JP5067917B2 - Separation and purification method of ADAMTS13 - 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酵素（ＡＤＡＭＴＳ１３）の分離精製方法、ＡＤＡＭＴＳ１３含有組成物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 method for separating and purifying von Willebrand factor cleaving enzyme (ADAMTS13), a method for producing an ADAMTS13-containing composition, 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。最近、ＴＴＰの病因としてＶＷＦ切断酵素（ＡＤＡＭＴＳ１３）の活性低下が報告された。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)。
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. Recently, decreased activity of VWF-cleaving enzyme (ADAMTS13) has been 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 a VWF-cleaving enzyme (Non-patent
先天性或いは後天性のＡＤＡＭＴＳ１３欠損症の治療は、主に新鮮凍結血漿の輸注或いは血漿交換等によって、予後が大幅に改善されるようになっている。しかしながら、先天性ＡＤＡＭＴＳ１３欠損症患者は約２週間毎に新鮮凍結血漿の輸注を欠かせないことから、輸血副作用が危惧されている。ＡＤＡＭＴＳ１３の濃縮製剤が供給されれば、そのような危惧も大幅に改善されると考えられる。 In the treatment of congenital or acquired ADAMTS13 deficiency, prognosis is greatly improved mainly by infusion or plasma exchange of fresh frozen plasma. However, since patients with congenital ADAMTS13 deficiency must infuse fresh frozen plasma every two weeks, there are concerns about blood transfusion side effects. If a concentrated preparation of ADAMTS13 is supplied, it is considered that such a concern is greatly improved.
ＡＤＡＭＴＳ１３は亜鉛型メタロプロテアーゼで、ＶＷＦサブユニットのＴｙｒ１６０５−Ｍｅｔ１６０６結合を特異的に切断する。この酵素は血漿中に約０．５−１μｇ／ｍＬの濃度で存在するといわれている。この酵素を精製する試みはいくつか報告されている（非特許文献６，７）しかしながら、何れの報告も複雑な工程が必要であり、収率も悪いものであった。そこで簡便な精製方法の開発が望まれていた。 ADAMTS13 is a zinc-type metalloprotease that specifically cleaves the Tyr1605-Met1606 bond of the VWF subunit. This enzyme is said to be present in plasma at a concentration of about 0.5-1 μg / mL. Several attempts to purify this enzyme have been reported (Non-patent Documents 6 and 7). However, each report requires a complicated process and the yield is poor. Therefore, development of a simple purification method has been desired.
免疫吸着体を用いた抗原又は抗体の精製方法は一般的に行われている方法である。その手段は目的物質から不要物の除去、目的物質の精製及び濃縮等に有用である。抗原を担体に固定化した抗原固定化担体を用いて特異抗体を吸着させた後、抗原抗体結合体を酸、アルカリ或いはロダン塩等を用いて解離させ、特異抗体を回収する方法等はよく知られた技術である。また、抗体固定化担体を用いて抗原を吸着させたのち、抗原抗体結合体を解離させて抗原を回収する方法も公知である。この場合には、抗原の性質によって解離剤を選択する必要があることも知られている（非特許文献８）。本発明の目的であるＡＤＡＭＴＳ１３の分離精製方法において、抗ＡＤＡＭＴＳ１３抗体を用いて効率よくＡＤＭＴＳ１３を含む画分を回収した報告はない。
本発明の目的は、ＡＤＡＭＴＳ１３の効率的な分離精製方法の提供ならびに当該方法により調製されたＡＤＡＭＴＳ１３含有組成物の産業的利用方法を提供することである。 An object of the present invention is to provide an efficient method for separating and purifying ADAMTS13, and to provide an industrial utilization method for an ADAMTS13-containing composition prepared by the method.
本発明者らは、ＡＤＡＭＴＳ１３の分離精製方法を鋭意研究し、抗ＡＤＡＭＴＳ１３抗体を用いてＡＤＡＭＴＳ１３分子を抗体に結合させた後、結合体より水溶性有機溶媒を用いてＡＤＡＭＴＳ１３を解離させる、或いは高濃度の塩化マグネシウムを用いて解離させることにより、効率よくＡＤＡＭＴＳ１３を含む画分を回収することができることを見出し、本発明を完成させた。 The inventors of the present invention diligently studied a method for separating and purifying ADAMTS13. After anti-ADAMTS13 antibody was used to bind ADAMTS13 molecule to the antibody, ADAMTS13 was dissociated from the conjugate using water-soluble organic solvent, or high concentration. It was found that a fraction containing ADAMTS13 can be efficiently recovered by dissociation using magnesium chloride, and the present invention was completed.
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酵素（ＡＤＡＭＴＳ１３）を含む試料よりＡＤＡＭＴＳ１３分子を含む画分を分離精製する方法であって、ＡＤＡＭＴＳ１３分子に特異的結合性を有する抗体とＡＤＡＭＴＳ１３を含む溶液中のＡＤＡＭＴＳ１３とを結合させた後、該結合を解離させる工程において、水溶性有機溶媒を含む解離剤を用いることを特徴とする分離精製方法。
２、水溶性有機溶媒を含む解離剤が、ジメチルスルオキシド、エチレングリコール、プロピレングリコール、エタノール、メタノール、アセトン、ジメチルホルムアミド及びグリセロールの中から選択された少なくとも１種の水溶性有機溶媒を含む解離剤を用いる上記１に記載のＡＤＡＭＴＳ１３の分離精製方法。
３、水溶性有機溶媒を少なくとも３（ｖ／ｖ）％含む解離剤を用いる上記１および２のいずれかに記載のＡＤＡＭＴＳ１３の分離精製方法。
４、ＡＤＡＭＴＳ１３分子に特異的結合性を有する抗体と試料中のＡＤＡＭＴＳ１３を結合させた後、０．１〜３Ｍの無機塩を含む緩衝液で結合物を含む画分を洗浄した後、
水溶性有機溶媒を含む解離剤を用いる上記１〜３のいずれか１に記載のＡＤＡＭＴＳ１３の分離精製方法。
５、ＡＤＡＭＴＳ１３を含む試料よりＡＤＡＭＴＳ１３分子を含む画分を分離精製する方法であって、ＡＤＡＭＴＳ１３分子に特異的結合性を有する抗体とＡＤＡＭＴＳ１３を含む溶液中のＡＤＡＭＴＳ１３とを結合させた後、該結合を解離させる工程において、塩化マグネシウムを含む解離剤を用いることを特徴とする分離精製方法。
６、塩化マグネシウムの濃度が少なくとも１mol/Lを含む解離剤を用いる上記５に記載のＡＤＡＭＴＳ１３の分離精製方法。
７、固相担体に担持させたＡＤＡＭＴＳ１３分子に特異的結合性を有する抗体を用いる上記１〜６のいずれか１に記載のＡＤＡＭＴＳ１３の分離精製方法。
８、上記１〜７のいずれか１に記載の分離精製方法と、ＡＤＡＭＴＳ１３分子の電気的性質、分子の大きさ、親水性又は疎水性、水素結合力、塩析又は塩溶、及び特異的リガンドとの結合性を利用した精製方法のうちから選択された少なくとも１つの精製工程を組み合わせるＡＤＡＭＴＳ１３の分離精製方法。
９、上記１〜８のいずれか１に記載の方法により分離精製されたＡＤＡＭＴＳ１３含有組成物。
１０、上記９に記載のＡＤＡＭＴＳ１３含有組成物を用いる、先天性或いは後天性ＡＤＡＭＴＳ１３減少症の治療薬。
１１、上記９に記載のＡＤＡＭＴＳ１３含有組成物を用いる、抗ＡＤＡＭＴＳ１３抗体の検査方法。
１２、上記１１に記載の検査方法に用いる検査試薬又はキット。
１３、上記９に記載のＡＤＡＭＴＳ１３含有組成物を免疫原として動物に免疫することにより抗ＡＤＡＭＴＳ１３抗体を調製する方法。
１４、上記１３に記載の方法により調製された抗体。
１５、上記１４に記載の抗体を含むＡＤＡＭＴＳ１３抗原検査用検査試薬又はキット。
The present invention has the following configuration.
1. A method for separating and purifying a fraction containing ADAMTS13 molecules from a sample containing von Willebrand factor cleaving enzyme (ADAMTS13), comprising an antibody having specific binding properties to ADAMTS13 molecules and ADAMTS13 in a solution containing ADAMTS13 A separation and purification method characterized by using a dissociator containing a water-soluble organic solvent in the step of dissociating the bond after the bond.
2. The dissociator containing at least one water-soluble organic solvent selected from dimethyl sulfoxide, ethylene glycol, propylene glycol, ethanol, methanol, acetone, dimethylformamide, and glycerol. 2. The method for separating and purifying ADAMTS13 as described in 1 above.
3. The method for separating and purifying ADAMTS13 according to any one of 1 and 2 above, using a dissociator containing at least 3 (v / v)% of a water-soluble organic solvent.
4. After binding the antibody having specific binding property to the ADAMTS13 molecule and ADAMTS13 in the sample, washing the fraction containing the bound substance with a buffer containing 0.1 to 3 M inorganic salt,
4. The method for separating and purifying ADAMTS13 according to any one of 1 to 3 above, using a dissociator containing a water-soluble organic solvent.
5. A method for separating and purifying a fraction containing ADAMTS13 molecules from a sample containing ADAMTS13, wherein an antibody having a specific binding property to ADAMTS13 molecules is bound to ADAMTS13 in a solution containing ADAMTS13, and then the binding is performed. A separation and purification method, wherein a dissociation agent containing magnesium chloride is used in the dissociation step.
6. The method for separating and purifying ADAMTS13 as described in 5 above, using a dissociator containing a magnesium chloride concentration of at least 1 mol / L.
7. The method for separating and purifying ADAMTS13 according to any one of 1 to 6 above, wherein an antibody having a specific binding property to an ADAMTS13 molecule supported on a solid phase carrier is used.
8. Separation and purification method according to any one of 1 to 7 above, and electrical properties of ADAMTS13 molecule, molecular size, hydrophilicity or hydrophobicity, hydrogen bonding force, salting out or salting, and specific ligand A method for separating and purifying ADAMTS13, which comprises combining at least one purification step selected from among purification methods utilizing the binding property to.
9. ADAMTS13-containing composition separated and purified by the method according to any one of 1 to 8 above.
10. A therapeutic agent for congenital or acquired ADAMTS13 reduction using the ADAMTS13-containing composition according to 9 above.
11. A test method for an anti-ADAMTS13 antibody using the ADAMTS13-containing composition according to 9 above.
12. A test reagent or kit for use in the test method according to 11 above.
13. A method for preparing an anti-ADAMTS13 antibody by immunizing an animal with the ADAMTS13-containing composition described in 9 above as an immunogen.
14. An antibody prepared by the method according to 13 above.
15. A test reagent or kit for testing ADAMTS13 antigen, comprising the antibody according to 14 above.
本発明のＡＤＡＭＴＳ１３の分離精製方法は、簡単に血漿などの体液及び細胞等の抽出液から効率よくＡＤＡＭＴＳ１３を含む画分を回収する方法を提供するものであり、血漿のごとく大多数の分子種、大量の夾雑物質を含む試料からＡＤＡＭＴＳ１３を高度に精製、濃縮する方法を提供するものである。当該ＡＤＡＭＴＳ１３の分離精製
方法により調製されたＡＤＡＭＴＳ１３組成物は治療薬、検査薬に応用できることから、
本発明は、ＡＤＡＴＳ１３含有組成物の利用方法にも及び産業上の利用価値も高い。
The method for separating and purifying ADAMTS13 of the present invention provides a method for easily recovering a fraction containing ADAMTS13 from a body fluid such as plasma and an extract of cells or the like, and a large number of molecular species such as plasma, A method for highly purifying and concentrating ADAMTS13 from a sample containing a large amount of contaminants is provided. Since the ADAMTS13 composition prepared by the ADAMTS13 separation and purification method can be applied to therapeutic drugs and test drugs,
The present invention has high industrial utility value as well as a method of using the ADATS13-containing composition.
本発明においてＡＤＡＭＴＳ１３を含む試料はヒト又は動物の血液、体液、組織或いは細胞の抽出液等特に限定されるものではない。また抗ＡＤＡＭＴＳ１３抗体はＡＤＡＭＴＳ１３分子と結合しうるものであればよく、その由来の動物種またモノクローナル抗体或いはポリクローナル抗体のいかなるものも含まれる。抗体は抗体分子全体でも、或いは抗体活性を有する分子の一部でもよい。抗体は液状のまま用いることも不可能ではないが、好適には固相担体に担持固定化された状態で用いられる。固相に担持固定化する方法は特に限定されることなく公知の方法が適用される。固相担体の体積当りの担持固定化される抗体の量はとりわけ限定されないが、０．１−１０ｍｇ／ｍＬ固相、より好適には０．５−５ｍｇ／ｍＬ固相である。 In the present invention, the sample containing ADAMTS13 is not particularly limited, such as human or animal blood, body fluid, tissue or cell extract. The anti-ADAMTS13 antibody may be any antibody as long as it can bind to the ADAMTS13 molecule, and includes any animal species derived therefrom, monoclonal antibodies or polyclonal antibodies. The antibody may be the whole antibody molecule or a part of a molecule having antibody activity. Although it is not impossible to use the antibody in a liquid state, the antibody is preferably used in a state of being supported and immobilized on a solid phase carrier. The method for carrying and immobilizing the solid phase is not particularly limited, and a known method is applied. The amount of the antibody immobilized on the solid phase volume is not particularly limited, but is 0.1-10 mg / mL solid phase, more preferably 0.5-5 mg / mL solid phase.
本発明の本質である抗原抗体結合体の解離剤は水溶性有機溶媒または塩化マグネシウムから選択される。水溶性有機溶媒として、ジメチルスルオキシド、エチレングリコール、プロピレングリコール、エタノール、メタノール、アセトン、ジメチルホルムアミド及びグリセロール等が好適に用いることができる。その濃度は用いる抗体の性質と溶媒の種類によって変動するが、少なくとも３（ｖ／ｖ）％の濃度で用いられる。より好適には、１０−５０（ｖ／ｖ）％の濃度で用いられる。水溶性有機溶媒を溶かす緩衝液は、ＡＤＡＭＴＳ１３の安定性を考慮し、中性付近のｐＨを有する緩衝液が用いられる。緩衝液の種類はＡＤＡＭＴＳ１３の安定性や活性を損なわないものであればよく、特に限定されないが、ＴｒｉｓやＨＥＰＥＳ等の公知の緩衝液が好適に用いられる。また、解離剤には、塩化ナトリウム、塩化カリウム、硫酸アンモニウム等の無機塩を０．１−３Ｍの濃度、より好適には０．５−２Ｍで適宜含ませることもある。塩化マグネシウムを解離剤として用いる場合には、前記の適当な緩衝液に少なくとも１Ｍを、より好適には２−５Ｍ含ませて用いる。 The dissociator for the antigen-antibody conjugate that is the essence of the present invention is selected from a water-soluble organic solvent or magnesium chloride. As the water-soluble organic solvent, dimethyl sulfoxide, ethylene glycol, propylene glycol, ethanol, methanol, acetone, dimethylformamide, glycerol and the like can be suitably used. The concentration varies depending on the nature of the antibody used and the type of solvent, but is used at a concentration of at least 3 (v / v)%. More preferably, it is used at a concentration of 10-50 (v / v)%. As the buffer solution for dissolving the water-soluble organic solvent, a buffer solution having a pH near neutral is used in consideration of the stability of ADAMTS13. The type of the buffer is not particularly limited as long as it does not impair the stability and activity of ADAMTS13, but a known buffer such as Tris or HEPES is preferably used. In addition, the dissociator may contain an inorganic salt such as sodium chloride, potassium chloride, or ammonium sulfate as appropriate at a concentration of 0.1-3M, more preferably 0.5-2M. When magnesium chloride is used as the dissociator, it is used by containing at least 1M, more preferably 2-5M in the appropriate buffer.
本発明のＡＤＡＭＴＳ１３の分離精製方法により調製されたＡＤＡＭＴＳ１３組成物は、さらに、ＡＤＡＭＴＳ１３の電気的性質、分子の大きさ、親水性又は疎水性、水素結合力、塩析又は塩溶、及び特異的リガンドとの結合性を利用した精製方法のうちから選択された方法により更に精製することも本発明に含まれる。電気的性質を利用した精製方法の例として、イオン交換クロマトグラフィ、等電点電気泳動などの手段、分子の大きさを利用した精製方法の例として、分子篩クロマトグラフィ、ゲル電気泳動、
限外ろ過膜による分離等の手段、親水性又は疎水性を利用した精製方法の例として、疎水性相互作用クロマトグラフィ等、水素結合を利用した精製方法の例として、水素結合クロマトグラフィ等、塩析又は塩溶を利用した精製方法の例として、硫酸アンモニウムや硫酸ナトリウムによる塩析操作や、塩析クロマトグラフィ等、特異的リガンドとの結合性を利用した精製方法の例として、ヘパリン固定化カラム等による群特異的親和性クロマトグラフィ等が例示される。
The ADAMTS13 composition prepared by the method for separating and purifying ADAMTS13 of the present invention further comprises the electrical properties, molecular size, hydrophilicity or hydrophobicity of ADAMTS13, hydrogen bonding force, salting out or salting, and specific ligand. Further purification by a method selected from among purification methods utilizing the binding property to is also included in the present invention. Examples of purification methods using electrical properties include means such as ion exchange chromatography and isoelectric focusing, examples of purification methods using molecular size include molecular sieve chromatography, gel electrophoresis,
Examples of methods such as separation using ultrafiltration membranes, purification methods utilizing hydrophilicity or hydrophobicity, hydrophobic interaction chromatography, etc., examples of purification methods utilizing hydrogen bonds, such as hydrogen bonding chromatography, salting out or Examples of purification methods using salt solution include salting-out operations with ammonium sulfate and sodium sulfate, salting-out chromatography, etc. Examples of purification methods that use binding properties with specific ligands include group-specific using a heparin-immobilized column, etc. Affinity chromatography and the like are exemplified.
本発明のＡＤＡＭＴＳ１３の分離精製方法により調製されたＡＤＡＭＴＳ１３組成物の利用方法として、先天性又は後天性にＡＤＡＭＴＳ１３が減少する疾患において、ＡＤＡＭＴＳ１３を補充するための治療薬が挙げられる。該組成物を免疫原として動物に投与し、公知の方法で抗ＡＤＡＭＴＳ１３抗体を調製するための抗原として利用することもできる。また、該組成物を用いて血液中の抗ＡＤＡＭＴＳ１３自己抗体の検査用原料としても利用できる。本発明には、上記の治療薬、抗体を作成する方法および作製した抗体、該抗体または該抗原を利用した検査方法、さらには該検査方法を実施するための検査試薬、キットも含まれる。 As a method of using the ADAMTS13 composition prepared by the method for separating and purifying ADAMTS13 of the present invention, a therapeutic agent for supplementing ADAMTS13 in a disease in which ADAMTS13 is decreased congenitally or acquiredly can be mentioned. The composition can be administered to an animal as an immunogen and used as an antigen for preparing an anti-ADAMTS13 antibody by a known method. The composition can also be used as a raw material for testing anti-ADAMTS13 autoantibodies in blood. The present invention also includes the above therapeutic agents, methods for producing antibodies, prepared antibodies, testing methods using the antibodies or antigens, and testing reagents and kits for carrying out the testing methods.
抗ＡＤＡＭＴＳ１３モノクローナル抗体（クローンＡ１０，受託番号 ＦＥＲＭ Ｐ−２０１８９のマウスハイブリドーマより産生されるモノクローナル抗体）を用いた本発明の方法を具体的に説明する。
１、抗ＡＤＡＭＴＳ１３モノクローナル抗体（Ａ１０抗体）を市販のアガロースゲルに公知の方法で固定化する。固定化する抗体の量は、ゲル体積１ｍＬ当たり約１ｍｇを目処に固定化する。この量は既に述べたように特に限定されるものではない。固定化したゲルを適当な大きさのカラムに充填し、０．１５Ｍ ＮａＣｌ含有２０ｍＭＴｒｉｓ−ＨＣｌ緩衝液、ｐＨ７．４、（ＴＢＳ緩衝液）のような適当な緩衝液で平衡化し、ヒトクエン酸加血漿をカラムに通過させる。所定量の血漿を通過させた後、カラムをＴＢＳ緩衝液により洗浄する。次いで４０（ｖ／ｖ）％ジメチルスルオキシドを含むＴＢＳ緩衝液をカラムに通し、吸着画分を溶出する。この操作により、ＡＤＡＭＴＳ１３を含む画分が回収される。
２、回収したＡＤＡＭＴＳ１３を含む画分を更に精製するために、回収した画分を２０ｍＭＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．４）に対して透析し、同緩衝液で平衡化したＤＥＡＥ−陰イオン交換体に吸着させた。吸着させたカラムを、食塩濃度を０−０．５Ｍの間で変化させることにより溶出し、ＡＤＡＭＴＳ１３を含む画分を回収する。
３、回収したＡＤＡＭＴＳ１３を含む画分を更に精製するために、上記２の工程で回収した画分を濃縮後、分子篩クロマトグラフィにより分画、精製する。
The method of the present invention using an anti-ADAMTS13 monoclonal antibody (a monoclonal antibody produced from a mouse hybridoma of clone A10, accession number FERM P-20189) will be specifically described.
1. An anti-ADAMTS13 monoclonal antibody (A10 antibody) is immobilized on a commercially available agarose gel by a known method. The amount of antibody to be immobilized is about 1 mg per mL of gel volume. This amount is not particularly limited as described above. The immobilized gel is packed in a column of an appropriate size, equilibrated with a suitable buffer such as 20 mM Tris-HCl buffer containing 0.15 M NaCl, pH 7.4, (TBS buffer), and human citrated plasma Is passed through the column. After passing a predetermined amount of plasma, the column is washed with TBS buffer. Next, TBS buffer containing 40 (v / v)% dimethyl sulfoxide is passed through the column to elute the adsorbed fraction. By this operation, the fraction containing ADAMTS13 is collected.
2. In order to further purify the fraction containing the collected ADAMTS13, the collected fraction was dialyzed against 20 mM Tris-HCl buffer (pH 7.4), and DEAE-anion exchanger equilibrated with the same buffer. It was made to adsorb to. The adsorbed column is eluted by changing the salt concentration between 0-0.5M, and the fraction containing ADAMTS13 is collected.
3. In order to further purify the fraction containing ADAMTS13 collected, the fraction collected in the
（解離剤の検索）
Ａ１０抗体を５μｇ／ｍＬの濃度に調製し、各々、１ウエル当たり１００μＬ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％ブロックエース（商品名）を含むリン酸緩衝液でブロッキングしＡ１０抗体固相化プレートを調製した。正常ヒト血漿５０μＬを固相プレートに添加し、３７℃で６０分間反応させた後、洗浄液で３回洗浄し、種々の解離剤を含む緩衝液１００μＬを加え、室温で１０分間放置した後、洗浄液で３回洗浄し、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ＡＤＡＭＴＳ１３モノクローナル抗体(クローンＣ７、受託番号 ＦＥＲＭ Ｐ−２０１９０)を３７℃で６０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１５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。各解離剤の固相抗体に対する影響を相殺するため、正常ヒト血漿を添加する前に、種々の解離剤で処理した後、洗浄後正常ヒト血漿を反応させたものを対照とした。解離剤により解離作用の判定は対照のウエルで得られた吸光度から試験ウエルの吸光度の差を求め、それを対照の吸光度で除した値を比較検討した。その結果を表１に示した。最下段の２０ｍM Tris＋１M NaClは解離剤を含まない対照である。解離剤を含まない２０ｍM Tris＋１M NaClでは試験ウエルと対照ウエルでの吸光度の差がなく、ウエルにＡ１０抗体を介して捕獲されたＡＤＡＭＴＳ１３がそのまま残存しているのに対して、エチレングリコール或いはジメチルスルオキシドを含む解離剤では対照ウエルの吸光度に比して試験ウエルの吸光度が何れも有意に減じていることから、ウエルに捕獲されているＡＤＡＭＴＳ１３が解離剤によって解離していることが判った。従って、この結果よりエチレングリコール或いはジメチルスルオキシドを含む解離剤を用いてＡ１０抗体−ＡＤＡＭＴＳ１３結合体よりＡＤＡＭＴＳ１３を解離できることが判った。
(Search for dissociation agent)
A10 antibody was prepared to a concentration of 5 μg / mL, and 100 μL per well was added to a microtiter plate and allowed to adsorb overnight. Then, phosphate buffer containing 0.05% Tween-20 (hereinafter referred to as a washing solution) The plate was washed five times with an abbreviation) and further blocked with a phosphate buffer containing 10% Block Ace (trade name) to prepare an A10 antibody-immobilized plate. After adding 50 μL of normal human plasma to a solid phase plate and reacting at 37 ° C. for 60 minutes, washing with a washing solution three times, adding 100 μL of a buffer solution containing various dissociating agents, and allowing to stand at room temperature for 10 minutes, washing solution And anti-ADAMTS13 monoclonal antibody (clone C7, accession number FERM P-20190) labeled with horseradish peroxidase (hereinafter abbreviated as HRP) was reacted at 37 ° C. for 60 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 15 minutes, and then the reaction was stopped with 2N sulfuric acid, and the absorbance was measured with an Eliza plate reader at a main wavelength of 492 nm. In order to offset the influence of each dissociation agent on the solid phase antibody, a treatment with normal human plasma after washing after treatment with various dissociation agents before adding normal human plasma was used as a control. The determination of the dissociation effect by the dissociating agent was carried out by calculating the difference in absorbance of the test well from the absorbance obtained in the control well, and comparing the value obtained by dividing it by the absorbance of the control. The results are shown in Table 1. The bottom 20 mM Tris + 1M NaCl is a control without dissociator. In 20 mM Tris + 1M NaCl containing no dissociator, there is no difference in absorbance between the test well and the control well, and ADAMTS13 captured via the A10 antibody remains in the well, whereas ethylene glycol or dimethyl sulfoxide remains. In the dissociation agent containing, the absorbance of the test wells was significantly reduced as compared to the absorbance of the control wells. Thus, it was found that ADAMTS13 captured in the wells was dissociated by the dissociation agent. Therefore, from this result, it was found that ADAMTS13 can be dissociated from the A10 antibody-ADAMTS13 conjugate using a dissociating agent containing ethylene glycol or dimethyl sulfoxide.
約５ｍｇのＡ１０抗体を約５ｍＬの市販のブルムシアン活性化セファロース４Ｂ（商品名）に公知の方法により固定化し、Ａ１０抗体固定化セファロースを調製した。調製したＡ１０抗体固定化セファロース、１ｍＬずつをカラムに充填し、ＴＢＳで洗浄、平衡化した後、各カラムに５ｍＬのクエン酸加ヒト血漿をカラムに通した。その後、カラムをＴＢＳで洗浄し、４．５Ｍ 塩化マグネシウムを含む２０ｍＭ Ｔｒｉｓ（ｐＨ７．４）緩衝液及び５０（ｖ／ｖ）％エチレングリコールと１ＭＮａＣｌを含む２０ｍＭ Ｔｒｉｓ（ｐＨ７．４）緩衝液でそれぞれカラムを溶出した。溶出画分を過剰のＴＢＳに透析した後、波長２８０ｎｍに於ける吸光度及びＡＤＡＭＴＳ１３活性を加藤らの方法（特願２００５−１５７５３０）により測定した。その結果を図１に示した。何れの解離剤でもＡＤＡＭＴＳ１３がカラムより溶出してくることがわかった。 About 5 mg of A10 antibody was immobilized on about 5 mL of commercially available Bloomian activated Sepharose 4B (trade name) by a known method to prepare A10 antibody-immobilized Sepharose. The prepared A10 antibody-immobilized sepharose, 1 mL each, was packed into a column, washed with TBS and equilibrated, and then 5 mL of citrated human plasma was passed through each column. Thereafter, the column was washed with TBS and washed with 20 mM Tris (pH 7.4) buffer containing 4.5 M magnesium chloride and 20 mM Tris (pH 7.4) buffer containing 50 (v / v)% ethylene glycol and 1 M NaCl, respectively. The column was eluted. After the elution fraction was dialyzed against excess TBS, the absorbance at a wavelength of 280 nm and ADAMTS13 activity were measured by the method of Kato et al. (Japanese Patent Application No. 2005-157530). The results are shown in FIG. It was found that ADAMTS13 was eluted from the column with any dissociator.
約５ｍｇのＡ１０抗体を約５ｍＬの市販のブルムシアン活性化セファロース４Ｂ（商品名）に公知の方法により固定化し、Ａ１０抗体固定化セファロースを調製した。調製したＡ１０抗体固定化セファロースをカラムに充填し、ＴＢＳで洗浄、平衡化した後、
９２０ｍＬのクエン酸加ヒト血漿をカラムに通した。その後、カラムをＴＢＳで洗浄し、
１Ｍ ＮａＣｌと１０（ｖ／ｖ）％ＤＭＳＯを含む２０ｍＭＴｒｉｓ（ｐＨ７．４）及び１Ｍ ＮａＣｌと３０（ｖ／ｖ）％ＤＭＳＯを含む２０ｍＭＴｒｉｓ（ｐＨ７．４）で溶出した。溶出画分を過剰のＴＢＳに透析した後、波長２８０ｎｍに於ける吸光度及びＡＤＡＭＴＳ１３活性を加藤らの方法（特願２００５−１５７５３０）により測定した。その結果、１０％ＤＭＳＯ溶出画分に約３％の収率で、一方３０％ＤＭＳＯ溶出画分に約２５％の収率でＡＤＡＭＴＳ１３活性が回収され、回収された画分の精製度は原料血漿に比して、それぞれ約８７倍及び約８７７倍であった。さらにこれらの画分をＤＥＡＥ−セファロースカラムによるイオン交換クロマトグラフィにより、更に精製度を
原料血漿に比して、それぞれ約１１９倍及び約３４６３倍まで高めることができた。この画分をセファロース６Ｂカラムによる分子篩クロマトグラフィにかけることにより、原料血漿に比して、それぞれ約１８５倍及び約７３００倍まで精製度を高めた画分を得た。
About 5 mg of A10 antibody was immobilized on about 5 mL of commercially available Bloomian activated Sepharose 4B (trade name) by a known method to prepare A10 antibody-immobilized Sepharose. After the prepared A10 antibody-immobilized sepharose is packed in a column, washed with TBS and equilibrated,
920 mL of citrated human plasma was passed through the column. The column is then washed with TBS,
Elution was performed with 20 mM Tris (pH 7.4) containing 1 M NaCl and 10 (v / v)% DMSO and 20 mM Tris (pH 7.4) containing 1 M NaCl and 30 (v / v)% DMSO. After the elution fraction was dialyzed against excess TBS, the absorbance at a wavelength of 280 nm and ADAMTS13 activity were measured by the method of Kato et al. (Japanese Patent Application No. 2005-157530). As a result, ADAMTS13 activity was recovered in a yield of about 3% in the fraction eluted with 10% DMSO, and in a yield of about 25% in the fraction eluted with 30% DMSO. Were about 87 times and about 877 times, respectively. Furthermore, these fractions could be further increased to about 119 times and about 3463 times, respectively, by ion exchange chromatography using a DEAE-Sepharose column as compared with the raw plasma. By subjecting this fraction to molecular sieve chromatography using a Sepharose 6B column, fractions having a degree of purification increased to about 185 times and about 7300 times, respectively, compared to the raw plasma.
Claims (6)
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