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

Abstract

Triticum mosaic virus (TriMV), an 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. Sensitive and high-throughput serology-based detection methods are crucial for the management of TriMV and germplasm screening in wheat breeding programs. In this study, TriMV coat protein (CP) was expressed in Escherichia coli, and polyclonal antibodies were generated against purified soluble native form 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 healthy extracts, and antiserum at a 1:10,000 dilution detected TriMV in crude sap up to 1:4860 dilution. Notably, rabbit anti-TriMV IgG and anti-TriMV IgG-alkaline phosphatase conjugate reacted positively with native virions in crude sap in a double antibody sandwich-ELISA, suggesting that these antibodies can be used as coating antibodies which is crucial for any ‘sandwich’ type of assays. Finally, the recombinant antibodies reacted positively in ELISA with representative TriMV isolates collected from fields, suggesting that antibodies generated against rCP can be used for sensitive, large-scale, and broad-spectrum detection of TriMV.