单环刺螠硫醌氧化还原酶间接竞争ELISA检测方法的建立

Establishment of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Urechis unicinctus Sulfide: Quinone Oxidoreductase

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。

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 immunosor- bent 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 opti- mized. 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% respective- ly. In conclusion, an indirect competitive ELISA for U. unicinctus SQR was established successfully.