Li Na, Chen Zhe, Liu Yan, Liu Yong, Zhou Xueping, Wu Jianxiang
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Virol J. 2015 Sep 4;12:136. doi: 10.1186/s12985-015-0367-4.
Barley yellow dwarf virus (BYDV) is one of the most devastating plant viruses and belongs to a ubiquitous plant virus group. In China, four BYDV strains (GPV, GAV, PAV and RMV) have been identified based on their specific aphid vectors and serological properties. Among the four identified strains, the GAV is the most common BYDV strain in China. To diagnose, forecast of BYDV GAV, two reliable serological assays for BYDV GAV detection were established.
We purified virion from a confirmed BYDV GAV source and used it as the immunogen to produce monoclonal antibodies against the virus. Using the hybridoma technology, three highly specific murine monoclonal antibodies were produced and two serological assays [antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot enzyme-linked immunosorbent assay (dot-ELISA)] were established for the BYDV GAV detection.
All three monoclonal antibodies reacted strongly and specifically with the BYDV GAV strain in crude leaf extracts. Titers of the monoclonal antibodies in ascitic fluids were up to 10(-7) by indirect-ELISA. These three monoclonal antibodies (18A1, 18A9 and 12A11) all belonged to the isotype IgG1, kappa light chain. The highest dilution points for the three antibodies during the ACP-ELISA using infected crude leaf extracts were 1:163,840, 1:81,920 and 1:81,920 (w/v, g · mL(-1)), respectively. Result of dot-ELISA showed a successful detection of BYDV GAV strain in 1:5,120 (w/v, g · mL(-1)) diluted wheat leaf crude extracts. Analysis of 22 field wheat leaf samples and 33 aphid samples from the Shaanxi Province in China, using the two newly developed assays confirmed the presence of BYDV GAV in about 80 % of the wheat samples and 18 % of the aphid samples.
All three monoclonal antibodies are highly sensitive and specific to the BYDV GAV. The two newly developed serological assays are simple and effective. These two assays, particularly the dot-ELISA, are useful for high throughput detection of BYDV GAV in host plants and aphid vectors.
大麦黄矮病毒（BYDV）是最具破坏性的植物病毒之一，属于一种广泛存在的植物病毒类群。在中国，已根据其特定的蚜虫传播介体和血清学特性鉴定出四种BYDV株系（GPV、GAV、PAV和RMV）。在已鉴定的四种株系中，GAV是中国最常见的BYDV株系。为了诊断和预测BYDV GAV，建立了两种可靠的用于检测BYDV GAV的血清学检测方法。
我们从确诊的BYDV GAV来源中纯化病毒粒子，并将其用作免疫原以生产针对该病毒的单克隆抗体。利用杂交瘤技术，制备了三种高度特异性的鼠单克隆抗体，并建立了两种用于检测BYDV GAV的血清学检测方法[抗原包被板酶联免疫吸附测定（ACP-ELISA）和斑点酶联免疫吸附测定（dot-ELISA）]。
所有三种单克隆抗体均与粗叶提取物中的BYDV GAV株系发生强烈且特异性的反应。通过间接ELISA法，腹水液中单克隆抗体的效价高达10(-7)。这三种单克隆抗体（18A1、18A9和12A11）均属于IgG1同种型、κ轻链。在使用感染粗叶提取物进行ACP-ELISA检测时，这三种抗体的最高稀释倍数分别为1:163,840、1:81,920和1:81,920（w/v，g·mL(-1)）。斑点ELISA结果显示，在1:5,120（w/v，g·mL(-1)）稀释的小麦叶粗提取物中成功检测到BYDV GAV株系。利用这两种新建立的检测方法对来自中国陕西省的22份田间小麦叶样本和33份蚜虫样本进行分析，证实约80%的小麦样本和18%的蚜虫样本中存在BYDV GAV。
所有三种单克隆抗体对BYDV GAV均具有高度敏感性和特异性。新建立的两种血清学检测方法简单有效。这两种检测方法，尤其是斑点ELISA，对于在寄主植物和蚜虫传播介体中高通量检测BYDV GAV很有用。
