| 温思思, 赵福运, 郭飘逸, 徐云飞, 赵伟春*.引起浙贝母黑斑病的交链格孢单抗及检测试纸条的制备[J].植物保护,2022,48(5):227-233. |
| 引起浙贝母黑斑病的交链格孢单抗及检测试纸条的制备 |
| Preparation of monoclonal antibody and test strips for Alternaria alternata causing black spot disease of Fritillaria thunbergii |
| 投稿时间：2021-08-08 修订日期：2021-11-08 |
| DOI：10.16688/j.zwbh.2021428 |
| 中文关键词: 浙贝母 黑斑病 交链格孢 单克隆抗体 试纸条 |
| 英文关键词:Fritillaria thunbergii black spot disease Alternaria alternata monoclonal antibody (McAb) test strip |
| 基金项目:浙江省基础公益研究计划(LGN21C140004, 2015C32076); 浙江省中医药科技计划(2020B060) |
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| 中文摘要: |
| 浙贝母黑斑病是危害浙贝母茎叶的重要真菌性病害之一, 该病侵染叶片产生叶斑?叶枯, 严重时全株枯死?本研究制备了针对浙贝母黑斑病致病菌交链格孢Alternaria alternata的单克隆抗体(单抗), 并开发了胶体碳免疫层析试纸条用于黑斑病早期精准检测?以交链格孢提取液免疫Bal b/c小鼠, 采用杂交瘤技术制备单抗, 间接ELISA法和Western blot分析单抗的效价?灵敏度?特异性?抗体类型及结合蛋白?以双抗夹心法制备胶体碳免疫层析试纸条?得到3种针对交链格孢的单抗及其杂交瘤细胞株AaA1?AaC2和AaC5?3种单抗对交链格孢均具有高度特异性, 灵敏度为12.21~24.41 ng/mL, 效价≥6.40×105, 抗体类型分别为IgG2a?IgG3和IgG3, 结合蛋白的分子量分别为37?62 kDa和66 kDa?以AaA1为碳标单抗, AaC2为划膜单抗的胶体碳试纸条检测交链格孢的方法快速简便?准确灵敏?经济环保?从田间采样到出结果用时少于15 min, 样品简单研磨后取汁液加入加样孔即完成全部操作, 对交链格孢抗原的检测灵敏度为6.25 μg/mL, 试纸条采用环保材料制备, 单个试纸条的成本约1.7元? |
| 英文摘要: |
| Black spot disease is one of the important fungal diseases that damage the stems and leaves of Fritillaria thunbergii. The disease infects leaves and produces leaf spot, leaf blight, and in severe cases, the whole plant dies. In this study, the monoclonal antibody (McAb) against Alternaria alternata, a pathogen of F.thunbergii, was prepared, and a colloidal carbon immunochromatographic test strip was developed for the early and accurate detection of black spot. Bal b/c mice were immunized with A. alternata extracts, McAb was prepared by hybridoma technology, and the titer, sensitivity, specificity, antibody type and binding protein of McAb were analyzed by indirect ELISA and Western blot, respectively. The colloidal carbon immunochromatographic test strip was prepared by double antibody sandwich method. Three McAbs against A. alternata and their hybridoma cell lines, AaA1, AaC2, and AaC5 were obtained separately. The McAbs were highly specific to A. alternata, with the sensitivities of 12.21-24.41 ng/mL and the titers of more than 6.40×105. The antibody types were IgG2a, IgG3, and IgG3, respectively, and the molecular weights of the binding proteins were 37 kDa, 62 kDa and 66 kDa, respectively. The colloidal carbon test strip with AaA1 as the carbon-labeled McAb and AaC2 as the scribing McAb can be used to detect A. alternata quickly, easily, accurately, sensitively, economically and environmentally. It takes less than 15 minutes from field sampling to the result, simply ground sample juice is added to the sample hole and the whole operation is complete. The minimum detection limit for A. alternata is 6.25 μg/mL. The test strip is made of environment-friendly materials, and the cost of a single test strip is about 1.7 yuan. |
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