JP4533995B2 - Anti-ADAMTS13 monoclonal antibody - Google Patents
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Description
本発明はフォンヴィルブランド因子切断プロテアーゼ（ADAMTS13）分子に対して特異的に親和性を有するモノクローナル抗体およびその製造方法ならびにそれらの用途に関するものである。 The present invention relates to a monoclonal antibody having specific affinity for a von Willebrand factor cleaving protease (ADAMTS13) molecule, a method for producing the same, and uses thereof.
血栓性血小板減少性紫斑病(thrombotic thrombocytopenic purpura; TTP)は、血小板減少、溶血性貧血、動揺性精神神経障害などを特徴とする症候群である。かつては約８０%の患者が3ヶ月以内に死亡する予後不良の疾患であった。現在では血漿交換によって、予後が大幅に改善されるようになっている。 Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterized by thrombocytopenia, hemolytic anemia, swaying neuropsychiatric disorder and the like. In the past, about 80% of patients had a poor prognosis that died within 3 months. Currently, plasma exchange has greatly improved prognosis.
最近、TTPの病因としてフォンヴィルブランド因子(VWF)切断酵素活性の低下が報告された。すなわち、後天性のTTPは、VWF切断酵素に対するIgG型インヒビターが産生されることによって酵素活性が低下することが原因であることが明らかにされた(非特許文献1及び２)。また先天的なTTPであるUpshaw-Schulman症候群(USS)では、遺伝的にVWF切断酵素が欠損していることが判明した(非特許文献３)。このVWF切断酵素をコードする遺伝子は、ADAMTS13であることが判明した(非特許文献４及び５)。 Recently, a decrease in von Willebrand factor (VWF) cleaving enzyme activity was reported as a cause of TTP. That is, it has been clarified that acquired TTP is caused by a decrease in enzyme activity due to production of an IgG-type inhibitor for VWF-cleaving enzyme (Non-patent Documents 1 and 2). In addition, Upshaw-Schulman syndrome (USS), an innate TTP, was found to be genetically deficient in VWF-cleaving enzyme (Non-patent Document 3). The gene encoding this VWF cleaving enzyme was found to be ADAMTS13 (Non-patent Documents 4 and 5).
ADAMTS13は亜鉛型メタロプロテアーゼで、VWFサブユニットのTyr８４２−Met８４３結合を特異的に切断する。この酵素活性の測定にはVWFを基質として、生じたVWFフラグメントの検出を電気泳動法で行うVWFマルチマー解析により行われている。この方法は、ADMATS13の活性を正確に測定できるという利点はあるが操作が煩雑であるため、とりわけ簡便な測定法の開発が望まれていた。
本発明の目的は、ADAMTS13に特異的に親和性を有する種々のタイプのモノクローナル抗体の提供ならびに当該モノクローナル抗体の用途を提供することにある。また、本発明の別の目的は、当該モノクローナル抗体を産生するハイブリドーマ細胞系を提供することである。 An object of the present invention is to provide various types of monoclonal antibodies having specific affinity for ADAMTS13 and to provide uses of the monoclonal antibodies. Another object of the present invention is to provide a hybridoma cell line that produces the monoclonal antibody.
本発明者らは、ADAMTS13の測定法、検査法又は機能解析法を鋭意研究し、ADAMTS13を特異的に認識するモノクローナル抗体（以下、ADAMTS13モノクローナル抗体）を得ることに成功し、当該モノクローナル抗体を産生するハイブリドーマ細胞系を確立し、さらに当該モノクローナル抗体の用途を見い出した。 The present inventors have intensively studied the measurement method, test method or functional analysis method of ADAMTS13 and succeeded in obtaining a monoclonal antibody that specifically recognizes ADAMTS13 (hereinafter referred to as ADAMTS13 monoclonal antibody), and produced the monoclonal antibody. A hybridoma cell line was established, and the use of the monoclonal antibody was found.
本発明は以下の構成からなる。
１、フォンヴィルブランド因子切断プロテアーゼ（ADAMTS13）分子に対して特異的に親和性を有し、ADAMTS13の酵素活性を少なくとも３０％阻害するモノクローナル抗体。
２、配列表配列番号１に記載のADAMTS13分子のアミノ酸配列の３８７番目よりN末端側の上流部分に特異的に親和性を有する前記１に記載のモノクローナル抗体。
３、配列表配列番号１に記載のADAMTS13分子のアミノ酸配列の２８５番目から３８７番目のアミノ酸までの領域に存在するエピトープを認識することを特徴とする前記１又は２に記載のモノクローナル抗体。
４、配列表配列番号２に記載のアミノ酸配列の全部または一部を有する蛋白質に対して特異的に親和性を有する前記２又は３に記載のモノクローナル抗体
。
５、配列表配列番号１に記載のADAMTS13分子のアミノ酸配列の１０１６番目よりC末端側の下流部分に特異的に親和性を有する前記１に記載のモノクローナル抗体。
６、配列表配列番号１に記載のADAMTS13分子の１０１６番目のアミノ酸から１１３５番目のアミノ酸までの領域に存在するエピトープを認識することを特徴とする前記１又は５に記載のモノクローナル抗体
７、配列表配列番号３に記載のアミノ酸配列の全部または一部を有する蛋白質に対して特異的に親和性を有する前記５又は６に記載のモノクローナル抗体
。
８、受託番号がＦＥＲＭ
Ｐ−２０１８９又はＦＥＲＭ Ｐ−２０１９０であるハイブリドーマにより産生される、前記１〜７のいずれかに記載のモノクローナル抗体。
９、前記１〜８のいずれかに記載のモノクローナル抗体を産生するハイブリドーマ。
１０、受託番号がＦＥＲＭ Ｐ−２０１８９又はＦＥＲＭ Ｐ−２０１９０である前記９に記載のハイブリドーマ。
１１、前記１に記載の少なくとも１種のモノクローナル抗体と試料とを反応させる工程を含む、試料中のADAMTS13の測定方法。
１２、前記２〜４のいずれかに記載のモノクローナル抗体と試料とを反応させる工程を含む、試料中のADAMTS13の測定方法。
１３、前記５〜７のいずれかに記載のモノクローナル抗体と試料とを反応させる工程を含む、試料中のADAMTS13の測定方法。
１４、前記２〜４のいずれかに記載のモノクローナル抗体及び請求項５〜７のいずれかに記載のモノクローナル抗体を組み合わせてもちいる試料中のADAMTS13の測定方法。
１５、前記１１〜１４のいずれかに記載の方法によって、血栓性疾患を検査する方法。
１６、 前記１〜８のいずれかに記載のモノクローナル抗体を含む試薬またはキット。
The present invention has the following configuration.
1. A monoclonal antibody having specific affinity for a von Willebrand factor cleaving protease (ADAMTS13) molecule and inhibiting the enzymatic activity of ADAMTS13 by at least 30%.
2. The monoclonal antibody according to 1 above, which has an affinity specifically for the upstream portion on the N-terminal side from the 387th position of the amino acid sequence of the ADAMTS13 molecule described in SEQ ID NO: 1 in Sequence Listing.
3. The monoclonal antibody according to 1 or 2 above, which recognizes an epitope present in the region from the 285th amino acid to the 387th amino acid of the amino acid sequence of the ADAMTS13 molecule described in SEQ ID NO: 1 in the sequence listing.
4. The monoclonal antibody according to 2 or 3 above, which has specific affinity for a protein having all or part of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
5. The monoclonal antibody according to 1 above, which has an affinity specifically for the downstream portion on the C-terminal side from the 1016th position of the amino acid sequence of the ADAMTS13 molecule described in SEQ ID NO: 1 in the sequence listing
6. The monoclonal antibody 7 according to 1 or 5 above, which recognizes an epitope present in the region from the 1016th amino acid to the 1135th amino acid of the ADAMTS13 molecule described in SEQ ID NO: 1 in the sequence listing 7. The monoclonal antibody according to 5 or 6 above, which has a specific affinity for a protein having all or part of the amino acid sequence set forth in SEQ ID NO: 3.
8. Acceptance number is FERM
The monoclonal antibody according to any one of 1 to 7 above, which is produced by a hybridoma which is P-20189 or FERM P-20190.
9. A hybridoma that produces the monoclonal antibody according to any one of 1 to 8 above.
10. The hybridoma according to 9 above, wherein the accession number is FERM P-20189 or FERM P-20190.
11. A method for measuring ADAMTS13 in a sample, comprising a step of reacting the sample with at least one monoclonal antibody according to 1 above.
12. A method for measuring ADAMTS13 in a sample, comprising a step of reacting the monoclonal antibody according to any one of 2 to 4 above and the sample.
13. A method for measuring ADAMTS13 in a sample, comprising a step of reacting the monoclonal antibody according to any one of 5 to 7 and a sample.
14. A method for measuring ADAMTS13 in a sample in which the monoclonal antibody according to any one of 2 to 4 and the monoclonal antibody according to any one of claims 5 to 7 are combined.
15. A method for examining a thrombotic disease by the method according to any one of 11 to 14 above.
16. A reagent or kit comprising the monoclonal antibody according to any one of 1 to 8 above.
本発明の抗ADMATS13モノクローナル抗体は、ADAMTS13酵素抗原分子を特異的に認識するモノクローナル抗体である。当該モノクローナル抗体を用いることにより、ADAMTS13酵素分子の測定、検査することが可能になり、さらにADAMTS13の機能を解析することが可能になる。即ち、本発明の抗ADAMTS13モノクローナル抗体を用いることにより、（１）ウエスタンブロット法によるADAMTS13の迅速な検出及び同定、（２）免疫沈降法(Immunoprecipitation法) による、発現したADAMTS１３の検出及び同定、さらに、ADAMTS１３と結合性を有する他の蛋白質の検出及び解析、（３）免疫化学的手段によるADAMTS１３の測定、検査（４）それらの成果に基づく治療薬、診断薬等の開発等に大きな意義を持つ。 The anti-ADMATS13 monoclonal antibody of the present invention is a monoclonal antibody that specifically recognizes the ADAMTS13 enzyme antigen molecule. By using the monoclonal antibody, ADAMTS13 enzyme molecules can be measured and tested, and the function of ADAMTS13 can be analyzed. That is, by using the anti-ADAMTS13 monoclonal antibody of the present invention, (1) rapid detection and identification of ADAMTS13 by Western blotting, (2) detection and identification of expressed ADAMTS13 by immunoprecipitation method (Immunoprecipitation method), , Detection and analysis of other proteins that bind to ADAMTS13, (3) measurement of ADAMTS13 by immunochemical means, testing (4) development of therapeutic drugs, diagnostic drugs, etc. based on the results .
ADAMTS13は、１４２７個のアミノ酸で構成される酵素蛋白質（配列表配列番号１）で、分子量は約２００KDである。その一次構造はマルチドメインからなり、N末端側よりプロペプチド、メタロプロテアーゼドメイン、ディスインテグリン様ドメイン、トロンボスポンディン−1モチーフ、システイン−リッチドメイン、スペーサードメイン、トロンボスポンディン−1モチーフの7回繰り返し構造、そして最後に２つのＣＵＢドメインが続いている。本発明のモノクローナル抗体は、ディスインテグリン様ドメインよりもN末端側を抗原認識している。ADAMTS１３に特異的に親和性を有することを特徴とする本発明のモノクローナル抗体（本発明の抗ADAMTS１３モノクローナル抗体）はフォンヴィルブランド因子切断プロテアーゼ（ADAMTS13）の酵素活性を少なくとも３０％阻害するモノクローナル抗体に関するものである。本発明の抗ADAMTS１３モノクローナル抗体はADAMTS13分子のディスインテグリン様ドメインを認識していることを特徴としている。本発明の抗ADAMTS１３モノクローナル抗体が認識するエピトープは、該モノクローナル抗体がADAMTS１３を特異的に認識し得る限り特に限定されないが、好ましくはADAMTS１３の２８５番目のアミノ酸から３８７番目のアミノ酸までの領域（配列表配列番号１）内に存在している。 ADAMTS13 is an enzyme protein composed of 1427 amino acids (SEQ ID NO: 1 in the sequence listing) and has a molecular weight of about 200 KD. Its primary structure consists of multiple domains. From the N-terminal side, the propeptide, metalloprotease domain, disintegrin-like domain, thrombospondin-1 motif, cysteine-rich domain, spacer domain, thrombospondin-1 motif are repeated seven times The structure, and finally two CUB domains follow. The monoclonal antibody of the present invention recognizes the antigen on the N-terminal side of the disintegrin-like domain. The monoclonal antibody of the present invention (anti-ADAMTS13 monoclonal antibody of the present invention) characterized by having specific affinity for ADAMTS13 relates to a monoclonal antibody that inhibits the enzyme activity of von Willebrand factor cleaving protease (ADAMTS13) by at least 30%. Is. The anti-ADAMTS13 monoclonal antibody of the present invention is characterized by recognizing the disintegrin-like domain of the ADAMTS13 molecule. The epitope recognized by the anti-ADAMTS13 monoclonal antibody of the present invention is not particularly limited as long as the monoclonal antibody can specifically recognize ADAMTS13. Preferably, the region from the 285th amino acid to the 387th amino acid of ADAMTS13 (Sequence Listing) Present in SEQ ID NO: 1).
また、本発明のモノクローナル抗体の別の態様としては、ADAMTS１３分子のトロンボスポンディン−1モチーフの7回繰り返し構造部位を特異的に認識していることを特徴としている。本発明の抗ADAMTS１３モノクローナル抗体が認識するエピトープは、該モノクローナル抗体がADAMTS１３を特異的に認識し得る限り特に限定されないが、好ましくはADAMTS１３の１０１６番目のアミノ酸から１１３５番目のアミノ酸までの領域（配列表配列番号２）内に存在している。 Another embodiment of the monoclonal antibody of the present invention is characterized by specifically recognizing the 7-fold structure site of the thrombospondin-1 motif of the ADAMTS13 molecule. The epitope recognized by the anti-ADAMTS13 monoclonal antibody of the present invention is not particularly limited as long as the monoclonal antibody can specifically recognize ADAMTS13. Preferably, the region from the 1016th amino acid to the 1135th amino acid of ADAMTS13 (Sequence Listing) Present in SEQ ID NO: 2).
本発明のモノクローナル抗体は、当分野で通常実施されている方法により調製される。即ち、ADAMTS１３に特異的に親和性を有する抗体産生細胞を、骨髄腫細胞と融合させて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、各蛋白質に特異的な抗体を産生するクローンを選択することによって製造される。 The monoclonal antibody of the present invention is prepared by a method commonly practiced in the art. That is, antibody producing cells having specific affinity for ADAMTS13 are fused with myeloma cells to form hybridomas, the hybridomas are cloned, and clones that produce antibodies specific for each protein are selected. Manufactured.
抗原としては目的とする特異性によって異なるが、ADAMTS１３蛋白質に対して特異的に親和性を有する抗体を得る場合には、ADAMTS１３の全部またはその一部を有するオリゴペプチド等が用いられる。又、抗原性を保持している限りは、当該抗原は、そのアミノ酸配列において、１乃至数個のアミノ酸の欠失や置換、若しくは付加といった変更がなされていてもよい。 Although antigens vary depending on the target specificity, oligopeptides or the like having all or part of ADAMTS13 are used to obtain antibodies having specific affinity for ADAMTS13 protein. Further, as long as the antigenicity is maintained, the antigen may be altered in its amino acid sequence such as deletion, substitution, or addition of one to several amino acids.
当該抗原は遺伝子工学的に、あるいは選択した部分的なアミノ酸配列に基づいて合成することによっても調製できる。感作抗原としては、得られたADAMTS１３等の蛋白質分子あるいは選択した部分的なアミノ酸配列に基づいたオリゴペプチドをリン酸緩衝液（ＰＢＳ）等の適当な緩衝液中に溶解、あるいは懸濁したものが用いられる。抗原溶液は通常抗原物質の量として５０〜５００μｇ／ｍｌ程度の濃度に調製すればよい。また、ペプチド抗原等、それだけでは抗原性が低い場合は、アルブミンやキーホールリンペットヘモシアニン等の適当なキャリヤータンパク質に架橋して用いることが好ましい。該抗原を免疫感作させる動物としてはマウス、ラット、ウマ、ヤギ、ウサギなどが例示される。好ましくはマウス、より好ましくはＢＡＬＢ／ｃマウスである。このとき、被免疫動物の抗原への応答性を高めるため、当該抗原溶液をアジュバントと混合して投与することができる。本発明において用いられるアジュバントとしては、フロイント完全アジュバント（ＦＣＡ）、フロイント不完全アジュバント（ＦＩＡ）、Ｒｉｂｉ（ＭＰＬ）、Ｒｉｂｉ（ＴＤＭ）、Ｒｉｂｉ（ＭＰＬ＋ＴＤＭ）、百日咳ワクチン(Bordetella pertussis vaccine)、ムラミルジペプチド（ＭＤＰ）、アルミニウムアジュバント（ＡＬＵＭ）、およびこれらの組合せが例示されるが、初回免疫時にＦＣＡ、追加免疫時にＦＩＡを使用する組み合わせが特に好ましい。 The antigen can also be prepared by genetic engineering or by synthesis based on a selected partial amino acid sequence. As a sensitizing antigen, a protein molecule such as ADAMTS13 obtained or an oligopeptide based on a selected partial amino acid sequence is dissolved or suspended in an appropriate buffer such as a phosphate buffer (PBS). Is used. The antigen solution is usually prepared to a concentration of about 50 to 500 μg / ml as the amount of the antigen substance. In addition, when a peptide antigen or the like alone has low antigenicity, it is preferably used after being crosslinked with an appropriate carrier protein such as albumin or keyhole limpet hemocyanin. Examples of animals to be immunized with the antigen include mice, rats, horses, goats, rabbits and the like. Preferred are mice, more preferred are BALB / c mice. At this time, in order to increase the responsiveness of the immunized animal to the antigen, the antigen solution can be mixed with an adjuvant and administered. Adjuvants used in the present invention include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL + TDM), pertussis vaccine (Bordetella pertussis vaccine), muramyl dipeptide (MDP), aluminum adjuvant (ALUM), and combinations thereof are exemplified, but a combination using FCA at the time of initial immunization and FIA at the time of booster immunization is particularly preferable.
免疫方法は、使用する抗原の種類やアジュバント混合の有無等により、注射部位、スケジュールなどを適宜変化させることができるが、例えば、被免疫動物としてマウスを用いる場合は、アジュバント混合抗原溶液０．０５〜１ｍｌ（抗原物質１０〜２００μｇ）を腹腔内、皮下、筋肉内または（尾）静脈内に注射し、初回免疫から約４〜１４日毎に１〜４回追加免疫を行い、さらに約１〜４週間後に最終免疫を行う。該抗原溶液をアジュバントを使用せずに投与する場合には、抗原量を多くして腹腔内注射してもよい。抗体価は追加免疫の約５〜６日後に採血して調べる。抗体価の測定は、後述の抗体アッセイに準じ、通常行われる方法で行うことができる。最終免疫より約３〜５日後、該免疫動物から脾細胞を分離して抗体産生細胞を得る。 The immunization method can appropriately change the injection site, schedule, etc. depending on the type of antigen to be used, the presence or absence of adjuvant mixing, etc. For example, when using a mouse as the immunized animal, an adjuvant mixed antigen solution 0.05 -1 ml (10 to 200 μg of antigenic substance) is injected intraperitoneally, subcutaneously, intramuscularly, or (tail) intravenously, and boosted 1 to 4 times about every 4 to 14 days from the first immunization, and further about 1 to 4 Final immunization is performed after a week. When administering the antigen solution without using an adjuvant, the antigen amount may be increased and injected intraperitoneally. The antibody titer is examined by collecting blood about 5 to 6 days after the booster immunization. The antibody titer can be measured by a commonly performed method according to the antibody assay described below. About 3 to 5 days after the final immunization, spleen cells are separated from the immunized animal to obtain antibody-producing cells.
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。例えばマウスミエローマＰ３Ｘ６３−Ａｇ８、Ｐ３Ｘ６３−Ａｇ８−Ｕ１、Ｐ３ＮＳ１−Ａｇ４、ＳＰ２／０−Ａｇ１４、Ｐ３Ｘ６３−Ａｇ８・６５３等が例示されるが、抗体産生細胞と骨髄腫細胞とは同種動物、特に同系統の動物由来であることが好ましい。骨髄腫細胞は凍結保存するか、ウマ、ウサギまたはウシ胎児血清を添加した一般的な培地で継代して維持することができる。細胞融合には対数増殖期の細胞を用いるのが好ましい。 As myeloma cells, those derived from mice, rats, humans and the like are used. Examples include mouse myeloma P3X63-Ag8, P3X63-Ag8-U1, P3NS1-Ag4, SP2 / 0-Ag14, P3X63-Ag8.653, and the like. It is preferably derived from an animal. Myeloma cells can be stored frozen or passaged and maintained in common media supplemented with horse, rabbit or fetal calf serum. For cell fusion, cells in the logarithmic growth phase are preferably used.
抗体産生細胞と骨髄腫細胞とを融合させてハイブリドーマを形成させる方法としては、ポリエチレングリコール（ＰＥＧ）を用いる方法、センダイウイルスを用いる方法、電気融合装置を用いる方法などが例示される。例えばＰＥＧ法の場合、約３０〜６０％のＰＥＧ（平均分子量１０００〜６０００）を含む適当な培地または緩衝液中に脾細胞と骨髄腫細胞を１〜１０：１、好ましくは５〜１０：１の混合比で懸濁し、温度約２５〜３７℃、ｐＨ６〜８の条件下で、約３０秒〜３分間程度反応させればよい。反応終了後、ＰＥＧ溶液を除いて培地に再懸濁し、セルウエルプレート中に播種して培養を続ける。 Examples of the method for fusing antibody-producing cells and myeloma cells to form hybridomas include a method using polyethylene glycol (PEG), a method using Sendai virus, and a method using an electrofusion device. For example, in the case of the PEG method, splenocytes and myeloma cells are placed in an appropriate medium or buffer containing about 30 to 60% PEG (average molecular weight 1000 to 6000), preferably 1 to 10: 1, preferably 5 to 10: 1. And the reaction may be carried out for about 30 seconds to 3 minutes under conditions of a temperature of about 25 to 37 ° C. and a pH of 6 to 8. After completion of the reaction, the PEG solution is removed, the suspension is resuspended in a medium, seeded in a cell well plate, and the culture is continued.
融合操作後の細胞を選択培地で培養して、ハイブリドーマの選択を行う。選択培地は、親細胞株が死滅し、融合細胞のみが増殖し得る培地であり、通常ヒポキサンチン−アミノプテリン−チミジン（ＨＡＴ）培地が使用される。ハイブリドーマの選択は、通常融合操作の１〜７日後に、培地の一部、好ましくは約半量を選択培地と交換することによって開始し、さらに２、３日毎に同様の培地交換を繰り返しながら培養することにより行う。顕微鏡観察によりコロニーが生育しているウエルを確認する。 The hybridoma is selected by culturing the cells after the fusion operation in a selective medium. The selection medium is a medium in which the parent cell line can be killed and only the fused cells can grow. Usually, hypoxanthine-aminopterin-thymidine (HAT) medium is used. Selection of hybridoma is usually started by exchanging a part of the medium, preferably about half of the medium, with the selective medium 1 to 7 days after the fusion operation, and further cultivating while repeating the same medium exchange every few days. By doing. Confirm wells where colonies are growing by microscopic observation.
生育しているハイブリドーマが所望の抗体を産生しているかどうかを知るには、培養上清を採取して抗体アッセイを行えばよい。抗体価は、例えば固相化したADAMTS１３酵素分子に該上清を加えて反応させ、さらに蛍光物質、酵素、ＲＩ等で標識した二次抗体（抗グロブリン、抗ＩｇＧ、抗ＩｇＭ血清等）を反応させて測定することができる。このようにして適切な抗体を産生しているウエルを得る。さらに限界希釈法、軟寒天法、蛍光励起セルソーターを用いた方法等により単一クローンを分離する。例えば限界希釈法の場合、ハイブリドーマコロニーを１細胞／ウエル前後となるように培地で段階希釈し、培養することにより目的とするモノクローナル抗体を産生するクローンを単離することができる。得られた抗体産生ハイブリドーマは、約１０％（ｖ／ｖ）ジメチルスルホキシド（ＤＭＳＯ）あるいはグリセリン等の凍結保護剤の共存下に凍結させて−７０〜−１９６℃で保存すると、約半年〜半永久的に保存可能である。細胞は用時３７℃前後の恒温槽中で急速融解して使用する。凍結保護剤の細胞毒性が残存しないようによく洗浄してから使用するのが望ましい。 In order to know whether the growing hybridoma is producing the desired antibody, the culture supernatant is collected and an antibody assay is performed. The antibody titer can be determined by, for example, reacting the supernatant with ADAMTS13 enzyme molecules that have been immobilized, and then reacting secondary antibodies (antiglobulin, anti-IgG, anti-IgM serum, etc.) labeled with fluorescent substances, enzymes, RI, etc. Can be measured. In this way, wells producing appropriate antibodies are obtained. Further, single clones are separated by a limiting dilution method, a soft agar method, a method using a fluorescence excitation cell sorter, or the like. For example, in the case of the limiting dilution method, a clone producing a target monoclonal antibody can be isolated by serially diluting a hybridoma colony with a medium so as to be about 1 cell / well and culturing. The obtained antibody-producing hybridoma is frozen for about 10% (v / v) dimethyl sulfoxide (DMSO) or in the presence of a cryoprotectant such as glycerin and stored at −70 to −196 ° C. Can be stored. Cells are used after being rapidly thawed in a thermostatic chamber at around 37 ° C. when used. It is desirable to use after thoroughly washing so that the cytotoxicity of the cryoprotectant does not remain.
上記の方法で得られる本発明のモノクローナル抗体は、具体的には、例えばマウス由来かつＩｇＧクラスのモノクローナル抗体であって、Ａ１０及びＣ７と命名されたものである。ハイブリドーマが産生する抗体のサブクラスを調べるためには、該ハイブリドーマを一般的な条件で培養し、その培養上清中に分泌された抗体のクラスを市販の抗体クラス・サブクラス判定用キットなどを用いて分析することにより知ることができる。 Specifically, the monoclonal antibody of the present invention obtained by the above method is, for example, a mouse-derived IgG class monoclonal antibody and designated as A10 and C7. In order to examine the subclass of the antibody produced by the hybridoma, the hybridoma is cultured under general conditions, and the class of the antibody secreted in the culture supernatant is determined using a commercially available antibody class / subclass determination kit or the like. You can know by analyzing.
本発明のモノクローナル抗体を産生するハイブリドーマのうち、モノクローナル抗体A10及びC7を産生するハイブリドーマA10株及びC7株は本発明者らによって新たに分離され、具体的には、ブタペスト条約に基づく国際寄託機関である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
（〒305-8566 茨城県つくば市東1-1-1 中央第6）に２００４年８月３１日付けで国内寄託され、受託番号としてＦＥＲＭ Ｐ−２０１８９及びＦＥＲＭ Ｐ−２０１９０が付されている。
Among the hybridomas producing the monoclonal antibodies of the present invention, the hybridoma A10 and C7 strains producing the monoclonal antibodies A10 and C7 were newly isolated by the present inventors, specifically, an international depository organization based on the Budapest Treaty. The National Institute of Advanced Industrial Science and Technology (AIST) was deposited in Japan on August 31, 2004 at the Patent Biological Deposit Center (Central 1-1, Higashi 1-1-1 Tsukuba City, Ibaraki Prefecture 305-8566). 201089 and FERM P-20190 are attached.
モノクローナル抗体の取得は、その必要量やハイブリドーマの性状等によってマウス腹水から取得するか、細胞培養によるか適宜選択できる。マウス腹腔内で増殖可能なハイブリドーマは腹水から数ｍｇ／ｍｌの高濃度で得ることができる。インビボで増殖できないハイブリドーマは細胞培養の培養上清から取得する。細胞培養によれば、抗体産生量はインビボより低いが、腹腔内に含まれる免疫グロブリンや他の夾雑物質の混入が少なく、精製が容易であるという利点がある。 Monoclonal antibodies can be appropriately selected from the ascites of the mouse or the cell culture depending on the required amount and the properties of the hybridoma. A hybridoma capable of growing in the mouse abdominal cavity can be obtained from ascites at a high concentration of several mg / ml. Hybridomas that cannot grow in vivo are obtained from the culture supernatant of the cell culture. According to cell culture, the amount of antibody production is lower than in vivo, but there is an advantage that purification is easy because there is little contamination with immunoglobulins and other contaminants contained in the abdominal cavity.
マウス腹腔内から取得する場合、例えば、予めプリスタン（２，６，１０，１４−テトラメチルペンタデカン）等の免疫抑制作用を有する物質を投与したＢＡＬＢ／ｃマウスの腹腔内へハイブリドーマ（約１０6
個以上）を移植し、約１〜３週間後に貯留した腹水を採取する。異種ハイブリドーマ（例えばマウスとラット）の場合には、ヌードマウス、放射線処理マウスを使用することが好ましい。
When the mouse is obtained from the intraperitoneal cavity, for example, a hybridoma (about 10 6 ) is injected into the intraperitoneal cavity of a BALB / c mouse to which a substance having an immunosuppressive action such as pristane (2,6,10,14-tetramethylpentadecane) is administered in advance.
Ascites collected after about 1 to 3 weeks is collected. In the case of heterologous hybridomas (for example, mice and rats), it is preferable to use nude mice or radiation-treated mice.
一方、細胞培養上清から抗体を取得する場合、例えば、細胞維持に用いられる静置培養法の他に、高密度培養方法あるいはスピンナーフラスコ培養方法などの培養法を用い、当該ハイブリドーマを培養し抗体を含有する培養上清を得る。血清には、他の抗体やアルブミン等の夾雑物が含まれ、抗体精製に不便な点が多いので培養液への添加は少なくすることが望ましい。 On the other hand, when the antibody is obtained from the cell culture supernatant, for example, in addition to the stationary culture method used for cell maintenance, the hybridoma is cultured by using a culture method such as a high-density culture method or a spinner flask culture method. A culture supernatant containing is obtained. Serum contains contaminants such as other antibodies and albumin, and there are many inconveniences in antibody purification, so it is desirable to reduce the addition to the culture medium.
腹水、培養上清からのモノクローナル抗体の精製は、免疫グロブリンの精製法として従来既知の硫酸アンモニウムや硫酸ナトリウムを用いた塩析による分画法、ポリエチレングリコール分画法、エタノール分画法、ＤＥＡＥイオン交換クロマトグラフィー法、ゲル濾過法等を応用することで、容易に達成される。さらに、本発明の抗ADAMTS１３モノクローナル抗体が、マウスＩｇＧ抗体である場合には、プロテインＡ結合担体あるいは抗マウスイムノグロブリン結合担体を用いたアフィニティークロマトグラフィーにより精製することが可能であり、簡便である。 Monoclonal antibody purification from ascites and culture supernatant is performed by conventional fractionation by salting out using ammonium sulfate or sodium sulfate, polyethylene glycol fractionation method, ethanol fractionation method, DEAE ion exchange. This can be easily achieved by applying a chromatography method, a gel filtration method or the like. Furthermore, when the anti-ADAMTS13 monoclonal antibody of the present invention is a mouse IgG antibody, it can be easily purified by affinity chromatography using a protein A binding carrier or an anti-mouse immunoglobulin binding carrier.
本発明の新規モノクローナル抗体を用いて試料中のADAMTS１３を迅速に測定することが可能である。本発明のモノクローナル抗体の1種又は2種以上を組み合わせて用いることが可能である。本発明の１種類のモノクローナル抗体と、もう一種類のモノクローナル抗体の組み合わせ、１種類の本発明のモノクローナル抗体とポリクローナル抗体の組み合わせ、さらには、本発明の一種類のモノクローナル抗体と本発明のモノクローナル抗体の機能以外の機能を有するモノクローナル抗体を組み合わせて用いることも本発明に含まれる。測定方法としては、通常当分野で行われる各種のイムノアッセイが利用され得る。該方法は検体と該抗体とを反応させ、形成される免疫複合体を測定する工程を含むものであれば特に限定されず、沈降反応や凝集反応を光学的に検出する免疫比濁法や、分別検出の容易な物質で標識した抗体を用いる標識化免疫測定法などがある。後者には、免疫複合体の検出に標識としてＲＩを用いるラジオイムノアッセイ、アルカリホスファターゼやパーオキシダーゼ等の酵素を用いるエンザイムイムノアッセイ、蛍光物質を用いる蛍光イムノアッセイなどが含まれる。標識する対象によって、検出すべき抗体を直接標識する直接法の他、検出すべき抗体の抗体つまり二次抗体を標識する間接法などがある。間接法を用いる場合、例えば本発明の抗ADAMTS１３モノクローナル抗体がマウスＩｇＧモノクローナル抗体である場合、二次抗体としては例えば抗マウスＩｇＧポリクローナル抗体等を使用すればよい。該二次抗体の調製法、並びに抗体の蛍光物質、ＲＩおよび酵素等による標識は、当分野で慣用の方法を用いて行うことができる。また、当該測定法にビオチン−アビジン（又はストレプトアビジン）の反応を利用する方法も可能であり、高い感度が要求される測定の場合には好ましく採用される。当該方法としては、例えばビオチンで標識した抗ADAMTS１３モノクローナル抗体と蛍光物質等で標識したストレプトアビジンとを組み合わせて用いるものが挙げられる。抗ADAMTS１３モノクローナル抗体のビオチンでの標識、ストレプトアビジンの蛍光物質等での標識は、当分野で通常行われる方法を用いて行うことができ、例えば蛍光物質等で標識したストレプトアビジンは商業的にも入手可能である。 ADAMTS13 in a sample can be rapidly measured using the novel monoclonal antibody of the present invention. One or more of the monoclonal antibodies of the present invention can be used in combination. Combination of one type of monoclonal antibody of the present invention and another type of monoclonal antibody, one type of combination of the monoclonal antibody of the present invention and a polyclonal antibody, and one type of monoclonal antibody of the present invention and the monoclonal antibody of the present invention Use of a combination of monoclonal antibodies having functions other than the above functions is also included in the present invention. As a measuring method, various immunoassays usually performed in this field can be used. The method is not particularly limited as long as it includes a step of reacting a specimen with the antibody and measuring the formed immune complex, an immunoturbidimetric method for optically detecting a precipitation reaction or an agglutination reaction, For example, a labeled immunoassay using an antibody labeled with a substance that can be easily detected separately. The latter includes a radioimmunoassay using RI as a label for detection of immune complexes, an enzyme immunoassay using enzymes such as alkaline phosphatase and peroxidase, and a fluorescent immunoassay using a fluorescent substance. In addition to the direct method of directly labeling the antibody to be detected depending on the target to be labeled, there are an indirect method of labeling the antibody of the antibody to be detected, that is, the secondary antibody. When the indirect method is used, for example, when the anti-ADAMTS13 monoclonal antibody of the present invention is a mouse IgG monoclonal antibody, an anti-mouse IgG polyclonal antibody or the like may be used as the secondary antibody. The method for preparing the secondary antibody and the labeling of the antibody with a fluorescent substance, RI, enzyme, and the like can be performed using a method commonly used in this field. In addition, a method using a biotin-avidin (or streptavidin) reaction is also possible for the measurement method, which is preferably employed in the case of measurement requiring high sensitivity. Examples of the method include a method using a combination of anti-ADAMTS13 monoclonal antibody labeled with biotin and streptavidin labeled with a fluorescent substance or the like. The labeling of the anti-ADAMTS13 monoclonal antibody with biotin and the labeling of streptavidin with a fluorescent substance or the like can be carried out using a method commonly used in this field. For example, streptavidin labeled with a fluorescent substance or the like is commercially available. It is available.
本発明の測定方法により測定される検体は特に制限はなく血液が一般的であるが、当該測定は細胞、組織レベルで測定可能である他、当該細胞、組織からの抽出物を用いても測定可能である。これらの測定方法は通常当分野で行われている方法を適用し得る。 The sample measured by the measurement method of the present invention is not particularly limited and blood is generally used. However, the measurement can be performed at the cell and tissue level, and the measurement can also be performed using an extract from the cell and tissue. Is possible. As these measurement methods, methods usually used in the art can be applied.
さらに本発明においては、本発明の新規モノクローナル抗体を試薬に含めることができる。ここでいう試薬には、例えば試料中のADAMTS１３の検出用試薬、キット、当該酵素分子に密接に関与する分子の検出用試薬、当該酵素分子の精製の為の試薬等が挙げられる。 Furthermore, in the present invention, the novel monoclonal antibody of the present invention can be included in the reagent. Examples of the reagent herein include a reagent for detecting ADAMTS13 in a sample, a kit, a reagent for detecting a molecule closely related to the enzyme molecule, and a reagent for purifying the enzyme molecule.
ADAMTS１３の検出用試薬としては、測定原理として直接法を用いる場合、該試薬は、例えばＲＩ、酵素、蛍光物質等で標識された該モノクローナル抗体を適当な緩衝液中に溶解した溶液の他、標識検出用試薬、ブロッキング液、洗浄液等から構成される。洗浄液としては、ドデシル硫酸ナトリウム（ＳＤＳ）やポリオキシエチレンソルビタンモノラウレートなどのイオン性または非イオン性界面活性剤あるいはゼラチン等を含有するＰＢＳ等の緩衝液（さらにアジ化ナトリウムを含有してもよい）が、ブロッキング液としてはウシ血清アルブミン（ＢＳＡ）や非イオン性界面活性剤（例えばポリオキシエチレンソルビタンモノラウレート等）などを含有するＰＢＳ等の緩衝液（さらにアジ化ナトリウムを含有してもよい）が例示される。バックグラウンドの高い試料を測定する場合などには、ブロッキング液はさらにヤギ等の動物由来血清を含んでいることが好ましい。一方、間接法を用いる場合には、該試薬は、例えば未標識の該モノクローナル抗体および該モノクローナル抗体に特異性を有する、ＲＩ、酵素、蛍光物質等で標識された二次抗体、ブロッキング液、洗浄液等から構成される。さらに、電気泳動法等と組み合わせて測定を行う場合、該電気泳動法に使用する試薬等を共に梱包しておくことも可能である。蛍光顕微鏡による蛍光測定を行う為の試薬には、試料（細胞や組織など）の封入剤を共に梱包しておくこともでき、かかる封入剤としては、グリセロール含有ＰＢＳ、ポリビニルアルコール含有ＰＢＳ等が好ましく例示される。 As a reagent for detecting ADAMTS13, when the direct method is used as a measurement principle, the reagent is, for example, a solution obtained by dissolving the monoclonal antibody labeled with RI, an enzyme, a fluorescent substance or the like in an appropriate buffer, or a label. It consists of a detection reagent, a blocking solution, a washing solution and the like. As a washing solution, a buffer solution such as PBS containing ionic or nonionic surfactant such as sodium dodecyl sulfate (SDS) or polyoxyethylene sorbitan monolaurate or gelatin (and also containing sodium azide) However, as a blocking solution, a buffer solution such as PBS (containing sodium azide) containing bovine serum albumin (BSA) or a nonionic surfactant (such as polyoxyethylene sorbitan monolaurate) may be used. May also be exemplified. When measuring a sample with a high background, the blocking solution preferably further contains animal-derived serum such as goat. On the other hand, when the indirect method is used, the reagent is, for example, an unlabeled monoclonal antibody and a secondary antibody labeled with RI, an enzyme, a fluorescent substance or the like having specificity for the monoclonal antibody, a blocking solution, and a washing solution. Etc. Furthermore, when measurement is performed in combination with electrophoresis or the like, it is possible to pack together reagents and the like used for the electrophoresis. The reagent for performing fluorescence measurement with a fluorescence microscope can be packed with a sample (cell, tissue, etc.) encapsulant, and as such encapsulant, glycerol-containing PBS, polyvinyl alcohol-containing PBS, etc. are preferable. Illustrated.
ADAMTS１３の精製用の試薬としては、例えば当該モノクローナル抗体の他に、当分野で一般的に行われているアフィニティー精製の方法に従って、必要なものを含めることができる。具体的には、本発明のモノクローナル抗体
の他、当該モノクローナル抗体を固定化する場合にはその為の担体や試薬、洗浄用や抗原溶出用の緩衝液等が挙げられる。
As a reagent for ADAMTS13 purification, for example, in addition to the monoclonal antibody, necessary reagents can be included according to the method of affinity purification generally performed in the art. Specifically, in addition to the monoclonal antibody of the present invention, when immobilizing the monoclonal antibody, carriers and reagents therefor, buffers for washing and antigen elution, and the like can be mentioned.
ADAMTS１３酵素分子に関与する分子の検出用試薬としては、目的とする分子の性質によっても異なるが、例えば、免疫沈降法を利用した検出法に用いられる試薬が挙げられる。具体的には、本発明のモノクローナル抗体の他、当該モノクローナル抗体を固定化する為の担体、洗浄用の緩衝液等が挙げられ、沈降後の当該分子及びADAMTS１３酵素分子の検出には通常の電気泳動用試薬や前述のウエスタンブロット法で用いる試薬と同様のものが例示される。 As a reagent for detecting a molecule involved in the ADAMTS13 enzyme molecule, for example, a reagent used in a detection method using an immunoprecipitation method may be mentioned, although it varies depending on the properties of the target molecule. Specifically, in addition to the monoclonal antibody of the present invention, a carrier for immobilizing the monoclonal antibody, a buffer for washing, and the like can be mentioned. Examples thereof include the same reagents for electrophoresis and the reagents used in the aforementioned Western blot method.
本発明はADAMTS１３測定による臨床検査にも適用される。適用される臨床検査は、血栓性血小板減少紫斑症（TTP）、溶血性***候群（HUS）、播種性血管内凝固症候群（DIC）、脳梗塞、慢性肝疾患、悪性腫瘍、HIV,心筋梗塞、自己免疫疾患、妊娠合併症、急性腎不全等の病状進展のリスクファクターの検査に適用される。 The present invention is also applied to clinical examinations using ADAMTS13 measurement. Laboratory tests applied include thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), disseminated intravascular coagulation (DIC), cerebral infarction, chronic liver disease, malignant tumor, HIV, myocardial infarction, Applicable to the examination of risk factors for disease progression such as autoimmune diseases, pregnancy complications, acute renal failure.
本発明のモノクローナル抗体は、ADAMTS13分子に対する機能が特定されているため、ADAMTS13の測定や検査法に適用されるばかりでなく、ADAMTS13の機能解析のツールとしても用いられる。例えば、基質であるVWFとADAMTS13の結合が正常に行われているか否かの機能解析、プロテアーゼ活性が有るか否かの機能解析等に用いることが可能である。さらには、後天的にADAMTS13分子に対する自己抗体出現により、ADAMTS13の活性が減少したり、抗原量が減少したりすることがあるが、このような場合においても、本発明のモノクローナル抗体を用いることにより、自己抗体の性質を検索するツールに用いることも可能である。 Since the monoclonal antibody of the present invention has a function for ADAMTS13 molecules, it is not only applied to ADAMTS13 measurement and testing methods, but also used as a function analysis tool for ADAMTS13. For example, it can be used for functional analysis of whether or not the binding between the substrate VWF and ADAMTS13 is normally performed, functional analysis of whether or not protease activity is present, and the like. Furthermore, ADAMTS13 activity may decrease or the amount of antigen may decrease due to the appearance of autoantibodies against ADAMTS13 molecules. In such cases, the monoclonal antibody of the present invention can be used. It can also be used as a tool for searching for the properties of autoantibodies.
本発明は、ADAMTS13を測定するための試薬、キット及び上述の検査用の試薬、キットにも及ぶ。試薬、キットの例としては、前記の免疫学的測定法に適したものを適宜選択して用いる。例えばEIA法であれば、抗体を固相化したマイクロプレートや磁性粒子等の固相試薬、標識抗体試薬、酵素基質試薬、標準液、洗浄液等を適宜組み合わせてキット化することが可能である。 The present invention extends to reagents and kits for measuring ADAMTS13, and the above-described test reagents and kits. As examples of reagents and kits, those suitable for the immunoassay described above are appropriately selected and used. For example, in the case of the EIA method, it is possible to prepare a kit by appropriately combining solid phase reagents such as microplates and magnetic particles on which antibodies are immobilized, labeled antibody reagents, enzyme substrate reagents, standard solutions, washing solutions and the like.
ハイブリドーマならびに抗ADAMTS１３モノクローナル抗体の作製
ハイブリドーマの作製方法
（１）マウス：７乃至８週令の近交系ＢＡＬＢ／ｃ系マウス雌を、動物飼育チェンバー内（２３±１℃、湿度７０％）で、標準ペレットを使用して飼育し、任意に給水して飼育した。
（２）免疫抗原：Ｃ末端フラッグ配列を有するＡＤＡＭＴＳ１３遺伝子をＰＣＲによりｃＤＮＡライブラリーより増幅させ、それを発現ベクターｐＣＡＧＣに繋ぎ、ＨｅＬａ細胞に導入した。細胞を無血清培地ＯＰＴＹＩＭＥＭ Ｉ 培地で44時間培養し、培養上清を集め抗ＦＬＡＧ Ｍ２モノクローナル抗体で精製したものをＡＤＡＭＴＳ１３抗原として用いた。
（３）免疫方法：
ADAMTS13抗原を１００μｇ／０．５ｍｌとなる様にＰＢＳで調製し、同量（０．５ｍｌ）のフロイント完全アジュバント（Freund's
complete adjuvant) （Ｄｉｆｃｏ社製）を混合して乳化した。この乳化状の抗原を７週令の４匹の雌のＢＡＬＢ／ｃマウスの腹腔に１匹あたり２００μｌ投与した。さらに２週間毎に、ＧＥＲＢＵＶＡＮＴ（ＧＥＲＢＵ
Ｂｉｏｔｅｃｈｎｉｋ，ＧｍｂＨ，Ｄ−６９０１ Ｇｕｉｂｅｒｇ，Ｇｅｒｍａｎｙ製）で１００μｇ／ｍｌとなるように調製した上記抗原をマウスあたり２０μｇずつ４回投与した。さらに１ヶ月後、ＧＥＲＢＵＶＡＮＴで１００μｇ／ｍｌとなるように調製した上記抗原を同様に追加免疫した後、マウスの抗体価を測定した。抗体価の高いマウスはさらに２週間後にＰＢＳで１００μｇ／ｍｌに調製したADAMTS13抗原を、マウス尾静脈より注射して最終免疫とした。尚、抗体価の測定は、当該抗原で免疫したマウスの血清を用いて、後述のスクリーニングの方法に準じて行った。
（４）細胞融合：最終免疫から３日後にＢＡＬＢ／ｃマウスの摘脾を行い、ＤＭＥＭ培養液中で脾細胞を浮遊させて、脾細胞の浮遊液を作製した。ついで、細胞数を算定し、１．９×１０8
個の脾細胞を得た。細胞融合は、２−アミノ−６−メルカプトプリン（６−チオグアニン［2-amino-6-mercaptopurine] ）耐性のＢＡＬＢ／ｃマウス由来骨髄腫培養細胞株（Ｐ３−Ｘ６３−Ａｇ８・６５３、以下Ｘ６３細胞ともいう）を親細胞株として用いた。Ｘ６３細胞は、牛胎児血清（fetal
calf serum: ＦＣＳ）１０％を含むDMEM培養液（5μｇ／ｍｌ、6-チオグアニン含有）で継代培養し、細胞融合の３日前より６−チオグアニンを含有しない１０％ＦＣＳ含有ＤＭＥＭ培養液でさらに培養し、対数増殖期の細胞を用いた。Ｘ６３細胞の細胞数を算定し、１．９×１０８個の生細胞を得た。ＤＭＥＭ培養液で、ポリエチレングリコール−１５００が５０（ｗ／ｖ）％濃度となるように溶解し、上記の脾細胞とＸ６３細胞の比が１：１となるように混合し、公知の方法（ケラーとミルスタイン共著，Nature,
第256 巻, 495-497 頁, 1975年、Eur. J. Immunol.
第6 巻, 511-519 頁, 1976年）に準じて細胞融合を行った。その後、１０％ＦＣＳおよび５％ブリクローン（Ａｒｃｈｐｏｒｔ社製）を添加したＤＭＥＭ培養液に、１×１０-4Ｍのヒポキサンチン、４×１０-7Ｍのアメソプテリン及び１．６×１０-5Ｍのチミジンを含有するＨＡＴ選択液を加え、脾細胞が２．０×１０6
個／ｍｌとなるように浮遊させた。ついで、この細胞浮遊液の１００μｌを、９６ウエルのマイクロタイタープレートの各ウエルに分注した後、ＣＯ2
無菌培養器において温度３７℃、湿度１００％、５％のＣＯ2条件下で培養を行った。培養開始後、３日目にＨＡＴ培地１００μＬを各ウエルに加え、その後３日おきに半分量のＨＡＴ培地を交換しながら培養を行った。その後、２〜３週間後に、目的の抗ADAMTS１３モノクローナル抗体を産生するクローンを、ADAMTS１３抗原を固相に吸着させたマイクロプレートを用いたエライザ法によるスクリーニングによって検索した。
（５）スクリーニング：上記ハイブリドーマ細胞の培養上清を用いて、ADAMTS１３抗原固定化エライザプレートとの反応により選択した。尚、非特異オリゴペプチド固相化エライザプレートに反応する非特異反応性クローンを除去し、ADAMTS１３抗原にのみ特異的に反応するクローンを選別した。抗原液として、ADAMTS13抗原を２μｇ／ｍｌの濃度に調製し、各々、１ウエル当たり５０μｌずつマイクロタイタープレートに添加し、一晩吸着させた後、Ｔｗｅｅｎ−２０を０．０５％含むリン酸緩衝液（以下洗浄液と略す）で５回洗浄し、さらに１０％スキムミルクを含むリン酸緩衝液でブロッキングしADAMTS１３抗原固相化プレートを調製した。上記で得られたハイブリドーマ細胞系の培養上清１００μＬを、当該固相化プレートに添加し、３７℃で３０分間反応させた後、洗浄液で５回洗浄し、さらにホースラディッシュペルオキシダーゼ（以下ＨＲＰと略す）標識した抗マウスイムノグロブリン抗体（ヤギ由来）を３７℃で３０分反応させた。この反応の後、洗浄液で４回洗浄し、基質液（ｏ−フェニレンジアミン0.4ｍｇ／ｍｌ及び０．０２％Ｈ2
Ｏ2 を含む）を３７℃で３０分間反応させた後、この反応を２Ｎ硫酸で停止させ、主波長４９２ｎｍでエライザ用プレートリーダーにて吸光度を測定した。ADAMTS１３抗原を固相化したエライザプレートに特異的に反応するハイブリドーマ細胞系を選別した後、限界機釈法によりクローニングし、受託番号FERM P-20189及びP-20190のハイブリドーマより、それぞれ抗ADAMTS１３モノクローナル抗体、クローンA10とC7を得た。
Production of Hybridoma and Anti-ADAMTS13 Monoclonal Antibody (1) Mice: Inbred BALB / c mice of 7 to 8 weeks old in an animal breeding chamber (23 ± 1 ° C., humidity 70%) The animals were reared using standard pellets, and were optionally fed with water.
(2) Immune antigen: The ADAMTS13 gene having a C-terminal flag sequence was amplified from a cDNA library by PCR, ligated to an expression vector pCAGC, and introduced into HeLa cells. Cells were cultured in serum-free medium OPTYIME I medium for 44 hours, and the culture supernatant was collected and purified with anti-FLAG M2 monoclonal antibody, and used as ADAMTS13 antigen.
(3) Immunization method:
ADAMTS13 antigen was prepared in PBS to a concentration of 100 μg / 0.5 ml, and the same volume (0.5 ml) of Freund's complete adjuvant (Freund's
complete adjuvant) (Difco) was mixed and emulsified. 200 μl of this emulsified antigen was administered to the abdominal cavity of four female BALB / c mice at 7 weeks of age. Every two weeks, GERBUVANT (GERBU
Biotechnik, GmbH, D-6901 (Guiberg, Germany)) and the above antigen prepared to 100 μg / ml were administered 4 times at 20 μg per mouse. One month later, the above antigen prepared to 100 μg / ml with GERBUVANT was boosted in the same manner, and then the antibody titer of the mice was measured. Mice with high antibody titers were further immunized after 2 weeks by injecting ADAMTS13 antigen prepared with PBS to 100 μg / ml from the tail vein of mice. The antibody titer was measured according to the screening method described later using the serum of a mouse immunized with the antigen.
(4) Cell fusion: BALB / c mice were excised 3 days after the final immunization, and the spleen cells were suspended in the DMEM culture solution to prepare a spleen cell suspension. Next, the number of cells was calculated and 1.9 × 10 8.
Individual splenocytes were obtained. Cell fusion was performed using a 2-cell-6-mercaptopurine-resistant BALB / c mouse-derived myeloma cell line (P3-X63-Ag8 · 653, hereinafter referred to as X63 cells). (Also referred to as parent cell line). X63 cells are fetal bovine serum (fetal
calf serum: FCS) is subcultured in a DMEM culture solution (5 μg / ml, containing 6-thioguanine) containing 10%, and further cultured in a DMEM culture solution containing 10% FCS not containing 6-thioguanine from 3 days before cell fusion. The cells in the logarithmic growth phase were used. The number of X63 cells was calculated and 1.9 × 10 8 viable cells were obtained. In a DMEM culture solution, polyethylene glycol-1500 is dissolved so as to have a concentration of 50 (w / v)%, and mixed so that the ratio of the above spleen cells to X63 cells is 1: 1. And Milstein, Nature,
256, 495-497, 1975, Eur. J. Immunol.
Cell fusion was carried out according to Vol. 6, 511-519 (1976). Thereafter, 1 × 10 −4 M hypoxanthine, 4 × 10 −7 M amesopterin and 1.6 × 10 −5 M were added to the DMEM culture medium supplemented with 10% FCS and 5% briclone (manufactured by Archport). HAT selection solution containing thymidine was added and splenocytes became 2.0 × 10 6.
It was made to float so that it might become a piece / ml. Next, 100 μl of this cell suspension was dispensed into each well of a 96-well microtiter plate, and then CO 2.
Cultivation was performed in a sterile incubator at 37 ° C., 100% humidity and 5% CO 2 . On the third day after the start of the culture, 100 μL of the HAT medium was added to each well, and then the culture was performed while changing half of the HAT medium every three days. Then, after 2 to 3 weeks, clones producing the desired anti-ADAMTS13 monoclonal antibody were searched for by the ELISA method using a microplate having the ADAMTS13 antigen adsorbed on a solid phase.
(5) Screening: Using the culture supernatant of the hybridoma cells, selection was made by reaction with an ADAMTS13 antigen-immobilized ELISA plate. In addition, non-specific reactive clones that react with the non-specific oligopeptide-immobilized ELISA plate were removed, and clones that specifically react only with the ADAMTS13 antigen were selected. As an antigen solution, ADAMTS13 antigen was prepared to a concentration of 2 μg / ml, added to each microtiter plate at 50 μl per well, adsorbed overnight, and then phosphate buffer containing 0.05% Tween-20. The plate was washed 5 times (hereinafter abbreviated as a washing solution), and further blocked with a phosphate buffer containing 10% skim milk to prepare an ADAMTS13 antigen-immobilized plate. 100 μL of the culture supernatant of the hybridoma cell line obtained above is added to the solid phased plate, reacted at 37 ° C. for 30 minutes, washed 5 times with a washing solution, and further horseradish peroxidase (hereinafter abbreviated as HRP). ) A labeled anti-mouse immunoglobulin antibody (derived from goat) was reacted at 37 ° C. for 30 minutes. After this reaction, it was washed 4 times with a washing solution, and a substrate solution (o-phenylenediamine 0.4 mg / ml and 0.02% H 2).
(Containing O 2 ) was allowed to react at 37 ° C. for 30 minutes, and then the reaction was stopped with 2N sulfuric acid, and the absorbance was measured at a main wavelength of 492 nm using an Eliza plate reader. After selecting a hybridoma cell line that specifically reacts with the ELISA plate on which the ADAMTS13 antigen is immobilized, the hybridoma cell line is cloned by the limiting method, and each of the anti-ADAMTS13 monoclonal antibodies is obtained from the hybridomas of accession numbers FERM P-20189 and P-20190. Clone A10 and C7 were obtained.
マウスイムノグロブリンサブクラスの同定：上記、クローニングにより単一クローンとして得られたハイブリドーマ細胞系の産生するモノクローナル抗体のマウスイムノグロブリンサブクラスを決定した。マウスイムノグロブリンサブクラスの同定には、各ハイブリドーマ細胞系の培養上清を用い、セロテック（Ｓｅｒｏｔｅｃ）社製Ｍｏｕｓｅ Ｍｏｎｏｃｌｏｎａｌ Ａｎｔｉｂｏｄｙ Ｉｓｏｔｙｐｉｎｇ ｋｉｔを用いた。その結果、クローンA10はIgG2ｂ−κ、クローンC7はIgG1-κであることが判った。 Identification of mouse immunoglobulin subclass: The mouse immunoglobulin subclass of the monoclonal antibody produced by the hybridoma cell line obtained as a single clone by cloning was determined as described above. For identification of the mouse immunoglobulin subclass, the culture supernatant of each hybridoma cell line was used, and the Mouse Monoclonal Antibody Isotyping kit manufactured by Serotech was used. As a result, it was found that clone A10 was IgG2b-κ and clone C7 was IgG1-κ.
マウスモノクローナル抗体の機能の確認：正常ヒト血漿２０μLに対してＩｇＧ濃度、０．２−２００μｇ／ｍLの実施例１で得たモノクローナル抗体を等量混合し、終濃度０．１−１００μｇ／ｍLとなるように調整し、３７℃で１２０分間インキュベートした後、ヒト血漿中に残存しているADAMTS13活性をVWFマルチマー解析法により測定した。その結果を図１に示した。クローンA10は10μｇ／ｍLの濃度で約５０％の阻害活性を示し、５０μｇ／ｍL以上では１００％ADAMTS13の活性を阻害した。一方、クローンC７は２５μｇ／ｍL以上の濃度でADAMTS１３の活性を約３５％阻害した。以上より、得られたモノクローナル抗体は何れもADMATS１３の活性を少なくとも３０％阻害することが判った。 Confirmation of the function of the mouse monoclonal antibody: 20 μL of normal human plasma was mixed with an equal amount of the monoclonal antibody obtained in Example 1 having an IgG concentration of 0.2-200 μg / mL to a final concentration of 0.1-100 μg / mL. After incubating at 37 ° C. for 120 minutes, ADAMTS13 activity remaining in human plasma was measured by VWF multimer analysis. The results are shown in FIG. Clone A10 showed about 50% inhibitory activity at a concentration of 10 μg / mL, and 100% ADAMTS13 activity was inhibited at 50 μg / mL or higher. On the other hand, clone C7 inhibited ADAMTS13 activity by about 35% at a concentration of 25 μg / mL or more. From the above, it was found that all of the obtained monoclonal antibodies inhibit ADMATS13 activity by at least 30%.
マウスモノクローナル抗体の特異性の確認：ADAMTS13分子のN末端アミノ酸より２８５番目までのポリペプチド、３８７番目までのポリペプチド、
４４９番目までのポリペプチド、６８８番目までのポリペプチド、７４６番目までのポリペプチド、８０８番目までのポリペプチド、８９７番目までのポリペプチド、１０１６番目までのポリペプチド、１１３５番目までのポリペプチド及び１４２７アミノ酸よりなるADAMTS13分子全体を用いて、ウエスタンブロット法により特異性を確認した。その結果を図２に示した。クローンA10とC7の両クローン共にADAMTS13分子と強く反応した。このことから両クローン共にADAMTS１３のウエスタンブッロト法による免疫化学的分析に有用であることが判った。そして、クローンA10は少なくともADAMTS13分子のN末端より３８７番目のアミノ酸よりもN末端側の上流部分にエピトープを有し、２８５番目までのポリペプチドでは反応を認めないことから、２８５番目と３８７番目の領域にエピトープを有していることが判った。一方、クローンC7は１０１６番目までのポリペプチドとは反応せずに、１１３５番目までのポリペプチドに反応することから１０１６番目よりC末端側下流にエピトープが存在し、１０１６番目から１１３５番目までの領域にエピトープが存在することが判った。
Confirmation of specificity of mouse monoclonal antibody: polypeptide up to 285th from N-terminal amino acid of ADAMTS13 molecule, polypeptide up to 387th,
Up to 449th polypeptide, up to 688th polypeptide, up to 746th polypeptide, up to 808th polypeptide, up to 897th polypeptide, up to 1016th polypeptide, up to 1135th polypeptide, and 1427 Specificity was confirmed by Western blotting using the entire ADAMTS13 molecule consisting of amino acids. The results are shown in FIG. Both clones A10 and C7 reacted strongly with ADAMTS13 molecule. From this, it was found that both clones are useful for immunochemical analysis of ADAMTS13 by Western blot method. Clone A10 has an epitope at the N-terminal upstream side of the 387th amino acid from the N-terminal of ADAMTS13 molecule, and no reaction is observed in the polypeptides up to 285, so that the 285th and 387th amino acids are not recognized. It was found that the region has an epitope. On the other hand, clone C7 does not react with the polypeptide up to the 1016th, but reacts with the polypeptide up to the 1135th, so that there is an epitope downstream of the C16 terminal from the 1016th, and the region from the 1016th to the 1135th It was found that an epitope exists.
本発明の抗ADAMTS13モノクローナル抗体を用いることにより、（１）ウエスタンブロット法によるADAMTS13の迅速な検出及び同定、（２）免疫沈降法(Immunoprecipitation法) による、発現したADAMTS１３の検出及び同定、さらに、ADAMTS１３と結合性を有する他の蛋白質の検出及び解析、（３）免疫化学的手段によるADAMTS１３の測定、検査（４）それらの成果に基づく治療薬、診断薬等の開発に利用できる。 By using the anti-ADAMTS13 monoclonal antibody of the present invention, (1) rapid detection and identification of ADAMTS13 by Western blotting, (2) detection and identification of expressed ADAMTS13 by immunoprecipitation method (Immunoprecipitation method), ADAMTS13 And (3) measurement of ADAMTS13 by immunochemical means, testing (4) development of therapeutic agents, diagnostic agents, etc. based on the results.
Claims (2)
A reagent or kit comprising the monoclonal antibody according to claim 1.
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