Production Of Monoclonal Antibodies Against Barley Yellow Dwarf Virus GAV And Wheat Yellow Mosaic Virus And Their Application

Wheat is the second food crop after rice in China, whose production safety related to people’s livelihood closely.In recent years, virus diseases on wheat are serious in China. Especially, wheat yellow dwarf disease caused by Barley yellow dwarf virus (BYDV) and wheat yellow mosaic disease caused by Wheat yellow mosaic virus (WYMV) cause severe yield and huge economic losses. At present, BYDV GAV has become a mainstream strain in China. In order to understand the occurrences and epidemic regularities of two wheat virus diseases and establish early detection and warning systems, rapid and efficient detection methods should be urgently developed. Monoclonal antibody-based serological methods for plant virus detection are simple, rapid, sensitive, specific and suitable to detect large-scale field samples. So it has been widely used in plant virus disease diagnosis, epidemic rule analyses, prediction and early warning, scientific prevention and control and resistive breeding. In this study, Monoclonal antibodies (MAbs) against BYDV GAV and WYMV were respectively produced, and two serological methods were developed for rapid and reliable virus detection.(1) MAbs against BYDV GAV and its application:The purified BYDV GAV virions were used as an immunogen to produce MAbs. Via cell fusion, cell culture, antibody detection and cell cloning, eight sensitive and specific murine MAbs against BYDV GAV (18A1,18A9,12A11,7H11.1G7,1F1,27E1and3F11) were produced. The titers of ascitic fluids of MAbs were up to10’6by indirect-ELISA. All MAbs were isotyped as IgG1, kappa light chain. Two serological methods of antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot enzyme-linked immunosorbent assay (dot-ELISA) were established for detecting BYDV GAV in field wheat and aphid samples. Specificities of the ACP-ELISA and dot-ELISA were confirmed by a positive reaction of detection with BYDV GAV-infected wheat plant tissues and negative reactions of detection with wheat plant infected by other detected viruses and healthy wheat plant. Dot-ELISA could specifically detect BYDV GAV in viruliferous aphids, and have a negative reaction of detection with aphids containing other detected viruses or non-viruliferous aphids. The ACP-ELISA and dot-ELISA could detect BYDV GAV in infected wheat tissue crude extracts diluted at1:81,920and1:5,120(w/v, g/mL), respectively.106wheat field samples from Hancheng in Shaanxi Province were screened for the presence of BYDV GAV using the two developed serological methods. The detection result showed that64of the106wheat field samples were infected by BYDV GAV, indicating further that BYDV GAV is prevalent in Hancheng.280aphid samples were also screened for the presence of BYDV GAV using the dot-ELISA. The result indicated that24of the280aphid samples were infected by BYDV GAV.(2) MAbs against WYMV and its application:Using the purified WYMV particles as the immunogen, three MAbs against WYMV (4E11,21D4and25B1) were produced with hybridoma technique. Titers of ascitic fluids of all three MAbs were up to10’6by indirect-ELISA. All MAbs were isotyped as IgG1, kappa light chain. Western blot analysis indicated that MAbs4E11and21D4could specifically react with the approximately32kDa coat protein of WYMV. Specificities of the ACP-ELISA and dot-ELISA were confirmed by a positive reaction of detection with WYMV-infected wheat plant tissues and negative reactions of detection with wheat plant infected by other detected viruses and healthy wheat plant. The ACP-ELISA and dot-ELISA could detect WYMV in infected wheat tissue crude extracts diluted at1:40,960and1:2,560(w/v, g/mL). respectively.90wheat field samples from Jining in Shandong Province were screened for the presence of WYMV using the two developed serological methods. The detection result demonstrated that39of the90wheat field samples were infected by WYMV, indicating further that WYMV is prevalent in Hancheng.