基于单克隆抗体的胶体金免疫层析条用于快速检测番茄环斑病毒。
Monoclonal antibody-based colloid gold immunochromatographic strip for the rapid detection of Tomato zonate spot tospovirus.
机构信息
College of Life Sciences, Shanxi Agricultural University, Taigu, 030801, China.
出版信息
Virol J. 2018 Jan 18;15(1):15. doi: 10.1186/s12985-018-0919-5.
BACKGROUND
Tomato zonate spot virus (TZSV), a new species of genus Tospovirus, caused significant losses in yield and problems in quality of many important vegetables and ornamentals in Southwest China and posed a serious threat to important economic crops for the local farmers. A convenient and reliable method was urgently needed for rapid detection and surveillance of TZSV.
METHODS
The nucleocapsid protein (N) of TZSV was expressed in Escherichia coli and purified, and was used as the antigen to immunize BALB/c mice. Three monoclonal antibodies (mAbs) 3A2, 5D2 and 5F7 against TZSV were obtained through the hybridoma technique. The mAb 3A2 was conjugated with colloid gold as detecting reagent; mAb 5D2 was coated on a porous nitrocellulose membrane as the detection line and protein A was coated as the control line respectively. The colloid gold immunochromatographic (GICA) strip was assembled.
RESULTS
The analysis of Dot-ELISA and Western blot showed that the obtained three independent lines of mAbs 3A2, 5D2 and 5F7 specifically recognized TZSV N. Based on the assembly of GICA strip, the detection of TZSV was achieved by loading the infected sap onto the test strip for visual inspection. The analysis could be completed within 5-10 min. No cross-reaction occurred between TZSV and other tested viruses. The visual detection limit of the test strip for TZSV was 800 fold dilutions of TZSV-infected leaf samples.
CONCLUSION
The mAbs were specific and the colloidal GICA strip developed in this study was convenient, fast and reliable for the detection of TZSV. The method could be applied for the rapid diagnosis and surveillance of TZSV in the field.
背景
番茄斑萎病毒（TZSV）是一新种，属于布尼亚病毒科（Bunyaviridae），番茄斑萎病毒属（Tospovirus），在中国西南地区的许多重要蔬菜和观赏植物中造成了严重的产量损失和质量问题，对当地农民的重要经济作物构成了严重威胁。迫切需要一种快速检测和监测 TZSV 的方便可靠方法。
方法
在大肠杆菌中表达 TZSV 的核衣壳蛋白（N）并进行纯化，将其作为抗原免疫 BALB/c 小鼠。通过杂交瘤技术获得了针对 TZSV 的 3 株单克隆抗体（mAbs）3A2、5D2 和 5F7。mAb 3A2 与胶体金偶联作为检测试剂；mAb 5D2 分别包被在多孔硝酸纤维素膜上作为检测线和蛋白 A 作为控制线。组装胶体金免疫层析（GICA）条。
结果
Dot-ELISA 和 Western blot 分析表明，获得的 3 株独立的 mAbs 3A2、5D2 和 5F7 特异性识别 TZSV N。基于 GICA 条的组装，通过将感染的汁液加载到测试条上进行目视检查来实现 TZSV 的检测。分析可以在 5-10 分钟内完成。TZSV 与其他测试病毒之间没有发生交叉反应。该测试条目视检测 TZSV 的下限为 TZSV 感染叶片样品的 800 倍稀释。
结论
所获得的 mAbs 具有特异性，本研究开发的胶体金 GICA 条方便、快速、可靠，可用于 TZSV 的快速诊断和田间监测。该方法可用于 TZSV 的快速诊断和田间监测。
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