The cost of worldwide crop losses due to plant diseases is estimated at $60 billion annually. The increase in international commerce could accelerate the spread of foreign diseases from one region to another. Early and accurate detection of pant viruses in infested seeds and plants is crucial to virus prevention, containment and eradication. Our mid-term objective is to develop these antibodies to evaluate their usefulness for the diagnosis of plant diseases. Our long-term goal is to explore broad applications of these nanobodies not only for disease detection and prevention but also for pest neutralization and eradication after they have infected the host. It is also within our objective to develop a core competency in this area to make nanobodies commercially available to the research community and industry at large because only through community-wide studies and the acceptance by key stakeholders could the full benefits of this exciting technology be harnessed and realized. (Nanobody is a registered trademark of Ablynx NV, Belgium.) OBJECTIVES: Specific Aim #1. Select a series of single domain antibodies to grapevine leafroll-associated virus type 3 (GLRaV-3) from immunized alpaca and express them in E. coli. Specific Aim #2. Compare them side-by-side with existing polyclonal and monoclonal antibodies in ELISA format to answer the critical questions of assay sensitivity and specificity under varying environmental conditions such as temperature, humidity and shelf-life. APPROACH: 1. Purification of GLRaV-3 Virions. Grapevine materials with high titer of GLRaV-3 are grinded in extraction buffer. GLRaV-3 virions will be purified by using antibody-coated tubes. Virus concentration can be determined by spectrophotometry; 2. Alpaca Immunization and Construction of the Phage Display HHV Library according to published methods; 3. Affinity Selection and Amplification of the Phage Displaying VHH according to published methods; 4. Characterization of Selected Phage Clones Specific for GLRaV-3 by Enzyme-linked Immunosorbent Assay (ELISA); 5. Soluble VHH Production by Standard Recombinant methodology; 6. Characterization of cross-reactivity of VHH by Direct ELISA
