2013 Annual Report
1a.Objectives (from AD-416):
To produce antibodies against bacterially expressed capsid proteins of four viruses from the leafroll complex to be able to develop triple-antibody sandwich (TAS) ELISA assays.
1b.Approach (from AD-416):
Polyclonal antibodies produced will be tested using protocols established at University of Idaho, Moscow, and USDA-ARS, Corvallis, against the reference isolates of GLRaV-1, -2, -4, and -5 maintained in grapevine hosts at Corvallis, OR. TAS-ELISA test protocols developed will be validated on field samples collected in Nez Perce and Latah counties.
This research was conducted in support of objective NP303 objective 2C of the parent project. More than a dozen viruses challenge production of wine and table grapes in the Pacific Northwest (PNW), including those from the so-called grapevine leafroll disease (GLD) complex. Main strategy to combat and control viruses from the GLD complex relies on certification of nursery material for planting, and maintaining acceptably low levels of these viruses. This strategy necessitates large-scale indexing and testing of mother plants used for propagation in the nurseries, and periodic tests of the plants in vineyards. Historically, RT-PCR in many different formats had been widely used in grapevine virus detection and differentiation, despite intrinsic problems due to natural sequence variation and relatively low scale-up potential. ELISA, a good sensitive method which is widely and successfully used in large-scale detection of many plant viruses, had not been used in grapevine virology to the same extent. The main limitation to the use of ELISA tests for grapevine viruses has been lack or scarcity of the key reagents for the test – specific, high-titer antibodies. Here, virus-specific antigens for the two grapevine viruses, GLRaV-1 and GLRaV-4 were produced in bacteria, affinity purified, and used for antibody production in laboratory animals. These antibodies were confirmed to be specific to GLRaV-1 and -4, and two immunodetection assays were developed utilizing the most advanced, triple-antibody sandwich (TAS) format for the ELISA tests. These TAS-ELISA based assays were highly specific and sensitive, utilizing working dilutions of the antisera of 1:10,000 both for coating and detecting antibodies when testing leaf tissue from infected grapevines. We are continuing to work on bacterial expression of the two other antigens, for GLRaV-2 and -5, and on development of the TAS-ELISA based assays in grapevine leaf tissue.