JP5690514B2 - アゾキシストロビン誘導体、アゾキシストロビンに対する抗体またはそのフラグメント、ならびにそれらの抗体またはフラグメントを用いた測定キットおよび測定方法 - Google Patents
アゾキシストロビン誘導体、アゾキシストロビンに対する抗体またはそのフラグメント、ならびにそれらの抗体またはフラグメントを用いた測定キットおよび測定方法 Download PDFInfo
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- JP5690514B2 JP5690514B2 JP2010149812A JP2010149812A JP5690514B2 JP 5690514 B2 JP5690514 B2 JP 5690514B2 JP 2010149812 A JP2010149812 A JP 2010149812A JP 2010149812 A JP2010149812 A JP 2010149812A JP 5690514 B2 JP5690514 B2 JP 5690514B2
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Description
本明細書でいう「アゾキシストロビン」とは、メトキシアクリレート骨格を有する殺菌剤であり、下記の式
一方、下記反応式のように、化合物8を得る。
(1)抗原を担体に固相化する。
用いる担体は、通常のELISAに用いる担体であれば特に制限されないが、96穴、48穴、192穴等のマイクロタイタープレートが好ましい。固相化は、例えば、固相化用抗原を含む緩衝液を担体上に載せ、インキュベーションすればよい。緩衝液中の抗原の濃度は、通常0.01μg/mLから100μg/mL程度である。緩衝液としては、検出手段に応じて公知のものを使用することができる。
2:1H-NMR (300 MHz, CDCl3):δ(ppm ) = 7.61(d, J = 15.9 Hz, 1H), 7.30 (m, 2H), 6.79 (d, J = 8.1 Hz, 1H), 6.29 (d, 16.2 Hz, 1H), 5.60 (s, 1H), 3.80 (s, 3H), 2.26 ppm (s, 3H)
13C-NMR(300MHz, CDCl3):δ(ppm ) = 168.34, 156.36, 145.21, 131.24, 127.80, 127.12, 124.70, 115.46, 114.91, 51.83, 15.92.
3:1H-NMR(300MHz, CDCl3):δ(ppm ) = 6.92 (d J-- = 8.1Hz, 2H), 6.68(d, J = 8.1 Hz, 1H), 5.56 (s, 1H), 3.68 (s, 3H), 2.86 (t, J= 7.5 Hz, 2H), 2.60 (t, J = 7.5 Hz, 2H), 2.22 (s, 3H).
13C-NMR(300MHz, CDCl3):δ(ppm ) = 174.13, 152.56, 132.33, 130.97, 126.67, 124.09, 114.96, 51.86, 36.23, 30.21, 15.93.
5:1H-NMR(300MHz, CDCl3):δ(ppm ) = 7.20-7.45 (m, 7H), 6.85 (m, 2H), 5.20 (brs, 2H), 3.65 (s, 2H), 3.62 (s, 3H).
7:1H-NMR(300MHz, CDCl3):δ(ppm ) = 7.62 (s, 1H), 7.22(d, J= 7.60, 1H), 6.90 (d, J=7.6, 1H), 6.70 (d, J=7.62, 1H), 3.65 (s, 2H), 3.62 (s, 3H).
8:1H-NMR(300MHz, CDCl3):δ(ppm ) =8.79 (s, 1H), 7.42 (s, 1H), 7.30-7.41 (m,, 3H), 7.18 (d, J=6.7Hz, 1H), 6.68 (s, 1H), 3.72 (s, 3H), 3.59 (s, 3H).
9:1H-NMR (300MHz, CDCl3):δ(ppm ) =8.40 (s, 1H), 7.42 (s, 1H), 7.02-7.41 (m, 6H), 6.85 (d, J=6.7 Hz, 1H), 6.20 (s, 1H), 3.73 (s, 3H) 3.65 (s, 3H), 3.49 (s, 3H), 2.92 (t, J=6.7 Hz,, 2H), 2.62 (t, J=6.7 Hz,, 2H), 2.12 (s, 3H)。
10:1H-NMR(300MHz, CDCl3):δ(ppm ) = 8.40 (s, 1H), 7.46 (s, 1H), 7.42-7.28 (m, 3H), 7.21 (m, 2H), 7.13-6.09 (m, 2H), 6.99-6.92 (m, 1H), 6.12 (s, 1H), 3.74 (s, 3H), 3.59 (s, 3H), 2.96 (t, J = 7.8 Hz, 2H), 2.70 (t, J = 7.8 Hz, 2H). 2.13ppm (s, 3H)
これをアゾキシストロビンハプテンとして抗体作製に供した。
免疫原としてスカシガイヘモシアニン(KLH)とBSAを用い、アゾキシストロビンとそれぞれの免疫原との複合体を、活性エステル法を用いて作製した。
実施例2で調製した免疫原を1mg/mLとなるようにPBSに溶解し、これに等量の完全アジュバント(商品名:フロイント完全アジュバント;FCA)を混合しエマルジョン化し、その100μLを6〜7週齢のメスのBALB/cマウスに腹腔投与した。これと同様の手順で、不完全アジュバント(商品名:フロイント不完全アジュバント;FICA)を等量混合した0.5mg/mLの免疫原100μLを2週間毎に追加免疫した。4回の免疫後、眼底から採血し、血清中の抗体力価が十分に上がっていることを間接ELISAにて確認した。
(1)実施例1で得られたアゾキシストロビンとBSAとの複合体を、PBSを用いて1μg/mLに希釈し、96穴マイクロプレートに100μL/ウェルずつ分注し、4℃で一晩放置することにより固相化した。次に液を吸引除去後、0.4%BSAおよびPBSを300μL/ウェル分注し、4℃で一晩静置することによりブロッキングを行った後、ブロッキング液を吸引除去した。
血中の抗体価が十分に高くなったマウスを用いて、最終免疫(20μg/マウス)した。その3日後に当該マウスから脾臓を摘出し細胞融合に供した。
実施例3で得られたAZS11A-10F-9H抗体産生ハイブリドーマ株を10%牛胎児血清入りRPMI1640で培養し、約2×106個の細胞をBALB/c マウスの腹腔内に注射し、腹水液を採取した。得られた腹水はプロテインG カラムによりIgG精製を行った。得られたモノクローナル抗体はサブクラスがIgG1であり、軽鎖はいずれもκ鎖である。
すなわち、抗マウスIgGウサギ抗体をPBS緩衝液(10mM NaPB、150mM NaCl)で10μg/mLに希釈し、96穴マイクロプレートに100μg/ウェルづつ分注し、4℃で一晩放置することにより固相化した。次に液を吸引除去後、PBS緩衝液(0.4%BSA、10mM NaPB、150mM NaCl、pH7.0)を300μg/ウェル分注し、4℃で一晩静置することによりブロッキングを行った後、ブロッキング液を吸引除去した。
表1に示すキットを用いて、アゾキシストロビン(和光純薬)を始めとする各種農薬の標品の濃度測定を行った。
Claims (6)
- 下記式(1):
(式中、R1は、シアノ基またはメチル基を表し、nは、0から10までのいずれかの整数を表す)で表わされる構造を有する化合物と高分子化合物との複合体からなるアゾキシストロビン抗原。 - 請求項1記載のアゾキシストロビン抗原に対するモノクローナル抗体またはそのフラグメント。
- 請求項2に記載の抗体を産生するハイブリドーマ。
- 前記ハイブリドーマが、AZS11A-10F-9H(受託番号FERM-P-21815の下に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に寄託)である、請求項3に記載のハイブリドーマ。
- 請求項2に記載のモノクローナル抗体またはそのフラグメントを含んでなるアゾキシストロビンの測定キット。
- 請求項2に記載のモノクローナル抗体またはそのフラグメントを試料に接触させる工程を含むアゾキシストロビンの測定方法。
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