PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS
Molecular Plant Pathology
2009 Annual Report
1a.Objectives (from AD-416)
We will produce a population of scFv molecules with activity against Ca. Liberibacter asiaticus strains found in Florida as well as other species. We will also produce monoclonal antibodies with activity against the same organisms.
1b.Approach (from AD-416)
Liberibacter will be obtained from infected citrus psyllids (or culture if available). Extracts will be prepared (Flordia strains first) and Liberibacter quantified by Q-PCR to provide a known inoculum to mice. Mice will be sacrificed and scFv antibody library in phage will be prepared following standard methods. The phage library will be enriched for Liberibacter specific scFv by several cycles of removing phage that bind healthy psyllid extracts followed by amplification of those that do not. Monoclonal antibody expressing cell lines will be prepared using hybridoma technology in parallel with the scFv work. The antibodies against Liberibacter will then be used to develop research and diagnostic tests needed to manage the disease beginning with ELISA and dot blot tests and continuing with tests in the 'dip stick' format.
Funds were received in April of 2009. Since that time citrus and other host plants of Candidatus Liberibacter asiaticus were established, propagated and infected with Liberibacter asiaticus at Beltsville for use in this project. Plants were confirmed to be positive for the pathogen by real time PCR assays. Colonies of the insect vector of the pathogen were also established and maintained at Lake Alfred, Florida and Ft Detrick, Maryland. Test shipments of insects were sent to Beltsville to confirm shipping procedures and to perform initial extracts for testing. Many administrative tasks were completed, including transfer of funds to subcontractors at the University of Florida, USDA ARS, Ft Detrick, MD and Agdia, Elkhardt, IN. Material transfer agreements were negotiated with Sigma-tau Pharmaceutical Company, Rome, Italy to acquire the specialized vector for phage display technologies and with Agdia, Elkhardt, IN for exchange of materials. Visiting scientists were recruited internationally, identified in China and Italy and arrangements were made for their study visits in Beltsville, to begin August 10 and October 20, 2009. Funding arrangements were made with the USDA Foreign Agriculture Service to support these visitors on this project’s funds.