JP6654580B2 - Tsh受容体に対する自己抗体の検出 - Google Patents
Tsh受容体に対する自己抗体の検出 Download PDFInfo
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- JP6654580B2 JP6654580B2 JP2016574174A JP2016574174A JP6654580B2 JP 6654580 B2 JP6654580 B2 JP 6654580B2 JP 2016574174 A JP2016574174 A JP 2016574174A JP 2016574174 A JP2016574174 A JP 2016574174A JP 6654580 B2 JP6654580 B2 JP 6654580B2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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Description
材料
TSH受容体キメラをクローニングするために、D.L.Segaloff博士(アイオワ大学、米国)のプラスミドpcDNA3−rLHR(B9)を用いた。
コンフルエントで安定したCHO−K1及びHEK293細胞をそれぞれプレート中で増殖させる。上清(約8mg/mL総タンパク質)を集め、−70℃に保った。このようにして得られた上清を、それぞれHSA2−TSHR ECD−B−330−mAb1、TSHR−SEAP又はTSHR/LH−CGR−SEAPとして決定方法に使用することができる。
融合TSH−Rキメラのトランケートされた細胞外ドメインは、それらを発現する細胞から培養上清中に分泌される。分泌された受容体タンパク質は、アッセイ緩衝液中で規定された希釈の形態で実験に直接使用される。例えば、培養上清5mL由来の細胞外SEAP−TSH−RキメラB 10μLを、1回の測定につき1:10の最終希釈で使用する。
トシル化Dynabeads(登録商標)M−280(Invitrogen Dynal AS)10mg(330μL)を回収し、毎回0.1Mホウ酸緩衝液(pH9.5)1mlで2回洗浄し、次いで30μLのホウ酸緩衝液に再懸濁する。このために、以下の手順を各々の洗浄工程で使用する:磁石を活性化し、そして溶液を常磁性粒子から除去する。次に磁石を失活させ、そして常磁性粒子に0.1Mホウ酸緩衝液(pH9.5)を1mL加え、そして数秒間よく振り混ぜる。これに続いて、磁石の活性化及び常磁性粒子からの洗浄溶液の除去が行われる。抗体4.3μg(0.43μg抗体/mg Dynabeads(登録商標))の添加を行う。このために、抗TSHRクローン3D7を回収し、そしてホウ酸緩衝液で30μLに希釈する。さらに、0.1Mホウ酸緩衝液(pH9.5)で処理した3Mアンモニウムサルフェート溶液のDynabeads(登録商標)100μL当たり30μLを添加し、完全に混合する。これに続いて、室温で20時間より長く(一晩)インキュベートし、そしてオーバーヘッドシェーカーで振とうする。次に、上記のように毎回ホウ酸緩衝液1mLで2回洗浄し、そして新たに調製した遮断型緩衝液(ホウ酸緩衝液、0.5%BSA、0.05%Tween 20)0.5mlを添加する。分散液を振とうしながら37℃で2時間インキュベートする。次に、新しく調製した洗浄緩衝液(PBS(pH7.4)、0.1%BSA、0.1%Tween 20)1mLを用いてDynabeads(登録商標)を上記のように2回洗浄する。次いで、上清を除去し、そしてアンカー抗体と結合したDynabeads(登録商標)を洗浄緩衝液500mg中に20mg/mLの濃度で再懸濁する。
緩衝液(PBS(pH7.4)、0.1%BSA、0.1%Tween20)中においてアンカー抗体とカップリングした20mg/mLのDynabeads(登録商標)を含む溶液30μLを洗浄緩衝液1mL(PBS(pH7.4)で4回洗浄し、次に、本発明によるアンカー受容体(HSAとN末端で融合しそしてモノクローナル抗体とC末端で融合した細胞外TSH受容体キメラ)の培養上清100μLを添加した。得られた溶液を振とうしながら室温で1時間インキュベートする。次に、上清を除去し、Dynabeads(登録商標)を洗浄バッファー1mLで2回洗浄し、洗浄バッファー30μLに再懸濁し、4℃で保存する。
血清又は血漿サンプルは、3時間以内に静脈血から産生される。4℃で7日間又は−20℃で1〜2年間の保存が可能である。二次抗体(上記参照)を−20℃に保ち、そして試験に使用する前に室温で解凍する。TSH受容体キメラをマイクロタイタープレート上に凍結乾燥して保存し、そして使用前にタンパク質安定化剤を含む緩衝液中で再構成する。
標準シリーズを作成するために、標準化された濃度の自己抗体を用いたWHO90/672標準溶液を使用した。標準の出発溶液は100IU/Lを含み、これを標準曲線を生成するために希釈した。ヌル値として、TXBW緩衝液中の自己抗体を有さないボランティアからの血清を使用した。
この実施例では、一方では、トランケートされた2つのTSH受容体キメラと、他方では純粋なホロ受容体キメラが、EP1929311B1によるシグナル受容体としてのアンカー受容体及びトランケートされた受容体キメラ(ECD)として使用された。アンカー受容体キメラをマイクロタイタープレートのプラスチック壁に固定化した。
Claims (4)
- 患者サンプルを、固相に結合した一次TSH受容体タンパク質と接触させて、前記患者サンプル中に存在する可能性がある、TSH受容体に対する刺激型自己抗体を前記一次TSH受容体タンパク質に結合させ、
得られた免疫複合体を二次TSH受容体タンパク質と反応させて、前記患者サンプル中に存在する可能性がある、TSH受容体に対する刺激型自己抗体を前記二次TSH受容体タンパク質にさらに結合させ、そして
前記自己抗体を含有する免疫複合体を検出するための反応を実行する、
診断分析器で実施可能な、TSH受容体に対する刺激型自己抗体の検出方法であって、前記TSH受容体タンパク質は、TSH受容体に対する中間型及び遮断型自己抗体のための結合エピトープを有さず、
常磁性粒子が前記固相として使用されていること、
前記一次及び前記二次TSH受容体タンパク質が、TSH受容体に対して刺激型の自己抗体に関する結合性エピトープを含み、前記TSH受容体タンパク質が配列番号3又は配列番号4と少なくとも90%以上同一であり、そして、TSH受容体の細胞外空間への分泌に適したタンパク質とN末端で融合していること、そのタンパク質が、ヒト血清アルブミン(HAS)及び分泌型アルカリホスファターゼ(SEAP)から選択されていること、並びに
前記二次TSH受容体タンパク質が、形成された前記免疫複合体を検出するためのシグナリングアミノ酸配列又は別のシグナリングマーカーを含む、前記検出方法。 - 前記シグナリングマーカーが、分泌型アルカリホスファターゼ、ホタル若しくはガウシアルシフェラーゼ、又はペルオキシダーゼである、請求項1に記載の方法。
- 前記シグナリングマーカーが、アクリジン若しくはルテニウム化合物、又はフルオレセインイソチオシアネート化合物である、請求項1に記載の方法。
- ヒト血清アルブミン(HAS)及び分泌型アルカリホスファターゼ(SEAP)から選択されるタンパク質と融合されたTSH受容体タンパク質の使用であって、
TSH受容体タンパク質はTSH受容体に対する刺激型自己抗体に関する結合性エピトープを含み、そして配列番号3又は配列番号4のアミノ酸配列の1つと少なくとも90%同一であり、そして前記TSH受容体タンパク質はTSH受容体に対する刺激型自己抗体の検出用二次モノクローナル又はポリクローナル抗体と使用される、前記TSH受容体タンパク質の使用。
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DE102014011653.0 | 2014-08-11 | ||
PCT/EP2015/063627 WO2015193387A1 (de) | 2014-06-20 | 2015-06-17 | Nachweis von autoantikörpern gegen den tsh-rezeptor |
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