Development of monoclonal antibodies and serological assays specific for Barley yellow dwarf virus GAV strain.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China. chaoyueziwo77@126.com.
Na
N
Li
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China. chenzhelily@163.com.
Zhe
Z
Chen
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. yliu@ippcaas.cn.
Yan
Y
Liu
Hunan Plant Protection Institute, Changsha, 410125, China. haoasliu@163.com.
Yong
Y
Liu
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China. zzhou@zju.edu.cn.
Xueping
X
Zhou
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China. wujx@zju.edu.cn.
Jianxiang
J
Wu
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D001042:Aphids / Q000821:virology*; D002681:China; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007303:Insect Vectors / Q000821:virology; D017793:Luteovirus / Q000302:isolation & purification*; D008807:Mice, Inbred BALB C; D010935:Plant Diseases / Q000821:virology*
D000911:Antibodies, Monoclonal