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ARS Home » Plains Area » Lincoln, Nebraska » Wheat, Sorghum and Forage Research » Research » Publications at this Location » Publication #287181
Title: Immunodetection of Triticum mosaic virus by DAS- and DAC-ELISA using antibodies produced against coat protein expressed in Escherichia coli: potential for high-throughput diagnostic methods

Author
item Tatineni, Satyanarayana - Ts
item Sarath, Gautam
item SEIFERS, DALLAS - Kansas State University
item French, Roy

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2013
Publication Date: 3/21/2013
Citation: Tatineni, S., Sarath, G., Seifers, D., French, R.C. 2013. Immunodetection of Triticum mosaic virus by DAS- and DAC-ELISA using antibodies produced against coat protein expressed in Escherichia coli: potential for high-throughput diagnostic methods. Journal of Virological Methods. 189: 196-203.

Interpretive Summary: Triticum mosaic virus (TriMV) is an emergent virus infecting wheat in the Great Plains region where most wheat is grown. Currently, there are no sensitive and reliable diagnostic methods available for TriMV. Efficient diagnostic methods are prerequisite for the management of TriMV and for large-scale screening of germplasm for virus resistance in wheat breeding programs. In this study, we produced polyclonal antibodies against bacterially expressed TriMV coat protein in rabbits. These antibodies sensitively detected TriMV in crude sap, but not with extracts from healthy plants and plants infected with Wheat streak mosaic virus. Additionally, these antibodies detected field collected TriMV isolates from different regions of the Great Plains, suggesting that these antibodies can be used for broad-spectrum detection of TriMV isolates. The availability of highly-specific and sensitive antibodies would provide a much-needed method for high-throughput detection for the management of TriMV and for the development of TriMV-resistant wheat cultivars. These antibodies can also be used to develop TriMV diagnostic kits by biotechnology companies.

Technical Abstract: Triticum mosaic virus (TriMV), a recently introduced economically important virus infecting wheat in the Great Plains region of the USA, is the type species of the Poacevirus genus in the family Potyviridae. TriMV elicits mosaic, mottling, and chlorotic streaks on wheat, which are indistinguishable from those of Wheat streak mosaic virus (WSMV) warranting effective diagnostic methods. Sensitive and high-throughput detection methods are crucial for the management of TriMV and for germplasm screening in wheat breeding programs. In this study, TriMV coat protein (CP) was overexpressed in Escherichia coli, and polyclonal antibodies were generated against purified soluble native form of bacterially expressed recombinant CP (rCP) in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western immuno-blot and enzyme-linked immunosorbent assays (ELISA). In direct antigen coating (DAC)-ELISA, antibodies reacted specifically, beyond 1:20,000 dilution, with TriMV in crude sap, but not with WSMV or healthy extracts, and antiserum at 1:10,000 dilution detected TriMV in crude sap up to 1:4,860 dilution. Furthermore, we demonstrated that rabbit anti-TriMV IgG and anti-TriMV IgG-ALP conjugate reacted positively with native virions in crude sap in a double antibody sandwich (DAS)-ELISA. Finally, the recombinant antibodies reacted positively with representative field collected TriMV isolates from different states of the Great Plains region by DAC- and DAS-ELISA, suggesting that antibodies generated against rCP can be used for sensitive, large-scale, and broad-spectrum detection of TriMV.