| 单环刺螠硫醌氧化还原酶间接竞争ELISA检测方法的建立 |
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| 引用本文： | 马玉彬,史晓丽,李阳,董英萍,张立涛,张志峰.单环刺螠硫醌氧化还原酶间接竞争ELISA检测方法的建立[J].中国海洋大学学报(自然科学版),2012(Z2):64-69. |
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| 作者姓名： | 马玉彬 史晓丽 李阳 董英萍 张立涛 张志峰 |
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| 作者单位： | 中国海洋大学海洋生物遗传育种教育部重点实验室;中国科学院青岛生物能源与过程研究所生物燃料重点实验室,山东省能源生物遗传资源重点实验室 |
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| 基金项目： | 国家自然科学基金项目(40776074;31072191)资助 |
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| 摘 要： | 硫醌氧化还原酶(Sulfide:quinone oxidoreductase,SQR),是线粒体硫化物代谢的关键酶。本研究以生活在潮间带下区及潮下带中的底栖生物—单环刺螠SQR重组蛋白为材料,建立了单环刺螠SQR间接竞争ELISA检测方法,为定量分析SQR蛋白水平表达奠定了基础。将SQR重组蛋白免疫新西兰大白兔获得多克隆抗体,检测效价高达1:64 000。采用免疫印迹实验,将该抗体与重组蛋白和体壁总蛋白杂交,均得到了单一的条带,表明该抗体特异性好。优化SQR间接竞争ELISA检测条件,得出最佳的抗原包被浓度为250ng/mL,一抗浓度为1∶20 000,二抗浓度为1∶12 000,此条件下建立的标准曲线检测范围为2.5～1 000ng/mL。精确度检测结果显示,批内差异在0.42%～7.27%、批间差异在2.58%～7.78%范围内,表明该条件下精确度具有良好的准确性和重复性。以上结果表明,已成功建立单环刺螠SQR间接竞争ELISA检测方法。
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| 关 键 词： | 单环刺螠 硫醌氧化还原酶 间接竞争ELISA 多克隆抗体 |
Establishment of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Urechis unicinctus Sulfide: Quinone Oxidoreductase |
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| MA Yu-Bin,SHI Xiao-Li,LI Yang,DONG Ying-Ping,ZHANG Li-Tao,ZHANG Zhi-Feng.Establishment of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Urechis unicinctus Sulfide: Quinone Oxidoreductase[J].Periodical of Ocean University of China,2012(Z2):64-69. |
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| Authors: | MA Yu-Bin SHI Xiao-Li LI Yang DONG Ying-Ping ZHANG Li-Tao ZHANG Zhi-Feng |
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| Institution: | 1(1.The Key Laboratory of Marine Genetics and Breeding,Ministry of Education,Ocean University of China,Qingdao 266003,China;2.Shandong Provincial Key Laboratory of Energy Genetics,Key Laboratory of Biofuels,Qingdao Institute of Bioenergy and Bioprocess Technology,Chinese Academy of Sciences,Qingdao 266101,China) |
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| Abstract: | Sulfide:quinone oxidoreductase(SQR) is a key enzyme in mitochondrial sulfide metabolism.Urechis unicinctus is a benthic organism,mostly living in sulfidic habitats including intertidal and subtidal zone.Using U.unicinctus SQR recombinant protein,an indirect competitive enzyme-linked immunosorbent assay(ELISA) was established which provided a quantitative method for detecting U.unicincuts SQR protein expression in vivo.Firstly,polyclonal antibody was obtained by immunizing New Zealand rabbits using purified recombinant SQR with a titer of 1:64 000.Western Blot showed that rabbit antisera reacted with purified recombinant SQR and body wall total protein,forming a single band which indicated good specificity for the polyclonal antibody.And then,indirect competitive ELISA conditions were optimized.The optimized coating antigen concentration was 250 ng/mL,while SQR antisera concentration was at 1:20 000 and second antibody concentration was at 1:12 000.The established standard curve showed a working range between 2.5 ng/mL and 1000 ng/mL.The ELISA demonstrated precision and reproducibility,with inter and intra-assay variations between 0.41%~7.27% and 2.58%~7.78% respectively.In conclusion,an indirect competitive ELISA for U.unicinctus SQR was established successfully. |
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| Keywords: | Urechis unicinctus sulfide quinone oxidoreductase indirect competitive enzyme-linked immunosorbent assay polyclonal antibody |
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