JP2022103480A - 甲状腺刺激ホルモン受容体阻害性自己抗体の活性測定法およびキット - Google Patents
甲状腺刺激ホルモン受容体阻害性自己抗体の活性測定法およびキット Download PDFInfo
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Abstract
Description
工程(a)の後、前記cAMPバイオセンサーの活性化レベルを測定する工程(b);及び、
工程(b)で測定した活性化レベルと、対照における活性化レベルとを比較することで、試料中のTSH受容体に対する阻害性自己抗体の活性を算定する工程(c);
の工程(a)~(c)を含む、哺乳動物細胞中のTSH受容体に対する阻害性自己抗体活性測定方法うに当たり、すべての工程をPEG濃度5%以下またはPEG非存在の条件下で行うことにより、TSHによる競合阻害原理を用いた測定法において短時間での阻害活性の検出が可能となることを見出し、本発明を完成させた。
cAMPバイオセンサー及びTSH受容体の両方が発現する哺乳動物細胞を、被験者から採取された血液試料およびTSHの存在下でインキュベートする工程(a);
工程(a)の後、前記cAMPバイオセンサーの活性化レベルを測定する工程(b);及び、
工程(b)で測定した活性化レベルと、対照における活性化レベルとを比較することで、試料中のTSH受容体に対する阻害性自己抗体の活性を算定する工程(c);
の工程(a)~(c)を含む、哺乳動物細胞中のTSH受容体に対する阻害性自己抗体活性測定方法において、すべての工程をPEG濃度5%以下の条件下で行うことを特徴とする、阻害性時抗体活性の測定法(以下、「本件測定方法」ということがある)に関するものである。
GNAS遺伝子(Gene ID2778)
GNAL遺伝子(Gene ID2774)
[A]試料及びTSH添加区(インキュベート用液+TSH+試料)
[B]試料及びTSH非添加区(インキュベート用液+試料)
[C]対照溶液及びTSH添加区(インキュベート用液+TSH)
[D]対照溶液及びTSH非添加区(インキュベート用液)
阻害率(%)={1-([A]活性化レベルー[B]活性化レベル})/([C]活性化レベル-[D]活性化レベル)}×100
[E]試料及びTSH添加区(インキュベート用液+TSH+試料)
[F]試料及びTSH非添加区(インキュベート用液+試料)
[G]対照試料及びTSH添加区(インキュベート用液+TSH+健常者由来試料)
[H]対照試料及びTSH非添加区(インキュベート用液+健常者由来試料)
阻害率(%)={1-([E]活性化レベルー[F]活性化レベル})/([G]活性化レベル-[H]活性化レベル)}×100
cAMPバイオセンサーの活性化レベルを指標として、TSH阻害性自己抗体の阻害活性を定量する際の、PEG有無による測定への影響を検討した。
公知文献(WO2020/050208)の記載を参考に、GloSensor cAMP、すなわちホタルルシフェラーゼ(firefly luciferase)の359~544番目のアミノ酸残基と、プロテインキナーゼA(PKA)の制御サブユニットのcAMP結合領域と、ホタルルシフェラーゼの4~355番目のアミノ酸残基とを含むタンパク質を発現するプラスミドベクター(pGloSenso-22F、Promega社製)と、ヒトTSH受容体タンパク質を発現するpcDNA3.1(+)(pcDNA3.1_hTSHR)とを、FuGENE HD Transfection Reagent(Promega社製)を用い、GloSensor cAMP Assay(Promega社製)に添付のプロトコルに従って、ヒト胎児腎細胞由来細胞(HEK293細胞)へトランスフェクションすることによって、測定に用いる組換HEK293細胞を作製した。
対象試料としては、TSBAb高値であることがすでに知られている血清試料6例(陽性P8,P9,P10,P11,P13,P14)およびTSBAb量が健常であることが知られる血清6例(健常275,276,277,278,280,282)を使用した。試験区としては、下記4パターンを調製した。
[B]試料各種及びTSH非添加区(インキュベート用液+試料)
[C]対照試料及びTSH添加区(インキュベート用液+TSH+健常者由来試料)
[D]対照試料及びTSH非添加区(インキュベート用液+健常者由来試料)
(1) 調製済の各試料を、96well white plate(Corning社製)に25μLずつ分注する。
(2) 前記96well white plateに、各濃度のrecombinantヒト由来TSH(rhTSH)を25μLずつ分注する。
(3) GloSensor cAMP Reagent含有希釈液2mLに、TSHR組換HEK293細胞液を1.25 x 105 cells/mLとなるように加えてよく混合し、調製済細胞液を作製する。
(4) 室温で遮光し、5分間静置する。
(5) 工程(3)で得た調製済細胞液を前記96well white plateに100μLずつ分注する。
(6) 800rpmで30秒間撹拌する。
(7) 25℃で遮光し、20分、30分、40分、60分または120分間静置して反応させた。それぞれの反応時間におけるルシフェラーゼ活性レベルを、Glomax発光プレートリーダー(Promega社)を用いて測定する。
Claims (3)
- cAMPバイオセンサー及びTSH受容体の両方が発現する哺乳動物細胞を、被験者から採取された血液試料およびTSHの存在下でインキュベートする工程(a);
工程(a)の後、前記cAMPバイオセンサーの活性化レベルを測定する工程(b);及び、
工程(b)で測定した活性化レベルと、対照における活性化レベルとを比較することで、試料中のTSH受容体に対する阻害性自己抗体の活性を算定する工程(c);
の工程(a)~(c)を含む、哺乳動物細胞中のTSH受容体に対する阻害性自己抗体活性測定方法であって、すべての工程をPEG濃度5%以下の条件下で行うことを特徴とする、阻害性自己抗体活性の測定法。 - cAMPバイオセンサー及びTSH受容体の両方が発現する哺乳動物細胞を、被験者から採取された血液試料およびTSHの存在下でインキュベートする工程(a);
工程(a)の後、前記cAMPバイオセンサーの活性化レベルを測定する工程(b);及び、
工程(b)で測定した活性化レベルと、対照における活性化レベルとを比較することで、試料中のTSH受容体に対する阻害性自己抗体の活性を算定する工程(c);
の工程(a)~(c)を含む、哺乳動物細胞中のTSH受容体に対する阻害性自己抗体活性測定方法であって、すべての工程をPEGの非存在下で行うことを特徴とする、阻害性自己抗体活性の測定法。 - 工程(a)のインキュベート時間が60分以下である、請求項1又は2記載の阻害性自己抗体活性の測定法。
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