基于 Luminex xMAP 的灵敏微球免疫分析方法的建立，用于特异性检测鸢尾黄斑驳病毒。

Development of a sensitive Luminex xMAP-based microsphere immunoassay for specific detection of Iris yellow spot virus.

机构信息

Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai, 200135, China.

Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, China.

出版信息

Virol J. 2018 Apr 4;15(1):62. doi: 10.1186/s12985-018-0952-4.

DOI:10.1186/s12985-018-0952-4

PMID:29615087

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5883346/

Abstract

BACKGROUND

Iris yellow spot virus (IYSV) is an Orthotospovirus that infects most Allium species. Very few approaches for specific detection of IYSV from infected plants are available to date. We report the development of a high-sensitive Luminex xMAP-based microsphere immunoassay (MIA) for specific detection of IYSV.

RESULTS

The nucleocapsid (N) gene of IYSV was cloned and expressed in Escherichia coli to produce the His-tagged recombinant N protein. A panel of monoclonal antibodies (MAbs) against IYSV was generated by immunizing the mice with recombinant N protein. Five specific MAbs (16D9, 11C6, 7F4, 12C10, and 14H12) were identified and used for developing the Luminex xMAP-based MIA systems along with a polyclonal antibody against IYSV. Comparative analyses of their sensitivity and specificity in detecting IYSV from infected tobacco leaves identified 7F4 as the best-performed MAb in MIA. We then optimized the working conditions of Luminex xMAP-based MIA in specific detection of IYSV from infected tobacco leaves by using appropriate blocking buffer and proper concentration of biotin-labeled antibodies as well as the suitable ratio between the antibodies and the streptavidin R-phycoerythrin (SA-RPE). Under the optimized conditions the Luminex xMAP-based MIA was able to specifically detect IYSV with much higher sensitivity than conventional enzyme-linked immunosorbent assay (ELISA). Importantly, the Luminex xMAP-based MIA is time-saving and the whole procedure could be completed within 2.5 h.

CONCLUSIONS

We generated five specific MAbs against IYSV and developed the Luminex xMAP-based MIA method for specific detection of IYSV in plants. This assay provides a sensitive, high-specific, easy to perform and likely cost-effective approach for IYSV detection from infected plants, implicating potential broad usefulness of MIA in plant virus diagnosis.

摘要

背景

虹膜黄斑病毒（IYSV）是一种感染大多数葱属物种的正呼肠孤病毒。迄今为止，只有极少数方法可用于从感染植物中特异性检测 IYSV。我们报告了一种基于高灵敏度 Luminex xMAP 的微球免疫分析（MIA）的开发，用于特异性检测 IYSV。

结果

克隆并在大肠杆菌中表达了 IYSV 的核衣壳（N）基因，以产生带有 His 标签的重组 N 蛋白。用重组 N 蛋白免疫小鼠产生了针对 IYSV 的单克隆抗体（MAb）。鉴定出针对 IYSV 的五株特异性 MAbs（16D9、11C6、7F4、12C10 和 14H12），并与针对 IYSV 的多克隆抗体一起用于开发基于 Luminex xMAP 的 MIA 系统。通过比较分析它们从感染烟草叶片中检测 IYSV 的敏感性和特异性，确定 7F4 是 MIA 中表现最好的 MAb。然后，我们通过使用适当的封闭缓冲液和适当浓度的生物素标记抗体以及抗体与链霉亲和素 R-藻红蛋白（SA-RPE）之间的适当比例，优化了基于 Luminex xMAP 的 MIA 特异性检测感染烟草叶片中 IYSV 的工作条件。在优化条件下，基于 Luminex xMAP 的 MIA 能够特异性地以比传统酶联免疫吸附测定（ELISA）更高的灵敏度检测到 IYSV。重要的是，基于 Luminex xMAP 的 MIA 省时，整个过程可以在 2.5 小时内完成。