JP7403119B2 - ステロイドの測定方法、ステロイドの測定キット及びストレス度の測定方法 - Google Patents
ステロイドの測定方法、ステロイドの測定キット及びストレス度の測定方法 Download PDFInfo
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- JP7403119B2 JP7403119B2 JP2019193718A JP2019193718A JP7403119B2 JP 7403119 B2 JP7403119 B2 JP 7403119B2 JP 2019193718 A JP2019193718 A JP 2019193718A JP 2019193718 A JP2019193718 A JP 2019193718A JP 7403119 B2 JP7403119 B2 JP 7403119B2
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Description
ハイドロキシステロイド脱水素酵素と、ジアフォラーゼと、ニコチンアミドアデニンジヌクレオチドおよび/またはニコチンアミドアデニンジヌクレオチドリン酸と、電子伝達能を有するメディエーターと、前記メディエーターとの間に電子移動を生じさせる電極と、を用いる(ただし、デヒドロゲナーゼとジアフォラーゼと、の会合タンパク質を用いるものを除く)、ことを特徴とする。
ハイドロキシステロイド脱水素酵素と、ジアフォラーゼと、ニコチンアミドアデニンジヌクレオチドおよび/またはニコチンアミドアデニンジヌクレオチドリン酸と、電子伝達能を有するメディエーターと、を含み(ただし、デヒドロゲナーゼとジアフォラーゼと、の会合タンパク質を用いるものを除く)、
前記メディエーターとの間に電子移動を生じさせる電極を備える、
ことを特徴とする。
ハイドロキシステロイド脱水素酵素と、ジアフォラーゼと、ニコチンアミドアデニンジヌクレオチドおよび/またはニコチンアミドアデニンジヌクレオチドリン酸と、電子伝達能を有するメディエーターと、該メディエーターとの間に電子移動を生じさせる電極と、を用いて対象サンプルの電流値を測定する工程と、
得られた電流値をステロイド濃度に変換する工程と、
得られたステロイド濃度をストレス度に変換する工程と、
を含む。
市販のGCEガラス状カーボン電極(直径3mm、長さ6mm、ビー・エー・エス株式会社製)を0.125μmのダイヤモンド粒子を用いて、電極表面が鏡面状態になるまで研磨した後、エタノール中で30秒超音波洗浄を行った。次に、10mM Tris-HCl buffer(pH 7.5)2μLと、4kU/mL(3mg/mL)に調製したジアフォラーゼ溶液(ニプロ社製)0.2μLと、200μg/mLに調製した11-β-ハイドロキシステロイド脱水素酵素type 2(HSD2)(MyBioSource社)2μLとを上記の電極上に載せて5~15分間、室温でインキュベートし、電極にジアフォラーゼとHSD2とを担持させた。次いで、予め蒸留水で洗浄しておいたセルロース透析膜(Por 4、スペクトラムラボラトリーズ社製)で上記電極を被覆し、作用電極を製造した。
市販のGCEガラス状カーボン電極(直径3mm、長さ6mm、ビー・エー・エス株式会社製)を0.125μmのダイヤモンド粒子を用いて、電極表面が鏡面状態になるまで研磨した後、エタノール中で30秒超音波洗浄を行った。次に、10mM Tris-HCl buffer(pH 7.5)2μLと、4kU/mL(3mg/mL)に調製したジアフォラーゼ溶液(ニプロ社製)0.2μLと、4.7mg/mL(329U/mL)の3α-ハイドロキシステロイド脱水素酵素(3αHSD)(オリエンタル酵母社製)10μLとを上記の電極上に載せて5~15分間、室温でインキュベートし、電極にジアフォラーゼと3αHSDとを担持させた。次いで、予め蒸留水で洗浄しておいたセルロース透析膜(Por 4、スペクトラムラボラトリーズ社製)で上記電極を被覆し、作用電極を製造した。
Claims (6)
- ハイドロキシステロイド脱水素酵素と、ジアフォラーゼと、ニコチンアミドアデニンジヌクレオチドおよび/またはニコチンアミドアデニンジヌクレオチドリン酸と、電子伝達能を有するメディエーターと、該メディエーターとの間に電子移動を生じさせる電極と、を用いる(ただし、デヒドロゲナーゼとジアフォラーゼと、の会合タンパク質を用いるものを除く)、
ことを特徴とするステロイドの測定方法。 - 前記ハイドロキシステロイド脱水素酵素は、3α型、3β型、11β型及び17β型からなる群より少なくとも1つ選択される、
ことを特徴とする請求項1に記載のステロイドの測定方法。 - 前記ステロイドは、テストステロン、デヒドロエピアンドロステロン、コルチゾール、エストラジオール、アンドロステロン、それらの酸化型ステロイド及びそれらの誘導体からなる群より少なくとも1つ選択される、
ことを特徴とする請求項1又は2に記載のステロイドの測定方法。 - 前記電極により電流値を測定する、
ことを特徴とする請求項1乃至3のいずれか1項に記載のステロイドの測定方法。 - ハイドロキシステロイド脱水素酵素と、ジアフォラーゼと、ニコチンアミドアデニンジヌクレオチドおよび/またはニコチンアミドアデニンジヌクレオチドリン酸と、電子伝達能を有するメディエーターと、を含み(ただし、デヒドロゲナーゼとジアフォラーゼと、の会合タンパク質を用いるものを除く)、
前記メディエーターとの間に電子移動を生じさせる電極を備える、
ことを特徴とするステロイドの測定キット。 - ハイドロキシステロイド脱水素酵素と、ジアフォラーゼと、ニコチンアミドアデニンジヌクレオチドおよび/またはニコチンアミドアデニンジヌクレオチドリン酸と、電子伝達能を有するメディエーターと、該メディエーターとの間に電子移動を生じさせる電極と、を用いて対象サンプルの電流値を測定する工程と、
得られた電流値をステロイド濃度に変換する工程と、
得られたステロイド濃度をストレス度に変換する工程と、
を含むストレス度の測定方法。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2000189188A (ja) | 1998-12-24 | 2000-07-11 | Asahi Chem Ind Co Ltd | 酵素を用いる分析方法 |
JP2002372511A (ja) | 2001-06-14 | 2002-12-26 | Matsushita Electric Ind Co Ltd | 測定装置 |
WO2005056824A1 (ja) | 2003-12-08 | 2005-06-23 | National Institute Of Advanced Industrial Science And Technology | 硫酸抱合型胆汁酸の検出方法、それに用いられる試験紙およびバイオセンサ |
JP2007163268A (ja) | 2005-12-13 | 2007-06-28 | Canon Inc | 酵素電極 |
WO2009017188A1 (ja) | 2007-08-01 | 2009-02-05 | Nipro Corporation | ストレス測定キット及びストレス測定方法 |
JP2014219353A (ja) | 2013-05-10 | 2014-11-20 | 株式会社日立製作所 | 分子鋳型及びその製造方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
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JP2000189188A (ja) | 1998-12-24 | 2000-07-11 | Asahi Chem Ind Co Ltd | 酵素を用いる分析方法 |
JP2002372511A (ja) | 2001-06-14 | 2002-12-26 | Matsushita Electric Ind Co Ltd | 測定装置 |
WO2005056824A1 (ja) | 2003-12-08 | 2005-06-23 | National Institute Of Advanced Industrial Science And Technology | 硫酸抱合型胆汁酸の検出方法、それに用いられる試験紙およびバイオセンサ |
JP2007163268A (ja) | 2005-12-13 | 2007-06-28 | Canon Inc | 酵素電極 |
WO2009017188A1 (ja) | 2007-08-01 | 2009-02-05 | Nipro Corporation | ストレス測定キット及びストレス測定方法 |
JP2014219353A (ja) | 2013-05-10 | 2014-11-20 | 株式会社日立製作所 | 分子鋳型及びその製造方法 |
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