JP2017057198A - Cd81 lel-specific monoclonal antibody - Google Patents
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Abstract
Description
本発明は、関節リウマチの早期診断薬に関する。詳細には、本発明は、関節リウマチの滑液細胞において高発現しているタンパク質ＣＤ８１のLarge extracellular loop (LEL)（以下において「ＣＤ８１ ＬＥＬ」と称することがある）に対する新規な特異的抗体、該抗体を用いたＣＤ８１の検出および測定、ならびに該抗体を用いた関節リウマチの診断キット、サンドイッチＥＬＩＳＡ等に関する。 The present invention relates to an early diagnostic agent for rheumatoid arthritis. Specifically, the present invention relates to a novel specific antibody against the large extracellular loop (LEL) of the protein CD81 highly expressed in synovial cells of rheumatoid arthritis (hereinafter sometimes referred to as “CD81 LEL”), The present invention relates to detection and measurement of CD81 using an antibody, rheumatoid arthritis diagnostic kit using the antibody, sandwich ELISA, and the like.
関節リウマチは多発性関節炎を特徴とする炎症性疾患であり、進行すると関節が変形、破壊される。関節リウマチにおいては、滑膜内のタンパク質「シノビオリン」が過剰に作用して症状が悪化することがわかっている。シノビオリンはユビキチンリガーゼの一種であり、ＲＥＡＤ小胞体関連分解機構の促進などによるアポトーシスを抑制することで関節チウマチ滑膜線維芽細胞の増殖を促進させている。一方、細胞膜上に存在する４回膜貫通型タンパク質テトラスパニンの一種であるＣＤ８１は関節リウマチ滑膜細胞で高発現しており、シノビオリン遺伝子発現調節に関与している。 Rheumatoid arthritis is an inflammatory disease characterized by polyarthritis. When it progresses, the joint is deformed and destroyed. In rheumatoid arthritis, it is known that the protein “Synoviolin” in the synovium acts excessively to worsen the symptoms. Synoviolin is a kind of ubiquitin ligase, and promotes proliferation of rheumatoid arthritis synovial fibroblasts by suppressing apoptosis due to promotion of READ endoplasmic reticulum-related degradation mechanism. On the other hand, CD81, which is a kind of four-transmembrane protein tetraspanin existing on the cell membrane, is highly expressed in rheumatoid arthritis synovial cells and is involved in the regulation of synoviolin gene expression.
従来の関節リウマチ治療は抗炎症剤と炎症性サイトカインの抑制による対症療法が主流であったが、ＣＤ８１の発現を抑制することがリウマチ根絶治療につながると期待されている。 Conventional rheumatoid arthritis treatment has mainly been symptomatic treatment by suppressing anti-inflammatory agents and inflammatory cytokines, but suppressing the expression of CD81 is expected to lead to rheumatoid eradication treatment.
関節ウマチは、治療が遅れれば遅れるほど関節の変形も進み、薬効も悪くなる。したがって、関節リウマチは早期診断、早期治療が肝要である。 In the case of arthritis, the later the treatment is delayed, the more the joint is deformed, and the medicinal effect becomes worse. Therefore, early diagnosis and early treatment are important for rheumatoid arthritis.
テトラスパニンは保存された残基を含む４つの膜貫通ドメイン、２つの細胞外ループおよび３つの細胞内ドメインから構成されている。ＣＤ８１の２つの細胞外ループのうち大型のものがＣＤ８１ ＬＥＬである。ＣＤ８１ ＬＥＬは、ほとんどのテトラスパニンと異なり、潜在的な糖鎖付加部位を含んでいないので、これを特異的に認識する抗体を見いだすことができれば、サンプル中の微量のＣＤ８１を正確に検出、測定でき、関節リウマチの早期診断、早期治療に資することになる。 Tetraspanins are composed of four transmembrane domains containing conserved residues, two extracellular loops and three intracellular domains. Of the two extracellular loops of CD81, the larger one is CD81 LEL. Unlike most tetraspanins, CD81 LEL does not contain a potential glycosylation site, so if an antibody that specifically recognizes this can be found, a minute amount of CD81 in the sample can be accurately detected and measured. This will contribute to early diagnosis and early treatment of rheumatoid arthritis.
ＣＤ８１に対する抗体の作成が報告されているが（非特許文献１）、ＣＤ８１ ＬＥＬに特異的な抗体を作成し、サンプル中のＣＤ８１ ＬＥＬを実際に検出、測定したという報告はない。 Although preparation of an antibody against CD81 has been reported (Non-Patent Document 1), there is no report that an antibody specific to CD81 LEL was prepared and CD81 LEL in a sample was actually detected and measured.
本発明が解決しようとする課題は、サンプル中の極めて微量のＣＤ８１であっても正確に検出、定量できるＣＤ８１ ＬＥＬ特異的モノクローナル抗体を得ること、これらの抗体を用いてサンドイッチＥＬＩＳＡなどの検出、測定系を構築し、関節リウマチの診断、とりわけ早期診断に用いることであった。 The problem to be solved by the present invention is to obtain a CD81 LEL-specific monoclonal antibody that can be accurately detected and quantified even with a very small amount of CD81 in a sample, and to detect and measure a sandwich ELISA using these antibodies. The system was constructed and used for diagnosis of rheumatoid arthritis, especially for early diagnosis.
本発明者は上記課題を解決すべく鋭意研究を重ね、ＣＤ８１ ＬＥＬ特異的モノクローナル抗体を作成することに成功した。さらに本発明者は、これらのモノクローナル抗体を用いて、ＤＣ８１ ＬＥＬ用のサンドイッチＥＬＩＳＡを構築し、極めて微量（ピコグラムオーダー）のＣＤ８１ ＬＥＬを特異的に高感度で検出、測定することに成功した。かくして、本発明は完成された。 The present inventor has intensively studied to solve the above problems and succeeded in producing a CD81 LEL-specific monoclonal antibody. Furthermore, the present inventors constructed a sandwich ELISA for DC81 LEL using these monoclonal antibodies, and succeeded in detecting and measuring extremely small amounts (picogram order) of CD81 LEL with high sensitivity. Thus, the present invention has been completed.
したがって、本発明は以下のものを提供する。
（１）ＣＤ８１ ＬＥＬに特異的なモノクローナル抗体。
（２）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に寄託され、受託番号ＮＩＴＥ Ｐ−０２０９２を付与されたハイブリドーマにより産生される（１）記載のモノクローナル抗体。
（３）標識されている（２）記載のモノクローナル抗体。
（４）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に寄託され、受託番号ＮＩＴＥ Ｐ−０２０９３を付与されたハイブリドーマにより産生される（１）記載のモノクローナル抗体。
（５）標識されている（４）記載のモノクローナル抗体。
（６）（２）もしくは（３）記載のモノクローナル抗体および／または（４）もしくは（５）記載のモノクローナル抗体を含む、ＣＤ８１検出キット。
（７）対象が関節リウマチにかかっている可能性を検査、あるいは対象の関節リウマチの症状の重さを検査するための（６）記載のキット。
（８）（２）もしくは（３）記載のモノクローナル抗体および（４）もしくは（５）記載のモノクローナル抗体を用いるサンドイッチＥＬＩＳＡを含む、（６）または（７）記載のキット。
（９）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に寄託され、受託番号ＮＩＴＥ Ｐ−０２０９２を付与されたハイブリドーマ。
（１０）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に寄託され、受託番号ＮＩＴＥ Ｐ−０２０９３を付与されたハイブリドーマ。
（１１）（９）記載のハイブリドーマを培養し、培養液からモノクローナル抗体を回収することを特徴とする、ＣＤ８１ ＬＥＬに特異的なモノクローナル抗体の製造方法。
（１２）（１０）記載のハイブリドーマを培養し、培養液からモノクローナル抗体を回収することを特徴とする、ＣＤ８１ ＬＥＬに特異的なモノクローナル抗体の製造方法。
Accordingly, the present invention provides the following.
(1) A monoclonal antibody specific for CD81 LEL.
(2) The monoclonal antibody according to (1), which is produced by a hybridoma deposited with the Patent Microorganism Depositary Center and assigned with the accession number NITE P-02092.
(3) The monoclonal antibody according to (2), which is labeled.
(4) The monoclonal antibody according to (1), which is produced by a hybridoma deposited with the Patent Microorganism Depository Center and given the accession number NITE P-02093.
(5) The monoclonal antibody according to (4), which is labeled.
(6) A CD81 detection kit comprising the monoclonal antibody according to (2) or (3) and / or the monoclonal antibody according to (4) or (5).
(7) The kit according to (6), wherein the possibility of the subject having rheumatoid arthritis is examined or the severity of symptoms of the subject's rheumatoid arthritis is examined.
(8) A kit according to (6) or (7), comprising a sandwich ELISA using the monoclonal antibody according to (2) or (3) and the monoclonal antibody according to (4) or (5).
(9) A hybridoma deposited with the Patent Microorganism Depositary Center and assigned the deposit number NITE P-02092.
(10) A hybridoma deposited with the Patent Microorganism Depositary Center and assigned the deposit number NITE P-02093.
(11) A method for producing a monoclonal antibody specific for CD81 LEL, comprising culturing the hybridoma according to (9) and recovering the monoclonal antibody from the culture solution.
(12) A method for producing a monoclonal antibody specific for CD81 LEL, comprising culturing the hybridoma according to (10) and recovering the monoclonal antibody from the culture solution.
本発明によれば、微量のＣＤ８１ ＬＥＬを特異的に高感度で検出、定量できるので、関節リウマチの早期診断が可能となる。 According to the present invention, a very small amount of CD81 LEL can be detected and quantified specifically with high sensitivity, and early diagnosis of rheumatoid arthritis becomes possible.
本発明は、１の態様において、ＣＤ８１ ＬＥＬに特異的なモノクローナル抗体に関するものである。ＣＤ８１ ＬＥＬに特異的なモノクローナル抗体とは、ＣＤ８１ ＬＥＬ中のアミノ酸配列を認識して、ＣＤ８１ ＬＥＬに結合するモノクローナル抗体をいう。ＣＤ８１ ＬＥＬは潜在的な糖鎖付加部位を含んでいないので、これを特異的に認識する本発明のモノクローナル抗体は、サンプル中の微量のＣＤ８１であっても正確に検出でき、関節リウマチの早期診断、早期治療に資するものである。 In one aspect, the present invention relates to a monoclonal antibody specific for CD81 LEL. The monoclonal antibody specific for CD81 LEL refers to a monoclonal antibody that recognizes the amino acid sequence in CD81 LEL and binds to CD81 LEL. Since CD81 LEL does not contain a potential glycosylation site, the monoclonal antibody of the present invention that specifically recognizes this can accurately detect even a small amount of CD81 in a sample, and can be used for early diagnosis of rheumatoid arthritis. Contributes to early treatment.
本発明のＣＤ８１ ＬＥＬに特異的なモノクローナル抗体は、ＣＤ８１ ＬＥＬの全体または一部のアミノ酸配列を有するペプチド（以下において「ＣＤ８１ ＬＥＬポリペプチド」と称することがある）を免疫原として用いて得ることができる。ＣＤ８１ ＬＥＬのアミノ酸配列は公知であるので、当業者は公知の遺伝子工学的手法および／または合成化学的手法を用いてＣＤ８１ ＬＥＬポリペプチドを作成することができる。 The monoclonal antibody specific for CD81 LEL of the present invention can be obtained using a peptide having the whole or a part of the amino acid sequence of CD81 LEL (hereinafter sometimes referred to as “CD81 LEL polypeptide”) as an immunogen. it can. Since the amino acid sequence of CD81 LEL is known, those skilled in the art can produce a CD81 LEL polypeptide using known genetic engineering techniques and / or synthetic chemical techniques.
先ず、ＣＤ８１ ＬＥＬポリペプチドを動物に投与して免疫を行う。動物への免疫方法、手段は当業者に公知である。動物への免疫は、例えば皮下注射、皮内注射、静脈注射、脾臓への直接注射などの公知の経路によってＣＤ８１ ＬＥＬポリペプチドを投与して行う。動物としては、ラット、マウス、モルモット、ウサギ、サル、などが例示されるが、これらに限定されない。ＣＤ８１ ＬＥＬポリペプチドを動物に投与する際にフロイントの完全または不完全アジュバント等のアジュバントを用いることが好ましい。アジュバントの種類や用法についても公知である。 First, CD81 LEL polypeptide is administered to an animal for immunization. Methods and means for immunizing animals are known to those skilled in the art. Animals are immunized by administering CD81 LEL polypeptide by a known route such as subcutaneous injection, intradermal injection, intravenous injection, or direct injection into the spleen. Examples of animals include, but are not limited to, rats, mice, guinea pigs, rabbits, monkeys, and the like. It is preferred to use an adjuvant, such as Freund's complete or incomplete adjuvant, when administering the CD81 LEL polypeptide to an animal. The types and usage of adjuvants are also known.
次いで、免疫された動物から脾臓細胞を取り出し、骨髄腫細胞と融合させてハイブリドーマを得る。ハイブリドーマの取得方法は当業者に公知である。細胞融合法も公知であり、例えばポリエチレングリコールを用いる方法、センダイウイルスを用いる方法、電気融合法などが挙げられるが、これらの方法に限定されない。目的の抗体を産生するハイブリドーマをスクリーニングし、ハイブリドーマのクローン化を行う。ハイブリドーマの選択方法、抗体産生ハイブリドーマのスクリーニング方法は当業者に公知である。抗体活性の測定方法についても、目的とする抗体の特性に応じて当業者が適宜選択することができる。ハイブリドーマのクローン化方法も公知である。上記の方法以外にも、所望のモノクローナル抗体を作成する方法が知られており、例えばファージディスプレイ法を用いてもよい。 Spleen cells are then removed from the immunized animal and fused with myeloma cells to obtain hybridomas. Methods for obtaining hybridomas are known to those skilled in the art. Cell fusion methods are also known, and examples thereof include a method using polyethylene glycol, a method using Sendai virus, and an electrofusion method, but are not limited to these methods. A hybridoma producing the target antibody is screened, and the hybridoma is cloned. Methods for selecting hybridomas and methods for screening antibody-producing hybridomas are known to those skilled in the art. The method for measuring antibody activity can also be appropriately selected by those skilled in the art depending on the characteristics of the target antibody. Hybridoma cloning methods are also known. Besides the above method, a method for producing a desired monoclonal antibody is known. For example, a phage display method may be used.
本発明のＣＤ８１ ＬＥＬモノクローナル抗体はＣＤ８１ ＬＥＬに対する特異性が高く、サンプル中の微量のＣＤ８１タンパク質であっても検出することができる。本発明のＣＤ８１ ＬＥＬモノクローナル抗体の好ましい例としては、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に寄託され、受託番号ＮＩＴＥ Ｐ−０２０９２を付与されたハイブリドーマにより産生されるモノクローナル抗体、および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に寄託され、受託番号ＮＩＴＥ Ｐ−０２０９３を付与されたハイブリドーマにより産生されるモノクローナル抗体が挙げられる。 The CD81 LEL monoclonal antibody of the present invention has high specificity for CD81 LEL, and even a trace amount of CD81 protein in a sample can be detected. Preferable examples of the CD81 LEL monoclonal antibody of the present invention include a monoclonal antibody produced by a hybridoma deposited at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation, and given the accession number NITE P-02092, and an independent government. Examples include a monoclonal antibody produced by a hybridoma deposited at the Patent Microorganism Depositary and granted the Deposit Number NITE P-02093.
本発明のモノクローナル抗体の変異体や改変体、例えばキメラ抗体、ヒト化抗体も本発明のモノクローナル抗体に包含される。変異体や改変体は、元の本発明のモノクローナル抗体の特徴を維持しているものである。変異体や改変体は、例えば、元の本発明のモノクローナル抗体と同程度の強さでＣＤ８１ ＬＥＬに結合するものであってもよい。また、変異体や改変体は、元の本発明のモノクローナル抗体と同じエピトープを認識するものであってもよい。変異体や改変体の重鎖の３つの相補性決定領域（重鎖のＣＤＲ１、ＣＤＲ２、ＣＤＲ３）および軽鎖の３つの相補性決定領域（軽鎖のＣＤＲ１、ＣＤＲ２、ＣＤＲ３）のアミノ酸配列が、元の本発明のモノクローナル抗体の重鎖の重鎖のＣＤＲ１、ＣＤＲ２、ＣＤＲ３および軽鎖のＣＤＲ１、ＣＤＲ２、ＣＤＲ３のアミノ酸配列と同じであってもよい。変異体や改変体の重鎖のＣＤＲ１、ＣＤＲ２、ＣＤＲ３および軽鎖のＣＤＲ１、ＣＤＲ２、ＣＤＲ３のアミノ酸配列が、元の本発明のモノクローナル抗体の重鎖の重鎖のＣＤＲ１、ＣＤＲ２、ＣＤＲ３および軽鎖のＣＤＲ１、ＣＤＲ２、ＣＤＲ３のアミノ酸配列と異なっていてもよいが、相補性決定領域のアミノ酸配列の相違は各ＣＤＲあたり１残基、２残基または３残基が好ましい。 Variants and modifications of the monoclonal antibodies of the present invention, such as chimeric antibodies and humanized antibodies, are also encompassed by the monoclonal antibodies of the present invention. Mutants and variants maintain the characteristics of the original monoclonal antibody of the present invention. For example, the mutant or modification may bind to CD81 LEL with the same strength as the original monoclonal antibody of the present invention. In addition, the mutant or the variant may recognize the same epitope as the original monoclonal antibody of the present invention. The amino acid sequences of the three complementarity determining regions of the heavy chain of the variant or variant (CDR1, CDR2, CDR3 of the heavy chain) and the three complementarity determining regions of the light chain (CDR1, CDR2, CDR3 of the light chain) The amino acid sequences of the heavy chain CDR1, CDR2, CDR3 and the light chain CDR1, CDR2, CDR3 of the original heavy chain of the monoclonal antibody of the present invention may be the same. The amino acid sequences of CDR1, CDR2, CDR3 of the heavy chain of the variant or variant and CDR1, CDR2, CDR3 of the light chain are the CDR1, CDR2, CDR3 and light chain of the heavy chain of the original monoclonal antibody of the present invention. The amino acid sequences of CDR1, CDR2 and CDR3 may be different from each other, but the difference in the amino acid sequence of the complementarity determining region is preferably 1 residue, 2 residues or 3 residues for each CDR.
本発明のモノクローナル抗体の変異体または改変体には、キメラ抗体やヒト化抗体も包含される。キメラ抗体およびヒト化抗体の作成法は当業者に公知であり、容易に作成することができる。 The monoclonal antibody mutants or modifications of the present invention include chimeric antibodies and humanized antibodies. Methods for producing chimeric antibodies and humanized antibodies are known to those skilled in the art and can be easily produced.
本発明のモノクローナル抗体を用いてＣＤ８１ ＬＥＬを検出、測定することによってＣＤ８１を検出、測定することができる。本発明のモノクローナル抗体を用いてＣＤ８１を検出、測定する態様の具体例の１つとしてＥＬＩＳＡ法が挙げられる。ＥＬＩＳＡ法は公知であり、直接吸着法、サンドイッチ法、競合法などを用いて行うことができる。これらの方法は当業者が容易に実施することができる。サンドイッチＥＬＩＳＡの手順を以下に簡単に説明する。本発明のモノクローナル抗体（捕獲抗体または固相抗体という）を固相（プレートのウェル）に吸着させ、これにサンプルおよび本発明の別のモノクローナル抗体（一次抗体という）を添加し、次いで、検出可能な標識を付した抗体（二次抗体という）を添加する。固相としては、プレートのウェルのほかチップやビーズなどが公知であり、使用可能である。様々な二次抗体が市販されているので、適宜選択して用いることができる。反応後、標識を検出し、定量することができる。捕獲抗体と一次抗体は異なるエピトープを認識するものである。ＣＤ８１の検出、測定のためのサンドイッチＥＬＩＳＡに用いることのできる好ましい本発明のモノクローナル抗体の例としては、受託番号ＮＩＴＥ Ｐ−０２０９２を付与されたハイブリドーマにより産生されるモノクローナル抗体、および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に寄託され、受託番号ＮＩＴＥ Ｐ−０２０９３を付与されたハイブリドーマにより産生されるモノクローナル抗体が挙げられる。これらのモノクローナル抗体のいずれか一方を捕獲抗体として用い、もう一方を一次抗体として用いることができる。したがって、本発明は、さらなる態様において、２種類の本発明のモノクローナル抗体を用いるサンドイッチＥＬＩＳＡを含む、ＣＤ８１検出キットを提供するものでもある。通常、キットには取扱説明書が添付される。 CD81 can be detected and measured by detecting and measuring CD81 LEL using the monoclonal antibody of the present invention. One specific embodiment of detecting and measuring CD81 using the monoclonal antibody of the present invention is the ELISA method. The ELISA method is known and can be performed using a direct adsorption method, a sandwich method, a competitive method, or the like. These methods can be easily performed by those skilled in the art. The procedure for sandwich ELISA is briefly described below. A monoclonal antibody of the present invention (referred to as capture antibody or solid phase antibody) is adsorbed to a solid phase (well of a plate), to which a sample and another monoclonal antibody of the present invention (referred to as primary antibody) are added, and then detectable Add an antibody with a proper label (referred to as secondary antibody). As the solid phase, in addition to plate wells, chips and beads are well known and can be used. Since various secondary antibodies are commercially available, they can be appropriately selected and used. After the reaction, the label can be detected and quantified. The capture antibody and the primary antibody recognize different epitopes. Examples of preferred monoclonal antibodies of the present invention that can be used in sandwich ELISA for detection and measurement of CD81 include monoclonal antibodies produced by hybridomas assigned accession number NITE P-02092, and independent administrative agency product evaluation Examples include monoclonal antibodies produced by hybridomas deposited at the Patent Microorganism Depositary and granted accession number NITE P-02093. One of these monoclonal antibodies can be used as a capture antibody and the other as a primary antibody. Accordingly, the present invention, in a further aspect, also provides a CD81 detection kit comprising a sandwich ELISA using two types of monoclonal antibodies of the present invention. Usually, an instruction manual is attached to the kit.
本発明のモノクローナル抗体を用いてＣＤ８１を検出、測定する態様のさらなる具体例としてはウエスタンブロットが挙げられる。ウエスタンブロットの一般的手順としては、目的のタンパク質を含むサンプルをＳＤＳ−ＰＡＧＥにて展開し、ニトロセルロース膜やＰＶＤＦ膜に転写し、この膜に対して免疫染色を行うことで、タンパク質を検出するものである。ウエスタンブロットは公知であり、当業者は容易に実施することができる。したがって、本発明は、さらなる態様において、本発明のモノクローナル抗体を含む、ＣＤ８１を検出するためのウエスタンブロット用キットを提供するものである。通常、キットには取扱説明書が添付される。本発明のモノクローナル抗体を用いてＣＤ８１を検出、測定する態様は上記のものに限定されず、様々な手段、方法が公知であり、当業者はそれらを適宜選択あるいは変形して用いることができる。 As a further specific example of the mode of detecting and measuring CD81 using the monoclonal antibody of the present invention, Western blotting can be mentioned. As a general procedure for Western blotting, a sample containing the protein of interest is developed by SDS-PAGE, transferred to a nitrocellulose membrane or PVDF membrane, and this membrane is immunostained to detect the protein. Is. Western blots are known and can be easily performed by those skilled in the art. Therefore, in a further aspect, the present invention provides a kit for Western blotting for detecting CD81, which comprises the monoclonal antibody of the present invention. Usually, an instruction manual is attached to the kit. The mode of detecting and measuring CD81 using the monoclonal antibody of the present invention is not limited to the above, and various means and methods are known, and those skilled in the art can appropriately select or modify them.
ＣＤ８１の検出、測定のために、本発明のモノクローナル抗体に標識を付してもよい。抗体に付する標識は公知であり、放射性同位元素、金粒子、蛍光色素、酵素などが例示されるが、これらに限らない。標識の検出方法も公知である。 For detection and measurement of CD81, the monoclonal antibody of the present invention may be labeled. Labels attached to antibodies are known, and examples include, but are not limited to, radioisotopes, gold particles, fluorescent dyes, enzymes, and the like. Methods for detecting labels are also known.
上述のごとく、本発明のモノクローナル抗体およびキットをサンプル中のＣＤ８１ ＬＥＬの検出、測定に用いることができる。したがって、本発明のモノクローナル抗体およびキットを用いて対象の関節液サンプル中のＣＤ８１の量を測定することによって、対象が関節リウマチ等の関節液中のＣＤ８１レベルの上昇に関連する疾患にかかっている可能性あるいはその症状の重さを、診断、検査または判定することができる。本発明の好ましい態様において、かかる症状は関節リウマチである。関節液サンプル中のＣＤ８１の量が多いほど、対象が関節リウマチにかかっている可能性が高く、あるいは対象の関節リウマチの症状が重いとすることができる。 As described above, the monoclonal antibody and kit of the present invention can be used for detection and measurement of CD81 LEL in a sample. Thus, by measuring the amount of CD81 in a subject's synovial fluid sample using the monoclonal antibodies and kits of the present invention, the subject suffers from a disease associated with elevated CD81 levels in synovial fluid such as rheumatoid arthritis. The likelihood or severity of the symptoms can be diagnosed, examined or determined. In a preferred embodiment of the invention, the symptom is rheumatoid arthritis. The greater the amount of CD81 in the synovial fluid sample, the more likely the subject has rheumatoid arthritis or the more severe the symptoms of rheumatoid arthritis in the subject.
本発明は、別の態様において、ＣＤ８１ ＬＥＬに特異的なモノクローナル抗体を産生するハイブリドーマに関する。本発明のＣＤ８１ ＬＥＬに特異的なモノクローナル抗体を産生するハイブリドーマの好ましい例としては、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に寄託され、受託番号ＮＩＴＥ Ｐ−０２０９２を付与されたハイブリドーマおよび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に寄託され、受託番号ＮＩＴＥ Ｐ−０２０９３を付与されたハイブリドーマが挙げられる。 In another aspect, the present invention relates to a hybridoma that produces a monoclonal antibody specific for CD81 LEL. Preferred examples of hybridomas that produce monoclonal antibodies specific for CD81 LEL of the present invention include hybridomas deposited with the Patent Microorganism Depositary of the National Institute of Technology and Evaluation, and assigned the accession number NITE P-02092. Examples include hybridomas deposited with the Patent Microorganism Depositary Center of the National Institute for Product Evaluation and Technology and given the accession number NITE P-02093.
本発明は、さらなる態様において、ＣＤ８１ ＬＥＬに特異的なモノクローナル抗体を産生するハイブリドーマを培養し、培養液からモノクローナル抗体を回収することを特徴とする、ＣＤ８１ ＬＥＬに特異的なモノクローナル抗体の製造方法に関する。ハイブリドーマの培養、培養液からのモノクローナル抗体の回収および精製は、当業者に公知の方法によって行うことができる。 In a further aspect, the present invention relates to a method for producing a monoclonal antibody specific for CD81 LEL, comprising culturing a hybridoma producing a monoclonal antibody specific for CD81 LEL and recovering the monoclonal antibody from the culture medium. . Hybridoma culture and monoclonal antibody recovery and purification from the culture medium can be performed by methods known to those skilled in the art.
以下に実施例を示して本発明をさらに詳細かつ具体的に説明するが、実施例は本発明を限定するものと解してはならない。 The present invention will be described in more detail and specifically with reference to the following examples. However, the examples should not be construed as limiting the present invention.
動物実験は就実大学薬学部動物実験指針に従って行った。大腸菌にて産生された組換え型ヒトＬＥＬポリペプチド（アミノ酸配列を配列番号：１に示す）を抗原として用いてＢａｌｂ／ｃマウスを免疫した。免疫したマウスから脾臓細胞を取り出し、ポリエチレングリコールを用いて１ｘ１０８個の脾臓細胞を１ｘ１０７個の骨髄腫細胞と融合させた。融合細胞を９６ウェルプレートに分注し、ＨＡＴ培地を用いてハイブリドーマ細胞を選択した。 Animal experiments were conducted in accordance with the guidelines for animal experiments at the Faculty of Pharmaceutical Sciences of Tosho University. Balb / c mice were immunized using recombinant human LEL polypeptide (amino acid sequence shown in SEQ ID NO: 1) produced in E. coli as an antigen. Spleen cells were removed from the immunized mice and 1 × 10 8 spleen cells were fused with 1 × 10 7 myeloma cells using polyethylene glycol. The fused cells were dispensed into 96-well plates, and hybridoma cells were selected using HAT medium.
ＥＬＩＳＡを用いて、ＬＥＬに対する抗体力価が高いハイブリドーマをスクリーニングした。抗原（組換え型ヒトＬＥＬポリペプチド（配列番号：１））を含む緩衝液を９６ウェルプレートの各ウェルに添加し、ＢＳＡ（bovine serum albumin）をウェルに添加した。ハイブリドーマによって産生されたモノクローナル抗体（一次抗体）を含む培養上清を適宜希釈してウェルに添加し、二次抗体（アルカリホスファターゼ標識ヤギ抗マウスＩｇＧ抗体、プロメガ社Ｓ３７２１）を添加した。次いで、パラニトロフェニルリン酸（ＰＮＰＰ）を用いて発色させることにより、抗体力価を測定し、抗体力価が高いハイブリドーマを選択した。 Hybridomas with high antibody titers against LEL were screened using ELISA. A buffer containing the antigen (recombinant human LEL polypeptide (SEQ ID NO: 1)) was added to each well of a 96-well plate, and BSA (bovine serum albumin) was added to the well. The culture supernatant containing the monoclonal antibody (primary antibody) produced by the hybridoma was appropriately diluted and added to the well, and a secondary antibody (alkaline phosphatase-labeled goat anti-mouse IgG antibody, Promega S3721) was added. Subsequently, color development was performed using paranitrophenyl phosphate (PNPP) to measure the antibody titer, and a hybridoma having a high antibody titer was selected.
選択したハイブリドーマを１０％ fetal calf serumを添加したＲＰＭＩ１６４０培地にて増殖させ、１０６個のハイブリドーマをプリスタン投与したＢａｌｂ／ｃマウスに腹腔内投与し、１０〜１４日後に腹水を回収した。プロテインＧセファロース４（GE Healthcare UK Ltd., UK）カラムを用いて腹水中のモノクローナル抗体（ＩｇＧ）を精製した。HiTrap IgM Purification HP (GE Healthcare UK Ltd., UK)カラム を用いて腹水中のモノクローナル抗体（ＩｇＭ）を精製した。 Selected hybridomas were the grown at the added RPMI1640 medium with 10% fetal calf serum, 10 6 hybridomas were intraperitoneally administered Balb / c mice pristane administration, ascites was collected after 10-14 days. Monoclonal antibody (IgG) in ascites was purified using a protein G Sepharose 4 (GE Healthcare UK Ltd., UK) column. Monoclonal antibody (IgM) in ascites was purified using a HiTrap IgM Purification HP (GE Healthcare UK Ltd., UK) column.
最も高い抗体力価を示したハイブリドーマをＦ８（ＩｇＧモノクローナル抗体産生）およびＢ３（ＩｇＭモノクローナル抗体産生）と命名した。ハイブリドーマＦ８を千葉県木更津市かずさ鎌足２−５−８ 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に寄託し、２０１５年８月２５日付けで受託番号ＮＩＴＥ Ｐ−０２０９２を付与された。ハイブリドーマＢ３を千葉県木更津市かずさ鎌足２−５−８ 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に寄託し、２０１５年８月２５日付けで受託番号ＮＩＴＥ Ｐ−０２０９３を付与された。以下において、ハイブリドーマＦ８が産生するモノクローナル抗体をマウスＩｇＧ抗体Ｆ８と、ハイブリドーマＢ３が産生するモノクローナル抗体をマウスＩｇＭ抗体Ｂ３と称する。 The hybridomas that showed the highest antibody titers were named F8 (IgG monoclonal antibody production) and B3 (IgM monoclonal antibody production). Hybridoma F8 was deposited with the Patent Microorganism Depositary Center, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, and was assigned the NITE P-02092 as of August 25, 2015. The hybridoma B3 was deposited at the Patent Microorganism Depositary Center, Kazusa Kamashizu, Kisarazu City, Chiba Prefecture, and was assigned the NITE P-02093 as of August 25, 2015. Hereinafter, the monoclonal antibody produced by hybridoma F8 is referred to as mouse IgG antibody F8, and the monoclonal antibody produced by hybridoma B3 is referred to as mouse IgM antibody B3.
１．実験方法
（１）固相抗体のプレートへの固相化
９６穴プレートを蒸留水で１回洗い、５０ｍＭ カーボネートバッファー（ｐＨ９．６）で固相抗体（実施例１で得たマウスＩｇＧ抗体Ｆ８：２．５１ｍｇ／ｍＬ）を１０００倍希釈した溶液を１００μｌずつ９６穴プレートに分注（外側の２つずつを外した４Ｘ８個のウェルを使用）した。これをサランラップ（登録商標）でおおい、室温で１時間放置した後、ウェル中の溶液をピペットにより除去し、さらにプレートを逆さにして液をよく切った。
1. Experimental method (1) Immobilization of solid phase antibody on a plate A 96-well plate was washed once with distilled water, and a solid phase antibody (mouse IgG antibody F8 obtained in Example 1) was obtained with 50 mM carbonate buffer (pH 9.6): (2.51 mg / mL) was diluted 1000-fold, and 100 μl each was dispensed into a 96-well plate (using 4 × 8 wells with the outside two removed). This was covered with Saran Wrap (registered trademark) and allowed to stand at room temperature for 1 hour, and then the solution in the well was removed by a pipette, and the plate was turned upside down to thoroughly drain the solution.
（２）ブロッキング
ＢＳＡ（３％）を各ウェルに分注し、サランラップ（登録商標）でおおい、室温で１時間放置した。その後、プレートを逆さにしてＢＳＡを廃棄し、各ウェルにＰＢＳ−Ｔを２５０μｌ分注して、逆さにして液をよく切った。この操作を３回繰り返して洗浄した。
(2) Blocking BSA (3%) was dispensed into each well, covered with Saran Wrap (registered trademark), and allowed to stand at room temperature for 1 hour. Thereafter, the plate was turned upside down to discard BSA, and 250 μl of PBS-T was dispensed into each well, and the solution was turned upside down. This operation was repeated 3 times for washing.
（３）抗原と一次抗体との反応
（ｉ）プレート１
抗原（組換え型ヒトＬＥＬポリペプチド（配列番号：１）：ＬＥＬ発現ベクターを導入した大腸菌からＬＥＬポリペプチドを培地５００ｍＬあたり６０μｇ調製した）をＴＢＳで各濃度に希釈し、一次抗体（実施例１で得たマウスＩｇＭ抗体Ｂ３：３．５４ｍｇ／ｍＬ）をＴＢＳで１０００倍希釈した。希釈した抗原と一次抗体を各５０μｌずつ各ウェルに分注、混合した。その後、サランラップ（登録商標）でおおい、室温で１時間放置した。その後、ウェル中の溶液をピペットで除去し、各ウェルにＰＢＳ−Ｔを２５０μｌ分注して、逆さにして液をよく切った。この操作を３回繰り返して洗浄した。
(3) Reaction between antigen and primary antibody (i) Plate 1
Antigen (recombinant human LEL polypeptide (SEQ ID NO: 1): 60 μg of LEL polypeptide prepared from E. coli introduced with LEL expression vector per 500 mL of medium) was diluted with TBS to various concentrations, and a primary antibody (Example 1) Mouse IgM antibody B3 obtained in (3): 3.54 mg / mL) was diluted 1000 times with TBS. The diluted antigen and the primary antibody were dispensed and mixed in 50 μl of each well. Thereafter, it was covered with Saran Wrap (registered trademark) and allowed to stand at room temperature for 1 hour. Thereafter, the solution in the wells was removed with a pipette, and 250 μl of PBS-T was dispensed into each well, and the liquid was thoroughly cut by inversion. This operation was repeated 3 times for washing.
（ｉｉ）プレート２
関節リウマチ（ＲＡ）ヒト患者の関節液および変形性関節症（ＯＡ）ヒト患者の関節液を４℃、３０００ｒｐｍで１０分遠心分離後、上清を回収した。これらの上清をＴＢＳで１／２および１／４に希釈した（表２において原液１／２、原液１／４と表示）。これらのサンプルからアルブミンを除去したサンプルも調製した（表２においてア１／２、ア１／４と表示）。これらのサンプルおよび一次抗体（ＴＢＳで１０００倍希釈したマウスＩｇＭ抗体Ｂ３）を用いて、上記（ｉ）プレート１と同様に操作した。
(Ii) Plate 2
The joint fluid from a rheumatoid arthritis (RA) human patient and the joint fluid from an osteoarthritis (OA) human patient were centrifuged at 3000 rpm for 10 minutes at 4 ° C., and the supernatant was collected. These supernatants were diluted 1/2 and 1/4 with TBS (designated as stock solution 1/2 and stock solution 1/4 in Table 2). Samples from which albumin was removed from these samples were also prepared (shown as a1 / 2 and a1 / 4 in Table 2). Using these samples and the primary antibody (mouse IgM antibody B3 diluted 1000-fold with TBS), the same operation as (i) plate 1 was performed.
（４）二次抗体反応
二次抗体（ペルオキシダーゼ標識ヤギ抗マウスＩｇＭ抗体、サンタクルーズ社ＳＣ−２０６４）をＴＢＳで１０００倍希釈し、１００μｌずつ各ウェルに分注後、サランラップ（登録商標）でおおい、室温で１時間放置した。その後、ウェル中の溶液をピペットで除去し、各ウェルにＰＢＳ−Ｔを２５０μｌ分注して、逆さにして液をよく切った。この操作を３回繰り返して洗浄した。
(4) Secondary antibody reaction A secondary antibody (peroxidase-labeled goat anti-mouse IgM antibody, Santa Cruz SC-2064) is diluted 1000 times with TBS, and 100 μl is dispensed into each well and covered with Saran Wrap (registered trademark). And left at room temperature for 1 hour. Thereafter, the solution in the wells was removed with a pipette, and 250 μl of PBS-T was dispensed into each well, and the liquid was thoroughly cut by inversion. This operation was repeated 3 times for washing.
（５）検出反応
Thermo Scientific社のペルオキシダーゼ用化学蛍光基質QuantaBluTM Fluorogenic Peroxidase Substrate Kitを使用した。キット中のQuantaBluTM Substrate SolutionとQuantaBluTM Stable Peroxidase Solutionを９：１で混合したもの（ＷＳ）を１００μｌずつ各ウェルに分注し、フルオロスキャンアセントＦＬで測定した。５０分後にQuantaBluTM Stop Solutionを１００μｌずつ各ウェルに分注した。
(5) Detection reaction
Thermo Scientific Chemical Fluorescent Substrate for Peroxidase QuantaBlu ™ Fluorogenic Peroxidase Substrate Kit was used. 100 μl of a 9: 1 mixture of QuantaBlu ™ Substrate Solution and QuantaBlu ™ Stable Peroxidase Solution (WS) in the kit was dispensed into each well and measured with fluoroscan ascent FL. After 50 minutes, 100 μl of QuantaBlu ™ Stop Solution was dispensed into each well.
２．結果
プレート１の結果を図１（曲線ａ）に示す。実験を４系並行して行い、平均値を得た。図１に示す結果から、本発明のモノクローナル抗体を用いたサンドイッチＥＬＩＳＡにおいて、１０ｆｇ／ウェル〜１００００ｆｇ／ウェル（０．２ｐｇ／ｍｌ〜２００ｐｇ／ｍｌ）の範囲の組換えＬＥＬポリペプチドに関して、濃度と吸光度に正の相関が見られた。本発明のモノクローナル抗体を用いて極めて微量（ピコグラムオーダー）のＣＤ８１ ＬＥＬを検出できることがわかった。低濃度サンプルの場合は、二次抗体あたりの酵素量（あるいは他の標識量）を増加させることによって測定感度を上げることが可能である。また、高濃度サンプルの場合は、サンプルの希釈倍率を上げることによって適切な測定が可能となる。
2. Results The results for plate 1 are shown in FIG. 1 (curve a). Experiments were performed in parallel for 4 systems to obtain an average value. From the results shown in FIG. 1, in the sandwich ELISA using the monoclonal antibody of the present invention, the concentration and absorbance of the recombinant LEL polypeptide in the range of 10 fg / well to 10000 fg / well (0.2 pg / ml to 200 pg / ml). A positive correlation was observed. It was found that a very small amount (picogram order) of CD81 LEL can be detected using the monoclonal antibody of the present invention. In the case of a low-concentration sample, the measurement sensitivity can be increased by increasing the amount of enzyme (or other amount of label) per secondary antibody. In the case of a high-concentration sample, appropriate measurement can be performed by increasing the dilution factor of the sample.
比較のため、上記と同様にして、市販ウサギ抗体（サンタクルーズ社ｓｃ−９１５８）と市販マウス抗体（モノクローナル抗体、サンタクルーズ社ｓｃ−１６６０２９）による測定（図１の曲線ｂ）、ならびに市販マウス抗体（モノクローナル抗体、サンタクルーズ社ｓｃ−１６６０２９）と市販マウス抗体（モノクローナル抗体、サンタクルーズ社ｓｃ−１６６０２８）による測定（図１の曲線ｃ）も行った。いずれの場合も、試験したＬＥＬ濃度領域においては測定不可能であった。 For comparison, measurement with a commercially available rabbit antibody (Santa Cruz sc-9158) and a commercially available mouse antibody (monoclonal antibody, Santa Cruz sc-166029) (curve b in FIG. 1), and a commercially available mouse antibody in the same manner as described above. (Monoclonal antibody, Santa Cruz sc-166029) and commercially available mouse antibody (monoclonal antibody, Santa Cruz sc-166028) (curve c in FIG. 1) were also measured. In either case, measurement was not possible in the LEL concentration region tested.
プレート２の結果を表１に示す。実験を４系並行して行い、平均値を得た。
以上の実験結果から、本発明のモノクローナル抗体を用いて、関節リウマチ（ＲＡ）あるいは変形性関節症（ＯＡ）患者の関節液サンプル中のＣＤ８１（またはＬＥＬ）を検出、測定することが可能であることがわかった。 From the above experimental results, it is possible to detect and measure CD81 (or LEL) in a joint fluid sample of a patient with rheumatoid arthritis (RA) or osteoarthritis (OA) using the monoclonal antibody of the present invention. I understood it.
本発明は、生物学、医学等の研究分野、関節リウマチの診断薬の分野、および関節リウマチの研究分野において利用可能である。 The present invention can be used in research fields such as biology and medicine, in the field of diagnostic agents for rheumatoid arthritis, and in the research field of rheumatoid arthritis.
本発明のモノクローナル抗体（マウスＩｇＧ抗体Ｆ８）を産生するハイブリドーマＦ８を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に寄託し、受託番号ＮＩＴＥ Ｐ−０２０９２を付与された。
本発明のモノクローナル抗体（マウスＩｇＭ抗体Ｂ３）を産生するハイブリドーマＢ３を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に寄託し、受託番号ＮＩＴＥ Ｐ−０２０９３を付与された。
The hybridoma F8 producing the monoclonal antibody (mouse IgG antibody F8) of the present invention was deposited with the Patent Microorganism Depositary, National Institute of Technology and Evaluation, and was assigned the deposit number NITE P-02092.
Hybridoma B3 that produces the monoclonal antibody of the present invention (mouse IgM antibody B3) was deposited with the Patent Microorganism Depositary, National Institute of Technology and Evaluation, and was assigned the deposit number NITE P-02093.
配列番号：１は、実施例１で用いた組換えＬＥＬポリペプチドのアミノ酸配列を示す。 SEQ ID NO: 1 shows the amino acid sequence of the recombinant LEL polypeptide used in Example 1.
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