC and to purify them from the culture supernatant. Alternatively, it can be prepared by artificially synthesizing OC or a partial polypeptide or peptide thereof. The thus-obtained OC or a part thereof is combined with a carrier protein typified by, for example, KLH (Keyhole Limeto Hemocyanin) or mixed with PVP (polyvinylpyrrolidone) and then mixed with Freund's adjuvant for use in animal immunization. To do. Alternatively, OC or a part thereof is directly mixed with Freund's adjuvant and used for immunization of animals. Immunization is carried out by administering 20 to 200 μg of an antigen-adjuvant mixture once subcutaneously, intramuscularly or intraperitoneally 3 to 7 times once every 2-3 weeks. About 3 to 5 days after the final immunization, antibody-producing cells are collected from the immunized animal.
骨髄腫細胞としてはマウス、ラット、ヒト等由来のものが使用される。細胞融合は例えばＧ．ケラー（Ｇ．Ｋｅｈｌｅｒ）「ネーチャー（Ｎａｔｕｒｅ）第２５６巻、第４９５頁（１９７５）」に記載の方法、又はこれに準ずる方法により行われる。この際、３０〜５０％ポリエチレングリコール（分子量１０００〜６０００）を用い、抗体産生細胞と骨髄腫細胞とを３０〜４０℃の温度下、約１〜３分間程度反応させる。細胞融合により得られたハイブリドーマはスクリーニングに付される。例えば、抗原としてラットＯＣを用いた酵素抗体法（ＥＩＡ）等により前記配列表の配列番号１で表される配列を含むＯＣと反応する抗体を生産するハイブリドーマのスクリーニングが行われる。さらに、配列表の配列番号２で表される配列を含むＯＣと反応しない抗体を生産するハイブリドーマのスクリーニングが行われる。得られた抗体産生ハイブリドーマは、例えば限界希釈法によりクローン化される。得られたクローンは、次いで目的とする高感度、高特異性のモノクローナル抗体を産生するクローンを選択するため、例えば酵素抗体法等によるスクリーニングに供される。
こうして選ばれたクローンは、例えばあらかじめプリスタン（２，６，１０，１４，−テトラメチルペンタデカン）やＦＩＡ（Ｆｒｅｕｎｄ ｉｎｃｏｍｐｌｅｔｅ ａｄｊｕｖａｎｔ）を投与したＢＡＬＢ／ｃマウスの腹腔内へ移植し、１０〜１４日後にモノクローナル抗体を高濃度に含む腹水を採取する。この腹水からのモノクローナル抗体の回収は、イムノグロブリンの精製法として従来既知の硫安分画法、ポリエチレングリコール分画法、イオン交換クロマトグラフ法、ゲルクロマトグラフ法、アフイニテイークロマトグラフ法等を応用することで達成される。
As myeloma cells, those derived from mice, rats, humans and the like are used. Cell fusion is described, for example, in G. It is carried out by the method described in G. Kehler “Nature, Vol. 256, p. 495 (1975)” or a method analogous thereto. At this time, 30 to 50% polyethylene glycol (molecular weight 1000 to 6000) is used, and the antibody-producing cells and myeloma cells are reacted at a temperature of 30 to 40 ° C. for about 1 to 3 minutes. The hybridoma obtained by cell fusion is subjected to screening. For example, a hybridoma that produces an antibody that reacts with OC containing the sequence represented by SEQ ID NO: 1 in the sequence listing is performed by an enzyme antibody method (EIA) using rat OC as an antigen. Furthermore, a hybridoma that produces an antibody that does not react with OC containing the sequence represented by SEQ ID NO: 2 in the sequence listing is screened. The obtained antibody-producing hybridoma is cloned by, for example, a limiting dilution method. The obtained clone is then subjected to screening by, for example, an enzyme antibody method in order to select a clone that produces a target highly sensitive and highly specific monoclonal antibody.
The clone thus selected is transplanted, for example, into the abdominal cavity of BALB / c mice to which pristane (2,6,10,14, -tetramethylpentadecane) or FIA (Freund incomplete adjuvant) has been administered. Ascites fluid with high concentration of monoclonal antibody is collected. For the recovery of monoclonal antibodies from this ascites, the conventionally known ammonium sulfate fractionation method, polyethylene glycol fractionation method, ion exchange chromatography method, gel chromatography method, affinity chromatography method, etc. can be applied as immunoglobulin purification methods. To be achieved.
本発明の一態様として、配列表の配列番号１で表されるラットＯＣのＣ末端部位と反応し、配列表の配列番号２で表されるウシＯＣのＣ末端部位と反応しないことを特徴とする抗ＯＣモノクローナル抗体は、ハイブリドーマ細胞ＲａｔＯＣ−Ｃ ９−１２Ｈにより産生されるモノクローナル抗体ＲａｔＯＣ−Ｃ ９−１２Ｈである。ハイブリドーマ細胞ＲａｔＯＣ−Ｃ ９−１２Ｈは、平成２０年１月３１日（原寄託日）より独立行政法人産業技術総合研究所特許生物寄託センター（茨城県つくば市東１丁目１番地１中央第６（郵便番号３０５−８５６６））にＦＥＲＭ Ｐ−２１４９６として寄託されている。このモノクローナル抗体ＲａｔＯＣ−Ｃ ９−１２Ｈは、配列番号４で表わされるマウスＯＣのＣ末端部位とも反応する。
この抗体を含有するＯＣ測定試薬は、配列表の配列番号１で表される配列を含むラットＯＣ、さらには配列番号４で表わされる配列を含むマウスＯＣと反応し、配列表の配列番号２で表される配列を含むウシＯＣと反応しないことから、広くウシＯＣとマウス、ラットＯＣの分別、測定に適用することができる。
As one aspect of the present invention, it reacts with the C-terminal site of rat OC represented by SEQ ID NO: 1 in the sequence listing and does not react with the C-terminal site of bovine OC represented by SEQ ID NO: 2 of the sequence listing. The anti-OC monoclonal antibody is the monoclonal antibody RatOC-C 9-12H produced by the hybridoma cell RatOC-C 9-12H. The hybridoma cell RatOC-C 9-12H is available from the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (January 1, 1st East, 1-chome Tsukuba, Ibaraki, Japan) on January 31, 2008 (original deposit date). No. 305-866)) is deposited as FERM P-21396. This monoclonal antibody RatOC-C 9-12H also reacts with the C-terminal site of mouse OC represented by SEQ ID NO: 4.
The reagent for measuring OC containing this antibody reacts with rat OC containing the sequence represented by SEQ ID NO: 1 in the sequence listing, and further with mouse OC containing the sequence represented by SEQ ID NO: 4, Since it does not react with bovine OC containing the represented sequence, it can be widely applied to the separation and measurement of bovine OC and mouse and rat OC.
本発明は前記の抗体を産生する細胞、すなわち本発明のモノクローナル抗体ＲａｔＯＣ−Ｃ ９−１２Ｈを産生するハイブリドーマ細胞ＲａｔＯＣ−Ｃ ９−１２Ｈを包含する。 The present invention includes cells that produce the above-described antibodies, that is, hybridoma cells RatOC-C 9-12H that produce the monoclonal antibody RatOC-C 9-12H of the present invention.
（２）本発明のＯＣ測定試薬
本発明のＯＣ測定試薬は、被験試料中のＯＣを測定する試薬において、前記（１）記載の抗ＯＣモノクローナル抗体を含むことを特徴とする。
本発明の一態様として、本発明はさらに下記より選択される少なくとも１つのモノクローナル抗体を含むＯＣ測定試薬である；
（Ａ）γ−カルボキシグルタミン酸残基を有するＯＣ（Ｇｌａ型：活性型ＯＣともいう）と反応し、γ−カルボキシグルタミン酸残基を脱炭酸してグルタミン酸残基としたＯＣ（Ｇｌｕ型：不活性型ＯＣともいう）とは反応しない抗ＯＣモノクローナル抗体；
（Ｂ）ＯＣの中央部位と反応する抗ＯＣモノクローナル抗体；
（Ｃ）ＯＣのＮ末端部位と反応する抗ＯＣモノクローナル抗体。
(2) OC Measuring Reagent of the Present Invention The OC measuring reagent of the present invention is a reagent for measuring OC in a test sample, characterized in that it contains the anti-OC monoclonal antibody described in (1) above.
As one aspect of the present invention, the present invention is an OC measurement reagent further comprising at least one monoclonal antibody selected from the following:
(A) OC (Glu type: inactive type) which reacts with OC having a γ-carboxyglutamic acid residue (Gla type: also referred to as active OC) and decarboxylates the γ-carboxyglutamic acid residue to form a glutamic acid residue Anti-OC monoclonal antibody that does not react with OC);
(B) an anti-OC monoclonal antibody that reacts with the central site of OC;
(C) An anti-OC monoclonal antibody that reacts with the N-terminal site of OC.
前記（１）の抗ＯＣモノクローナル抗体を、前記（Ａ）〜（Ｃ）の抗ＯＣモノクローナル抗体と組み合わせ、それぞれ多数の組み合わせをさらにスクリーニングし、高感度で再現性よくＯＣを測定できるモノクローナル抗体の組み合わせを決定することにより、本発明のＯＣ測定試薬が調製される。 The anti-OC monoclonal antibody of (1) above is combined with the anti-OC monoclonal antibodies of (A) to (C) above, and a combination of monoclonal antibodies capable of measuring OC with high sensitivity and high reproducibility by further screening a large number of combinations. Is determined to prepare the OC measurement reagent of the present invention.
本発明の測定試薬は、配列表の配列番号１で表されるラットＯＣのＣ末端部位と反応し、配列表の配列番号２で表されるウシＯＣのＣ末端部位と反応しないため、配列番号２で表される配列を含むウシＯＣが混入する被検試料、例えばウシ又はウシ胎仔由来の血液、血清、血漿を含む試料であっても、ウシＯＣの影響を受けることなく、配列表の配列番号１で表される配列を含むＯＣ、例えばラットＯＣを特異的かつ高感度に測定することが可能である。さらに、本発明の測定試薬は、配列番号４で表わされるマウスＯＣのＣ末端部位を含むマウスＯＣを測定することが可能である。 The measurement reagent of the present invention reacts with the C-terminal site of rat OC represented by SEQ ID NO: 1 in the sequence listing and does not react with the C-terminal site of bovine OC represented by SEQ ID NO: 2 in the sequence listing. Even in the case of a test sample contaminated with bovine OC containing the sequence represented by 2, for example, a sample containing blood, serum, or plasma derived from bovine or fetal bovine, the sequence of the sequence listing is not affected by bovine OC. It is possible to measure the OC containing the sequence represented by No. 1, for example, rat OC specifically and with high sensitivity. Furthermore, the measurement reagent of the present invention can measure mouse OC containing the C-terminal site of mouse OC represented by SEQ ID NO: 4.
さらに、（Ａ）Ｇｌａ型ＯＣと反応し、Ｇｌｕ型ＯＣとは反応しない抗ＯＣモノクローナル抗体を含む本発明の測定試薬は、Ｇｌａ型ＯＣと反応しＧｌｕ型ＯＣと反応しない。ＯＣは骨芽細胞で合成され、細胞内でビタミンＫ依存性カルボキシラーゼによりＧｌａ型ＯＣとなる。このＧｌａ型ＯＣが骨のヒドロキシアパタイトと結合し、骨基質中に蓄積して骨形成が進行するため、Ｇｌａ型ＯＣは骨形成マーカーとして機能する。一方、Ｇｌａ残基を有しないＯＣ又は脱炭酸されたＧｌｕ型ＯＣは、骨基質との親和性が弱く血中に放出されるため、Ｇｌｕ型ＯＣは骨吸収マーカーとして機能する。つまり、本発明の測定試薬は骨形成マーカーであるＧｌａ型ＯＣを特異的かつ高感度に測定することが可能である。したがって、この態様の測定試薬は、Ｇｌａ型ＯＣとＧｌｕ型ＯＣを、ヒドロキシアパタイトへの吸着などの分画操作により分画せずに測定することが可能である。さらに、Ｎ末端を欠損するＯＣであっても測定することができ、有用である。 Furthermore, (A) the measurement reagent of the present invention containing an anti-OC monoclonal antibody that reacts with Gla-type OC and does not react with Glu-type OC reacts with Gla-type OC and does not react with Glu-type OC. OC is synthesized in osteoblasts and becomes Gla-type OC by vitamin K-dependent carboxylase in the cell. Since this Gla-type OC binds to bone hydroxyapatite and accumulates in the bone matrix to promote bone formation, the Gla-type OC functions as a bone formation marker. On the other hand, OC that does not have a Gla residue or decarboxylated Glu-type OC has a weak affinity for the bone matrix and is released into the blood, so that the Glu-type OC functions as a bone resorption marker. That is, the measurement reagent of the present invention can specifically and highly measure Gla-type OC, which is a bone formation marker. Therefore, the measurement reagent of this aspect can measure Gla-type OC and Glu-type OC without fractionation by fractionation operations such as adsorption to hydroxyapatite. Furthermore, even OCs lacking the N-terminus can be measured and are useful.
本発明の一態様としての、本発明の測定試薬の（Ａ）の抗ＯＣモノクローナル抗体としては、ハイブリドーマＯＣ ４−３０により産生されるモノクローナル抗体ＯＣ ４−３０が挙げられる（特開平２−２４２６９６号公報）。ハイブリドーマ細胞ＯＣ ４−３０は、平成元年２月２８日（原寄託日）より独立行政法人産業技術総合研究所特許生物寄託センター（茨城県つくば市東１丁目１番地１中央第６（郵便番号３０５−８５６６））にＦＥＲＭ ＢＰ−２７２５として寄託されている。 The anti-OC monoclonal antibody (A) of the measurement reagent of the present invention as one embodiment of the present invention includes monoclonal antibody OC 4-30 produced by hybridoma OC 4-30 (Japanese Patent Laid-Open No. Hei 2-242696). Publication). Hybridoma cell OC 4-30 has been established since February 28, 1989 (original deposit date), National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, Japan) -8566)) is deposited as FERM BP-2725.
また、（Ｂ）ＯＣの中央部位と反応する抗ＯＣモノクローナル抗体を含む本発明の測定試薬は、Ｍｉｄ Ｐｏｒｔｉｏｎと言われる中央部位を有するフラグメント化されたＯＣを選択的に測定することができる。この態様の測定試薬は、新生ＯＣなどの全長を有するＯＣのみならず、既存の骨基質から破骨細胞より溶出したフラグメント化ＯＣも測定することが可能である。ＯＣの中央部位とは、配列番号５のラットＯＣのアミノ酸番号１５〜３５位、好ましくは１８〜３３位、特に好ましくは２１〜３１位である。 Further, (B) the measurement reagent of the present invention containing an anti-OC monoclonal antibody that reacts with the central site of OC can selectively measure fragmented OC having a central site called Mid Portion. The measuring reagent of this embodiment can measure not only OC having a full length such as nascent OC but also fragmented OC eluted from osteoclasts from existing bone matrix. The central site of OC is amino acid number 15 to 35, preferably 18 to 33, particularly preferably 21 to 31 of rat OC of SEQ ID NO: 5.
本発明の一態様としての、本発明の測定試薬の（Ｂ）の抗ＯＣモノクローナル抗体としては、ＯＣの２１〜３１位を認識するハイブリドーマＯＣ−Ｇ３により産生されるモノクローナル抗体ＯＣ−Ｇ３である（特開平１−１６０４９３号公報）。ハイブリドーマ細胞ＯＣ−Ｇ３は、昭和６２年１１月５日（原寄託日）より独立行政法人産業技術総合研究所特許生物寄託センター（茨城県つくば市東１丁目１番地１中央第６（郵便番号３０５−８５６６））にＦＥＲＭ ＢＰ−２０７８として寄託されている。 The anti-OC monoclonal antibody (B) of the measurement reagent of the present invention as one aspect of the present invention is a monoclonal antibody OC-G3 produced by a hybridoma OC-G3 that recognizes positions 21 to 31 of OC ( JP-A-1-160493). The hybridoma cell OC-G3 was established on November 5, 1987 (original deposit date), the National Institute of Advanced Industrial Science and Technology (AIST), Patent Biological Deposit Center (Higashi 1-chome, 1-chome, Tsukuba-shi, Ibaraki) 8566)) is deposited as FERM BP-2078.
さらに、（Ｃ）ＯＣのＮ末端部位と反応する抗ＯＣモノクローナル抗体を含む本発明の測定試薬は、新生ＯＣなどの全長を有するＯＣを測定することができる。ＯＣのＮ末端部位とは、配列番号５のラットＯＣのアミノ酸番号１〜２５位、好ましくは１〜１５位、特に好ましくは１〜１０位である。 Furthermore, (C) the measurement reagent of the present invention comprising an anti-OC monoclonal antibody that reacts with the N-terminal site of OC can measure OC having a full length such as nascent OC. The N-terminal site of OC is amino acid number 1 to 25, preferably 1 to 15 and particularly preferably 1 to 10 in the rat OC of SEQ ID NO: 5.
本発明の測定試薬は、０．２５ｎｇ／ｍＬの濃度で存在するＯＣを検出することが可能である。 The measurement reagent of the present invention can detect OC present at a concentration of 0.25 ng / mL.
本発明の測定試薬は、測定方法として二抗体サンドイッチ法などの固相酵素免疫検定（ＥＬＩＳＡ）法を含むエンザイムイムノアッセイに用いることができる、ＯＣの測定試薬である。一態様として、被験試料は血漿、血清などの体液又は細胞抽出物及び細胞の培養上清等が挙げられる。前記被検試料は、本発明の測定試薬により測定可能な試料であれば特に限定はされないが、例えばラット又はマウス由来の試料が挙げられる。 The measurement reagent of the present invention is an OC measurement reagent that can be used in enzyme immunoassays including solid-phase enzyme immunoassay (ELISA) methods such as the two-antibody sandwich method. As one aspect, the test sample includes body fluids such as plasma and serum, or cell extracts and cell culture supernatants. The test sample is not particularly limited as long as it is a sample that can be measured by the measurement reagent of the present invention, and examples thereof include samples derived from rats or mice.
本発明の測定試薬の一態様として、上記（１）に記載する抗ＯＣモノクローナル抗体と、上記（Ａ）〜（Ｃ）より選択される１つの抗ＯＣモノクローナル抗体の２種のモノクローナル抗体を構成成分とするが、これらの抗体のうち、一方は固相抗体（１次抗体）、他方は標識抗体（２次抗体）として使用することができる。ここで、固相抗体とは適切な不溶性担体に固定化された抗体を意味し、標識抗体とは適切な標識物質により標識化された抗体を意味する。固相抗体はＯＣとの抗原抗体反応によって、対象試料中の被検物質であるＯＣをトラップするために使用され、トラップされた前記の被検物質を検出するために標識抗体が使用される。特に（１）に記載する抗ＯＣモノクローナル抗体を固相抗体、（Ａ）〜（Ｃ）より選択される抗ＯＣモノクローナル抗体を標識抗体とする測定試薬が好適に使用できる。この態様の測定試薬は、ウシＯＣを含む試料であっても、ラットＯＣ又はマウスＯＣを特異的にトラップすることが可能であり、固相抗体がウシＯＣによりマスクされることなく、また、標識抗体の種交差性に影響を受けずにラットＯＣ又はマウスＯＣを特異的に測定することが可能である。 As one aspect of the measuring reagent of the present invention, the anti-OC monoclonal antibody described in (1) above and two types of monoclonal antibodies selected from the above (A) to (C) are used as components. However, one of these antibodies can be used as a solid phase antibody (primary antibody) and the other as a labeled antibody (secondary antibody). Here, the solid phase antibody means an antibody immobilized on an appropriate insoluble carrier, and the labeled antibody means an antibody labeled with an appropriate labeling substance. The solid phase antibody is used for trapping OC, which is a test substance in a target sample, by antigen-antibody reaction with OC, and a labeled antibody is used for detecting the trapped test substance. In particular, a measurement reagent using the anti-OC monoclonal antibody described in (1) as a solid phase antibody and the anti-OC monoclonal antibody selected from (A) to (C) as a labeled antibody can be preferably used. The measurement reagent of this embodiment can specifically trap rat OC or mouse OC even if it is a sample containing bovine OC, and the solid phase antibody is not masked by bovine OC. It is possible to specifically measure rat OC or mouse OC without being affected by the species cross-reactivity of the antibody.
本発明に使用されるモノクローナル抗体は、特に限定されるものではないが、放射性同位元素、酵素、蛍光物質、発光物質、タンパク質などを用いて標識化され、標識抗体が作製される。放射性同位元素としては、特に限定されるものではないが、例えば［１２５Ｉ］、［１３１Ｉ］、［３Ｈ］、［１４Ｃ］などが好ましい。酵素としては、特に限定されるものではないが、安定で比活性の大きなものが好ましく、例えばβ−ガラクトシダーゼ、β−グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが挙げられる。蛍光物質としては、特に限定されるものではないが、例えばフルオレスカミン、フルオレッセインイソチオシアネートなどが挙げられる。発光物質としては、特に限定されるものではないが、例えばルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが挙げられる。さらに、ビオチンのような化合物を用いることができる。本発明の測定試薬において、標識抗体は溶液又は凍結乾燥等の各種形態で提供することができる。 The monoclonal antibody used in the present invention is not particularly limited, but is labeled using a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, a protein, or the like to produce a labeled antibody. Radioisotopes include, but are not particularly limited, for example, [125 I], [131 I ], [3 H], [14 C] , etc. is preferable. Although it does not specifically limit as an enzyme, A stable thing with large specific activity is preferable, for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase etc. are mentioned. Although it does not specifically limit as a fluorescent substance, For example, a fluorescamine, a fluorescein isothiocyanate, etc. are mentioned. Although it does not specifically limit as a luminescent substance, For example, luminol, a luminol derivative, luciferin, lucigenin etc. are mentioned. In addition, compounds such as biotin can be used. In the measurement reagent of the present invention, the labeled antibody can be provided in various forms such as a solution or lyophilization.
固相抗体は、ビーズ、マイクロタイタープレート、試験管、ニトロセルロース膜、ナイロン膜等の担体表面に、当業者には公知の方法によって、ＯＣを認識する本発明の測定試薬を結合させることによって調製される。また、固相抗体を調製するための抗体、担体及び固相化に必要な試薬を、固定化する前の段階で提供しても良い。前記目的に使用される抗体も、本発明の固相抗体に含まれる。 The solid-phase antibody is prepared by binding the measurement reagent of the present invention that recognizes OC to a carrier surface such as a bead, a microtiter plate, a test tube, a nitrocellulose membrane, or a nylon membrane by a method known to those skilled in the art. Is done. Further, an antibody for preparing a solid phase antibody, a carrier, and a reagent necessary for immobilization may be provided at a stage before immobilization. The antibody used for the above purpose is also included in the solid phase antibody of the present invention.
本発明の測定試薬の態様としては、ＯＣを認識する２種のモノクローナル抗体に加えて、様々な試薬、材料、器具等を適宜含有させることができる。本発明の測定試薬を構成するモノクローナル抗体を吸着させるための吸着プレートを含んでいてもよい。また、標識した抗体を検出するための試薬、使用する際にコントロールとなる試薬を含んでいてもよい。 As an aspect of the measurement reagent of the present invention, various reagents, materials, instruments and the like can be appropriately contained in addition to the two monoclonal antibodies recognizing OC. An adsorption plate for adsorbing the monoclonal antibody constituting the measurement reagent of the present invention may be included. Moreover, the reagent for detecting the labeled antibody and the reagent used as a control when using it may be included.
（３）本発明のＯＣの測定方法
本発明のＯＣの測定方法は、前記（１）に記載の抗ＯＣモノクローナル抗体又は前記（２）に記載のＯＣ測定試薬を使用することを特徴とする方法である。例えば、本発明のモノクローナル抗体を使用して、競合的免疫検定法によりＯＣを測定することができる。また、２種の抗ＯＣモノクローナル抗体を含む態様の本発明の測定試薬を使用して、２つのモノクローナル抗体のうち、一方を固相抗体、他方を標識抗体とする二酵素サンドイッチ法の場合、被検物質と固相抗体を接触させ、これに標識抗体をさらに接触させ、これらのモノクローナル抗体と被検物質の複合体を検出することにより、ＯＣを測定することができる。さらに、被検物質と接触させた後に固相抗体を洗浄する工程及び／又は被検物質と結合しなかった標識抗体を洗浄により除去する工程を含んでいてもよい。また、固相抗体及び標識抗体は、前記（２）に記載の通り固相化及び標識化の操作により調製される。本発明の一態様として、モノクローナル抗体ＲａｔＯＣ−Ｃ ９−１２Ｈ、モノクローナル抗体ＯＣ４−３０の使用が好適であり、さらに、固相抗体としてモノクローナル抗体ＲａｔＯＣ−Ｃ ９−１２Ｈ、標識抗体としてモノクローナル抗体ＯＣ４−３０の組み合わせの測定系が特に好適である。本発明の方法においては、定性的な測定と、定量的な測定が含まれる。一態様として、被験試料は血漿、血清などの体液又は細胞培養物等が挙げられる。
(3) Method for Measuring OC of the Present Invention The method for measuring OC of the present invention uses the anti-OC monoclonal antibody described in (1) above or the reagent for measuring OC described in (2) above. It is. For example, OC can be measured by a competitive immunoassay using the monoclonal antibody of the present invention. In the case of the two-enzyme sandwich method in which the measurement reagent of the present invention including two types of anti-OC monoclonal antibodies is used and one of the two monoclonal antibodies is a solid phase antibody and the other is a labeled antibody, OC can be measured by bringing a test substance into contact with a solid phase antibody, further contacting with a labeled antibody, and detecting a complex of these monoclonal antibody and test substance. Further, it may include a step of washing the solid phase antibody after contacting with the test substance and / or a step of removing the labeled antibody not bound to the test substance by washing. In addition, the solid phase antibody and the labeled antibody are prepared by the operation of solid phase and labeling as described in (2) above. As one aspect of the present invention, it is preferable to use monoclonal antibody RatOC-C 9-12H and monoclonal antibody OC4-30. Furthermore, monoclonal antibody RatOC-C 9-12H is used as a solid phase antibody, and monoclonal antibody OC4- is used as a labeled antibody. A measurement system of 30 combinations is particularly suitable. In the method of the present invention, qualitative measurement and quantitative measurement are included. As one aspect, the test sample includes body fluids such as plasma and serum, or cell cultures.
本発明の方法は、０．２５ｎｇ／ｍＬの濃度で存在するＯＣを検出することが可能である。本発明の方法は、ＯＣの異なる部位を認識する２種のモノクローナル抗体を使用することにより、極めて高い特異性を有する識別が可能である。 The method of the present invention can detect OC present at a concentration of 0.25 ng / mL. The method of the present invention enables discrimination with extremely high specificity by using two monoclonal antibodies that recognize different sites of OC.
以下に、本発明を実施例により更に具体的に説明するが、本発明は以下の実施例の範囲のみに限定されるものではない。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited only to the scope of the following examples.
実施例１ 抗体の作製
（１）抗原免疫・細胞融合
ラットＯＣのＮ末端ペプチド（配列番号５のアミノ酸番号１〜６、配列番号３）、Ｃ末端ペプチド（配列番号５のアミノ酸番号３８〜５０、配列番号１）を調製し、１ｍｇ／ｍＬの抗原溶液とした。両抗原溶液を、それぞれＢＡＬＢ／ｃマウス４匹に５０μｇ／ｓｈｏｔ／ｂｏｄｙの投与量で、初回免疫ではフロイント完全アジュバントとエマルジョンを形成させてから腹腔投与し、２回目以降は市販の水溶性アジュバント（ＲＩＢＩ Ａｄｊｕｖａｎｔ）と混合して、２週間隔合計４回の投与を行った。その後、眼窩静脈血清中のラットＯＣに対する抗体価の上昇を確認した上で、すべての個体に最終免疫を実施した。最終免疫３日後に、４匹のうち２個体の免疫動物の脾臓を摘出し、無血清培地で分散・洗浄した後、細胞融合用ミエローマ（Ｐ３Ｕ１）と５：１（脾臓：ミエローマ）の割合で混合し遠心上清を除いた細胞ペレットとした。この細胞混合物に適温に保温した５０％ＰＥＧ溶液１ｍＬを一定速度で、軽い振とうを加えながら混合し、合計３ｍＬまで加え、そのあとは、無血清培地７ｍＬを同様に一定速度で加え、１０ｍＬにフィルアップし、この操作で細胞融合を実施した。約２週間の時差をつけて、残る２匹のマウスも同様の最終免疫をさらに１回追加して、３日後に細胞融合に供した。
２回のチャレンジで、多数の融合細胞を取得した。この幅広い母集団より抗原に特異的な抗体をスクリーニングした。
Example 1 Antibody Production (1) Antigen Immunization / Cell Fusion Rat OC N-terminal peptide (amino acids 1 to 6 of SEQ ID NO: 5, SEQ ID NO: 3), C-terminal peptide (amino acids 38 to 50 of SEQ ID NO: 5, SEQ ID NO: 1) was prepared and used as a 1 mg / mL antigen solution. Both antigen solutions were administered to 4 BALB / c mice at a dose of 50 μg / shot / body, and in the first immunization, an intraperitoneal administration was carried out after forming an emulsion with Freund's complete adjuvant. RIBI Adjuvant) was administered for a total of 4 doses at 2-week intervals. Thereafter, after confirming an increase in antibody titer against rat OC in orbital vein serum, final immunization was performed on all individuals. Three days after the final immunization, the spleens of two immunized animals out of four were removed, dispersed and washed with serum-free medium, and then at a ratio of myeloma for cell fusion (P3U1) and 5: 1 (spleen: myeloma). The cell pellet was obtained by mixing and removing the centrifugal supernatant. To this cell mixture, 1 mL of 50% PEG solution kept at an appropriate temperature was mixed at a constant speed while adding light shaking, and added to a total of 3 mL. After that, 7 mL of serum-free medium was added at a constant speed in the same manner to 10 mL. After filling up, cell fusion was performed by this operation. With a time difference of about 2 weeks, the remaining two mice were further added with the same final immunization once and subjected to cell fusion after 3 days.
A large number of fused cells were obtained in two challenges. Antibodies specific for the antigen were screened from this broad population.
（２）ＨＡＴ選択
融合細胞のスクリーニングには、クローニング培地（三光純薬社製）にＨＡＴ（Ｈ：ヒポキサンチン、Ａ：アミノプテリン、Ｔ：チミジン）を加えたものを用意し、融合日の翌日から３回の培地交換をこのＨＡＴ培地で行った。この培地交換操作で、成長してきた細胞は、脾臓由来のｄｅ ｎｏｖｏ合成系を持ちかつ不死化した融合細胞であった。
(2) HAT selection For fusion cell screening, a cloning medium (manufactured by Sanko Junyaku Co., Ltd.) with HAT (H: hypoxanthine, A: aminopterin, T: thymidine) is prepared, and the day after the day of fusion. The medium was changed three times from this HAT medium. Cells that grew by this medium exchange operation were fused cells having a de novo synthesis system derived from spleen and immortalized.
（３）スクリーニング
免疫原のラットＯＣのＮ末端ペプチド又はＣ末端ペプチドを、５μｇ／ｍＬ ＰＢＳ、５０μＬ／ｗｅｌｌで、イムノプレート（ナルジェヌンク社製）上に添加し、４℃で一晩放置して物理吸着させた。ブランク用抗原として、ウシ骨由来天然型の全長ＯＣをブランク抗原としてスクリーニングの際の評価用タンパクとした。５μｇ／ｍＬ ＰＢＳ、５０μＬ／ｗｅｌｌで、同様にコーティングした。翌日、抗原溶液を捨てて、５０％Ｂｌｏｃｋｅｒ Ｃａｓｅｉｎ（ピアス社製）を２００μＬ／ｗｅｌｌになるように加えて、室温（２０〜３０℃）で１時間放置して、ブロッキング操作を行った。その後、ブロッキング溶液を捨て、上記（２）で得られた融合細胞の培養上清（原液使用）を、ナンバリングした上で抗原プレートに投入し、一次反応を室温（２０〜３０℃）で１時間行った。ＰＢＳで反応が終了した各ウェルを３回洗浄し、ペーパータオルで充分に液を切った。検出には、抗マウスＩｇＧラットモノクローナル抗体カクテル−ペルオキシダーゼ標識抗体を使用した。前記抗体を１μｇ／ｍＬ、５０μＬ／ｗｅｌｌで添加し、室温（２０〜３０℃）で１時間反応を行った。その後、標識抗体液を捨て、ウェルをＰＢＳで４回洗浄した。ペーパータオルで、洗浄液を充分に除き、ペルオキシダーゼ基質であるＡＢＴＳ［２，２’−アジノビス（３−エチルベンゾチアゾリン−６−スルホン酸）］溶液（ピアス社製）を５０μＬ／ｗｅｌｌで投入し、室温で１５〜３０分発色させ、強弱ある陽性株を幅広く厳格に検出した。等量の１５０ｍＭ シュウ酸を加えて反応を停止させた後、肉眼及びプレートリーダーで、陽性株を確認し、ブランク抗原に反応せず、目的のラットＯＣに特異的に反応する株の選抜を試みた。
(3) Screening N-terminal peptide or C-terminal peptide of rat OC as an immunogen is added to an immunoplate (Nalgenunk) at 5 μg / mL PBS, 50 μL / well, and left at 4 ° C. overnight to physically Adsorbed. As a blank antigen, bovine bone-derived natural full-length OC was used as a blank antigen and used as a protein for evaluation during screening. The same coating was performed with 5 μg / mL PBS, 50 μL / well. On the next day, the antigen solution was discarded, 50% Blocker Casein (Pierce) was added to 200 μL / well, and allowed to stand at room temperature (20 to 30 ° C.) for 1 hour to perform a blocking operation. Thereafter, the blocking solution is discarded, and the culture supernatant (using the stock solution) of the fused cells obtained in (2) above is numbered and placed on the antigen plate, and the primary reaction is performed at room temperature (20-30 ° C.) for 1 hour. went. Each well that had been reacted with PBS was washed three times, and the solution was thoroughly drained with a paper towel. For detection, an anti-mouse IgG rat monoclonal antibody cocktail-peroxidase labeled antibody was used. The antibody was added at 1 μg / mL and 50 μL / well and reacted at room temperature (20-30 ° C.) for 1 hour. Thereafter, the labeled antibody solution was discarded, and the wells were washed 4 times with PBS. Using a paper towel, thoroughly remove the washing solution, add ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] solution (Pierce), which is a peroxidase substrate, at 50 μL / well at room temperature. The color was developed for 15 to 30 minutes, and strong and weak positive strains were detected extensively and strictly. After stopping the reaction by adding an equal amount of 150 mM oxalic acid, the positive strain was confirmed with the naked eye and a plate reader, and an attempt was made to select a strain that does not react with the blank antigen but reacts specifically with the target rat OC. It was.
（４）陽性株選抜とクローニング、株樹立
（３）に示す厳格なスクリーニングの結果、ブランク抗原に反応せず、目的のラットＯＣに特異的に反応する株は、９６０ウェルに含まれる２０００以上の株中ＯＣのＣ末端ペプチドに反応するわずかに２株であり、ＯＣのＮ末端ペプチドに反応する株は得られなかった。この２株を用い、直ちに限界希釈法によりクローニングを行った。クローニングされた抗ラットＯＣ抗体産生ハイブリドーマ２種類について、それぞれクローンを各２種（本株・亜株）確保した。
(4) Positive strain selection and cloning, strain establishment As a result of the strict screening shown in (3), more than 2000 strains contained in 960 wells did not react with the blank antigen and reacted specifically with the target rat OC. Only two strains reacting with the OC C-terminal peptide of the strain were obtained, and no strain reacting with the OC N-terminal peptide was obtained. Using these two strains, cloning was immediately performed by the limiting dilution method. For the two types of cloned anti-rat OC antibody-producing hybridomas, two clones (this strain and substrain) were secured.
（５）マウス腹水採取
特異性の高い前記ハイブリドーマクローンは、本株・亜株ともに凍結細胞としてマスター細胞を保管後、ほぼ並行しながら、本株をＢＡＬＢ／ｃマウスの腹腔内で大量培養し、腹水として粗精製抗体を得た。腹水は、１個体あたりおよそ３〜５ｍＬであった。
(5) Collection of mouse ascites The hybridoma clone having high specificity is obtained by culturing the strain in a large amount in the peritoneal cavity of a BALB / c mouse while storing the master cell as a frozen cell in both the strain and sub-strain, in parallel. Crude purified antibody was obtained as ascites. Ascites was approximately 3-5 mL per individual.
（６）抗体精製
得られた腹水は、５０％飽和硫安塩析・透析を行い、その画分をＰｒｏｔｅｉｎ Ａ カラムに供した。平衡化緩衝液は、３Ｍ ＮａＣｌ、１．５Ｍ ｇｌｙｃｉｎ−ＮａＯＨ緩衝液（ｐＨ８．９）の高塩濃度のものを調製し、どのサブクラスのＩｇＧであっても良好に結合する条件を採用した。平衡化緩衝液で２倍希釈した腹水硫安塩析画分を腹水液量とほぼ等量の容積のＰｒｏｔｅｉｎ Ａ樹脂にアプライし、吸光度Ａ２８０がほぼゼロになるまで平衡化緩衝液でカラムを洗った。その後、クエン酸緩衝液（ｐＨ４．０）とクエン酸緩衝液（ｐＨ３．０）の２段階で溶出を行った。溶出画分は、ただちに１Ｍ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ９．０）で中和し、硫安塩析もしくは、遠心限外ろ過濃縮を行った。最終抗体は、ＰＢＳで透析し、０．２２μｍフィルターろ過により無菌化した。その結果、１株からのみ抗体が得られた。抗体の純度は、１０％ＳＤＳ−ＰＡＧＥ（還元加熱条件）により分析し、Ｈ鎖とＬ鎖以外のものがない良好な純度の抗体であることを確認した。
(6) Antibody Purification The obtained ascites was subjected to 50% saturated ammonium sulfate salting out and dialysis, and the fraction was applied to a Protein A column. As the equilibration buffer, a high salt concentration of 3M NaCl, 1.5M glycin-NaOH buffer (pH 8.9) was prepared, and a condition that satisfactorily binds any IgG of any subclass was adopted. Ascites ammonium sulfate salted-out fraction diluted 2-fold with equilibration buffer was applied to Protein A resin having a volume approximately equal to the amount of ascites fluid, and the column was washed with equilibration buffer until absorbance A280 was almost zero. . Thereafter, elution was carried out in two stages of citrate buffer (pH 4.0) and citrate buffer (pH 3.0). The eluted fraction was immediately neutralized with 1M Tris-HCl buffer (pH 9.0), and subjected to ammonium sulfate salting out or centrifugal ultrafiltration concentration. The final antibody was dialyzed against PBS and sterilized by 0.22 μm filter filtration. As a result, antibodies were obtained only from one strain. The purity of the antibody was analyzed by 10% SDS-PAGE (reduction heating condition), and it was confirmed that the antibody had good purity with no other than H and L chains.
実施例２ ＯＣ測定系構築
（１）抗体修飾
抗ＯＣ抗体ＯＣ４−３０（特開平２−２４２６９６号公報、ＦＥＲＭ−ＢＰ２７２５）１ｍｇに、過ヨウ素酸法によるペルオキシダーゼ標識を施した。ＯＣ４−３０抗体はＧｌａ型ＯＣと反応し、Ｇｌｕ型ＯＣとは反応しないモノクローナル抗体である。過ヨウ素酸法は、ペルオキシダーゼの糖鎖ジオールを脱水素酸化させシッフベースを形成させ、抗体側のアミノ基と結合する方法である。抗原との結合活性を保持した状態で酵素標識体とすることができた。
Example 2 Construction of OC Measurement System (1) Antibody Modification 1 mg of anti-OC antibody OC4-30 (JP-A-2-242696, FERM-BP2725) was subjected to peroxidase labeling by the periodic acid method. The OC4-30 antibody is a monoclonal antibody that reacts with Gla type OC and does not react with Glu type OC. The periodic acid method is a method in which a sugar chain diol of peroxidase is dehydrogenated to form a Schiff base, which is bound to an amino group on the antibody side. It was possible to obtain an enzyme label while maintaining the binding activity with the antigen.
（２）ＯＣ測定系
固相に実施例１−（６）で得られたの抗体を用い、最も高感度にＯＣを定量できる系を試験した。当初、固相抗体にモノクローナル抗体ＯＣ４−３０、標識抗体にモノクローナル抗体ＲａｔＯＣ−Ｃ ９−１２Ｈを用いる組み合わせでは、ラットＯＣの測定感度が低かった。逆に、固相抗体にモノクローナル抗体ＲａｔＯＣ−Ｃ ９−１２Ｈ、標識抗体にモノクローナル抗体ＯＣ４−３０を用いる組み合わせで初めて測定系が確立した。確立した測定系を使用した測定方法を以下に示す。
（Ａ）イムノプレート器材（ナルジェヌンク製）に、ＰＢＳで１０μｇ／ｍｌに希釈したモノクローナル抗体ＲａｔＯＣ−Ｃ ９−１２Ｈ溶液を１００μＬ／ｗｅｌｌで投入し、４℃で一晩放置する。
（Ｂ）翌日、抗体溶液を捨て、２５％ブロックエース＋０．３Ｍマルトース／ＰＢＳ溶液（ブロッキング溶液）を２００μＬ／ｗｅｌｌで投入し、４℃一晩放置し、前記抗体が結合しなかった部分をタンパク質ブロックする。
翌日、ブロッキング溶液を捨て、以下の測定に使用する。
（Ｃ）各濃度の検体を１００μＬずつマイクロピペットで各ｗｅｌｌに２連ずつ加え、室温（２０〜３０℃）で１時間反応させる。（第一反応）
（Ｄ）反応液を捨て、０．１％Ｔｗｅｅｎ２０含有ＰＢＳで３回洗浄後、モノクローナル抗体ＯＣ４−３０標識抗体液を１００μＬずつ各ｗｅｌｌに加え、室温（２０〜３０℃）で一時間反応させる。（第二反応）
（Ｅ）反応液を捨て、０．１％Ｔｗｅｅｎ２０含有ＰＢＳで４回洗浄後、３，３’，５，５’−テトラメチルベンジジン（ＴＭＢＺ）溶液（ＢｉｏＦＸ社製）を１００μＬずつ各ｗｅｌｌに加え、室温（２０〜３０℃）で１５分反応させる。（発色反応）
（Ｆ）１Ｎ 硫酸を１００μＬずつ、ＴＭＢＺ溶液を入れた順番に各ｗｅｌｌに加え、反応を停止させた後よく混和する。
蒸留水を対照としてマイクロプレートリーダーをブランク補正し、波長４５０ｎｍで吸光度を測定する。
標準曲線を作成し、検体の吸光度から対応するＯＣ濃度を読み取る。
(2) OC Measurement System Using the antibody obtained in Example 1- (6) as the solid phase, a system capable of quantifying OC with the highest sensitivity was tested. Initially, in the combination using the monoclonal antibody OC4-30 as the solid phase antibody and the monoclonal antibody RatOC-C 9-12H as the labeled antibody, the measurement sensitivity of rat OC was low. Conversely, a measurement system was established for the first time with a combination using the monoclonal antibody RatOC-C 9-12H as the solid phase antibody and the monoclonal antibody OC4-30 as the labeled antibody. The measurement method using the established measurement system is shown below.
(A) Monoclonal antibody RatOC-C 9-12H solution diluted to 10 μg / ml with PBS is added at 100 μL / well to immunoplate equipment (Narugenunk), and left overnight at 4 ° C.
(B) The next day, the antibody solution is discarded, and 25% Block Ace + 0.3 M maltose / PBS solution (blocking solution) is added at 200 μL / well and left at 4 ° C. overnight. To block.
The next day, discard the blocking solution and use it for the following measurements.
(C) 100 μL of each concentration specimen is added to each well in duplicate by a micropipette, and reacted at room temperature (20-30 ° C.) for 1 hour. (First reaction)
(D) The reaction solution is discarded, and after washing 3 times with PBS containing 0.1% Tween 20, 100 μL of monoclonal antibody OC4-30 labeled antibody solution is added to each well and reacted at room temperature (20-30 ° C.) for 1 hour. (Second reaction)
(E) Discard the reaction solution, wash 4 times with PBS containing 0.1% Tween 20, and add 100 μL of 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) solution (manufactured by BioFX) to each well. And react at room temperature (20-30 ° C.) for 15 minutes. (Coloring reaction)
(F) Add 100 μL of 1N sulfuric acid to each well in the order in which the TMBZ solution was added, and mix well after stopping the reaction.
A microplate reader is blank-corrected using distilled water as a control, and the absorbance is measured at a wavelength of 450 nm.
Create a standard curve and read the corresponding OC concentration from the absorbance of the sample.
上記測定系により、ラット骨由来天然型ＯＣをラットＧｌａ型ＯＣ標準品として測定した結果を表１に示す。必要サンプル量は、１００μｌ／ｗｅｌｌであり、０．２５ｎｇ／ｍＬのラットＧｌａ型ＯＣであっても検出可能であった。 Table 1 shows the results of measuring rat bone-derived natural OC as a rat Gla-type OC standard by the above measurement system. The necessary sample amount was 100 μl / well, and even a rat Gla-type OC of 0.25 ng / mL was detectable.
（３）同時再現性試験
実施例２−（２）記載の測定系により、ラットＧｌａ型ＯＣを希釈して作成した３種類の濃度コントロールを用いて同時再現性試験を行った。表２に結果を示すように、ＣＶ値は良好な同時再現性を示した。
(3) Simultaneous reproducibility test Using the measurement system described in Example 2- (2), a simultaneous reproducibility test was performed using three types of concentration controls prepared by diluting rat Gla-type OC. As shown in Table 2, the CV value showed good simultaneous reproducibility.
（４）日差再現性試験
実施例２−（２）記載の測定系で、三日間にわたり３種類の濃度コントロールを定量して日差再現性試験を行った。表３に結果を示すように、良好な日差再現性を示した。
(4) Day difference reproducibility test In the measurement system described in Example 2- (2), three kinds of concentration controls were quantified over three days, and a day difference reproducibility test was performed. As shown in Table 3, good day difference reproducibility was shown.
（５）添加回収試験
実施例２−（２）記載の測定系でラットＧｌａ型ＯＣの添加回収試験を行った。各種濃度のラットＯＣ検体２種を等量に混合したサンプルを測定し、実測値からラットＯＣ量の理論値との差（＝回収率）を求めた。表４に結果を示すように、添加回収率８７％から１１９％（平均値１００．７８％）と良好な結果が得られた。
(5) Addition / recovery test An addition / recovery test for rat Gla-type OC was performed using the measurement system described in Example 2- (2). Samples in which two types of rat OC specimens of various concentrations were mixed in equal amounts were measured, and the difference (= recovery rate) from the theoretical value of the rat OC amount was determined from the actually measured values. As shown in Table 4, good results were obtained with an addition recovery rate of 87% to 119% (average value 100.78%).
（６）交差反応性
実施例２−（２）記載の測定系で交差反応性試験を行った。各種の動物由来のＯＣは、正常動物血清（コスモバイオ社製）をＯＣ含有物とした。これらのＯＣ含有物を、５、２５、１２５、６２５倍に希釈して使用した。表５に吸光度Ａ４５０の測定結果から算出したＧｌａ型ＯＣ濃度（ｎｇ／ｍＬ）を示す。表５に示すように、ラットＧｌａ型ＯＣを高感度かつ特異的に検出可能であることが示された。さらに、マウスを免疫して調製した抗体にも関わらず、マウスＧｌａ型ＯＣも高感度かつ特異的に検出可能であることが示された。
(6) Cross-reactivity A cross-reactivity test was performed using the measurement system described in Example 2- (2). As for OC derived from various animals, normal animal serum (manufactured by Cosmo Bio) was used as the OC-containing material. These OC-containing materials were used after being diluted 5, 25, 125, and 625 times. Table 5 shows the Gla type OC concentration (ng / mL) calculated from the measurement result of absorbance A450. As shown in Table 5, it was shown that rat Gla-type OC can be detected with high sensitivity and specificity. Furthermore, it was shown that mouse Gla-type OC can be detected with high sensitivity and specificity in spite of antibodies prepared by immunizing mice.
（７）市販製品との比較
ウシ血清サンプルＡ〜Ｇの７種類の血清サンプル、インフォームドコンセントの得られたヒト健常人ドナーの血清サンプルを入手し、５倍又は２５倍に希釈して使用した。各サンプルを市販製品Ｏｓｔｅｏｃａｌｓｉｎ，Ｒａｔ，ＥＩＡ Ｋｉｔ［ＢＴＩ（Ｂｉｏｍｅｄｉｃａｌ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｉｎｃ．）社製、製品コード：ＢＴ−４９０］及び実施例２−（２）記載の測定系でＯＣ濃度を測定した。表６に吸光度Ａ４５０の測定結果から算出したＯＣ濃度（ｎｇ／ｍＬ）を示す。表６に示すように、上記市販製品ではウシ及びヒト抗原への交差反応性が見られるが、実施例２−（２）記載の測定系では、ヒト及びウシに交差しないことが示された。なお、上記市販製品の測定に要する時間は５時間又は一晩であるが、実施例２−（２）記載の測定系の測定に要する時間は３時間前後であった。さらに、上記市販製品に比べて実施例２−（２）記載の測定系の操作工程は少なく、簡便であった。
(7) Comparison with commercially available products Seven serum samples of bovine serum samples A to G and serum samples of healthy human donors with informed consent were obtained and used after being diluted 5 times or 25 times. . The OC concentration of each sample was measured using the measurement system described in the commercially available products Osteocalsin, Rat, EIA Kit [manufactured by BTI (Biomedical Technologies Inc.), product code: BT-490] and Example 2- (2). Table 6 shows the OC concentration (ng / mL) calculated from the measurement result of absorbance A450. As shown in Table 6, the commercial product showed cross-reactivity to bovine and human antigens, but the measurement system described in Example 2- (2) showed that it did not cross human and bovine. In addition, although the time required for the measurement of the commercial product is 5 hours or overnight, the time required for the measurement of the measurement system described in Example 2- (2) was around 3 hours. Furthermore, compared with the said commercial product, there were few operation processes of the measurement system of Example 2- (2) description, and it was simple.
（８）サンプルの希釈直線
ラット血清サンプルＡ〜Ｄ、ラット腹水サンプルＥ〜Ｈを入手し、５０、１００、２００、４００倍に希釈して、実施例２−（２）記載の測定系でＧｌａ型ＯＣ濃度を測定した。サンプルの希釈によるラットＧｌａ型ＯＣ濃度の直線性を表７に示す。表７に示すように、良好な希釈直線性が示された。
(8) Sample dilution line Rat serum samples A to D and rat ascites samples E to H were obtained and diluted to 50, 100, 200, and 400 times, and Gla was measured using the measurement system described in Example 2- (2). The mold OC concentration was measured. Table 7 shows the linearity of rat Gla type OC concentration by dilution of the sample. As shown in Table 7, good dilution linearity was shown.
実施例３ ラットＧｌａ型ＯＣの検出
（１）ラット骨髄細胞の骨芽細胞への分化誘導の検出
正常ＳＤラット（オス、８週齢）から定法に従い骨髄細胞を採取し、２４ウェル培養プレートに、１ウェル当たり１０６ｃｅｌｌｓ／ウェルとなるように、２５μｇ／ｍＬ アスコルビン酸、１０％ＦＣＳ（ウシ胎仔血清）含有ＲＰＭＩ１６４０培地（シグマ社製）２ｍＬに懸濁した。さらに、タカラバイオ社製の骨芽細胞誘導試薬（製品コードＭＫ４３０）を取扱説明書に従い使用した。また、対照として前記骨芽細胞誘導試薬を添加しないものを用意し、同時に５％ＣＯ２インキュベーター内で３７℃で培養した。培養開始後、経時的に培養上清を採取し、実施例２−（２）記載の測定系によりＧｌａ型ＯＣ濃度を測定した。そのＧｌａ型ＯＣ濃度を表８に示す。表８に示すように、ウシ血清を含む培地による培養上清でも、ラットＧｌａ型ＯＣを特異的に測定することができ、骨芽細胞の誘導をモニタリングすることが可能であることが示された。
Example 3 Detection of Rat Gla-type OC (1) Detection of differentiation induction of rat bone marrow cells into osteoblasts Bone marrow cells were collected from normal SD rats (male, 8 weeks old) according to a conventional method, and placed in a 24-well culture plate. The suspension was suspended in 2 mL of RPMI 1640 medium (manufactured by Sigma) containing 25 μg / mL ascorbic acid and 10% FCS (fetal calf serum) so as to be 10 6 cells / well per well. Furthermore, an osteoblast induction reagent (product code MK430) manufactured by Takara Bio Inc. was used according to the instruction manual. In addition, as a control, one without the osteoblast induction reagent was prepared, and simultaneously cultured at 37 ° C. in a 5% CO 2 incubator. After the start of culture, the culture supernatant was collected over time, and the Gla-type OC concentration was measured by the measurement system described in Example 2- (2). The Gla-type OC concentration is shown in Table 8. As shown in Table 8, it was shown that the rat Gla-type OC can be specifically measured even in the culture supernatant of the medium containing bovine serum, and the induction of osteoblasts can be monitored. .
（２）卵巣摘出によるラットＧｌａ型ＯＣ量への影響
正常ＳＤラット（メス、８週齢）から卵巣を摘出し、１条件３個体で経過観察を行った。４週間経過後、８週間経過後、１０週間経過後、それぞれ同一卵巣摘出期間を経た３個体の大腿骨、骨髄を一括採取し、凍結細胞として保管した。最終骨髄調製後に、同時に復元し、それぞれの骨髄細胞を１ウェル当たり２×１０６ｃｅｌｌｓ／ウェルとなるように懸濁すること以外は実施例３−（１）と同様に培養した。培養開始後、経時的に培養上清を採取し、実施例２−（２）記載の測定系によりＧｌａ型ＯＣ濃度を測定した。そのＧｌａ型ＯＣ濃度を表９に示す。表９に示すように、卵巣摘出後の時間が経過するほど、骨髄の骨芽細胞誘導によるＧｌａ型ＯＣの産生が低下する傾向がみられた。Ｇｌａ型ＯＣは骨芽細胞マーカーであることから、当該測定系は、骨芽細胞への分化率の程度を数値化することを可能にしていると考えられた。
(2) Effect of ovariectomy on rat Gla-type OC amount The ovaries were removed from normal SD rats (female, 8 weeks old), and their follow-up was performed on 3 animals under 1 condition. After 4 weeks, 8 weeks, and 10 weeks, the femurs and bone marrows of 3 individuals that had undergone the same ovariectomy period were collected at one time and stored as frozen cells. After preparation of the final bone marrow, the cells were reconstituted at the same time and cultured in the same manner as in Example 3- (1) except that each bone marrow cell was suspended at 2 × 10 6 cells / well per well. After the start of culture, the culture supernatant was collected over time, and the Gla-type OC concentration was measured by the measurement system described in Example 2- (2). The Gla type OC concentration is shown in Table 9. As shown in Table 9, there was a tendency that the production of Gla-type OC due to bone marrow osteoblast induction decreased as the time after ovariectomy passed. Since Gla-type OC is an osteoblast marker, the measurement system was considered to be capable of quantifying the degree of differentiation into osteoblasts.
（３）細胞抽出物中のＧｌａ型ＯＣの測定
実施例３−（２）の骨髄細胞培養開始後、２１日目の培地上清と細胞を採取した。ウェルに含まれる全細胞２×１０６ｃｅｌｌｓを５００μＬの１％ＮＰ−４０溶液に移し、ピペッティングにより細胞を破壊して遠心上清を細胞抽出物とした。この培養上清と骨髄細胞抽出物を実施例２−（２）記載の測定系によりＧｌａ型ＯＣ濃度を測定した。そのＧｌａ型ＯＣ濃度を表１０に示す。表１０に示すように、細胞抽出物中のＧｌａ型ＯＣであっても測定することができた。
(3) Measurement of Gla-type OC in cell extract After starting bone marrow cell culture in Example 3- (2), the culture supernatant and cells on day 21 were collected. Total cells 2 × 10 6 cells contained in the well were transferred to 500 μL of a 1% NP-40 solution, the cells were disrupted by pipetting, and the centrifuged supernatant was used as a cell extract. The Gla-type OC concentration of this culture supernatant and bone marrow cell extract was measured by the measurement system described in Example 2- (2). The Gla-type OC concentration is shown in Table 10. As shown in Table 10, even the Gla type OC in the cell extract could be measured.
以上の結果から、本発明のモノクローナル抗体及び測定試薬を使用する検出系は、ラット及び／又はマウスＧｌａ型ＯＣを特異的に高感度で測定し、性能的にも安定な系であることを示している。
当該ＯＣを測定して得られる結果は、骨代謝をモニタリングする上で有用な情報を提供し、病勢判断や薬剤による治療効果判定、これらの予知、診断に有用な情報を提供する。
From the above results, it is shown that the detection system using the monoclonal antibody and the measurement reagent of the present invention is a system that specifically measures rat and / or mouse Gla type OC with high sensitivity and is stable in performance. ing.
The results obtained by measuring the OC provide information useful for monitoring bone metabolism, and provide information useful for disease state determination, therapeutic effect determination by drugs, prediction and diagnosis thereof.
SEQ ID NO:1: Amino acid sequence of rat osteocalcin C-terminus peptide.
SEQ ID NO:2: Amino acid sequence of bovine osteocalcin C-terminus peptide.
SEQ ID NO:3: Amino acid sequence of rat osteocalcin N-terminus peptide.
SEQ ID NO:4: Amino acid sequence of mouse osteocalcin C-terminus peptide.
SEQ ID NO:5: Amino acid sequence of rat osteocalcin.
SEQ ID NO: 1: Amino acid sequence of rat osteocalcin C-terminus peptide.
SEQ ID NO: 2: Amino acid sequence of bovine osteocalcin C-terminus peptide.
SEQ ID NO: 3: Amino acid sequence of rat osteocalcin N-terminus peptide.
SEQ ID NO: 4: Amino acid sequence of mouse osteocalcin C-terminus peptide.
SEQ ID NO: 5: Amino acid sequence of rat osteocalcin.
Claims (8)
（１）γ−カルボキシグルタミン酸残基を有するオステオカルシンと反応し、γ−カルボキシグルタミン酸残基を脱炭酸してグルタミン酸残基としたオステオカルシンとは反応しない抗オステオカルシンモノクローナル抗体；
（２）オステオカルシンの中央部位と反応する抗オステオカルシンモノクローナル抗体；
（３）オステオカルシンのＮ末端部位と反応する抗オステオカルシンモノクローナル抗体。 The osteocalcin measurement reagent according to claim 3, further comprising at least one monoclonal antibody selected from the following (1) to (3):
(1) an anti-osteocalcin monoclonal antibody that reacts with osteocalcin having a γ-carboxyglutamic acid residue and does not react with osteocalcin obtained by decarboxylating the γ-carboxyglutamic acid residue into a glutamic acid residue;
(2) an anti-osteocalcin monoclonal antibody that reacts with the central site of osteocalcin;
(3) An anti-osteocalcin monoclonal antibody that reacts with the N-terminal site of osteocalcin.
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