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.
Plants infected with the HLB pathogen were established and propagated in Florida and Maryland. Some of these plants were used to raise large numbers of psyllids (insects) to be used to immunize mice. Others were grown and used as plant extracts to screen antibody libraries. Visiting scientists were recruited from Sigma-tau Pharmaceuticals SA (Rome, Italy) and from Luzhou Medical College, (Luzhou, PRC). A large number of psyllids from Ft. Detrick and Lake Alfred have been screened for the presence of the HLB pathogen, and the 3% of the insects with the highest titer of bacteria were used to inject special mice. This required the development of a rapid and efficient method to make insect extracts which could be assayed by q-PCR without DNA extraction so that intact bacteria were available for injection into the mice. Each injection contained extracts of 1-2 insects at 200-500 million pathogens per insect. Two sets of mice were immunized. One set was taken to Agdia after a series of four immunizations and used to create standard monoclonal antibodies using hybridoma technology. The second set of mice received an additional immunization and was used to create an antibody library in a special vector, pKM19. Promising antibody expressing cell lines have been identified at Agdia. These antibodies appear to react with the outer membrane protein of the pathogen. These antibodies are being purified and characterized further. While screening these hybridomas we observed cross reactions to phloem extracts from healthy sweet orange fruit. This was not expected since the mice were immunized with insect and not plant extracts. The nature of the cross reacting antigen is presently unknown. An antibody library with activity against the HLB pathogen has also been prepared at Beltsville. The library was screened by ‘biopanning’ against extracts of rough lemon plants. This was not successful due to low concentration of the pathogen in these plants. We therefore are screening against insect extracts with known high titer, and are also using methods to concentrate the target bacteria in these extracts. We expect to have antibodies with specificity against the HLB pathogen in the near future.
Some other results should be noted. In the process of assaying the psyllids for the concentration of the HLB pathogen, we obtained a dataset of it’s concentration in 686 psyllids. In separate research this dataset may lead to insights into relations among other commensal bacteria. Also, at the beginning of the project, while plant materials were graft inoculated and insects were multiplying on HLB plants, we immunized mice with Xylella fastidiosa strain 9a5c (citrus variegated chlorosis) mixed with psyllid extracts. We did this to become familiar with process of creating the antibody libraries and screening them for desired antibodies. Interestingly, we have identified an antibody which reacts strongly to strain Xylella fastidiosa 9a5c but which do not react at all to strains of Xylella fastidiosa associated with Pierce’s disease of grapevine. These scFv fragments have interesting potential for diagnostics.