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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #303209
Title: Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca. Liberibacter asiaticus’

Author
item YUAN, QING - Luzhou Medical College
item Jordan, Ramon
item BRLANSKY, RONALD - University Of Florida
item MINENKOVA, OLGA - Sigma Tau Pharmaceutical
item Hartung, John

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/23/2015
Publication Date: 12/29/2015
Publication URL: http://handle.nal.usda.gov/10113/60332
Citation: Yuan, Q., Jordan, R.L., Brlansky, R.H., Minenkova, O., Hartung, J.S. 2015. Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca. Liberibacter asiaticus’. Journal of Microbiological Methods. 122:1-7.

Interpretive Summary: ‘Ca. Liberibacter asiaticus’ is a bacterium that causes citrus greening, the most serious disease of citrus worldwide. The pathogen is transmitted by insects, attacks all varieties of citrus, and is widespread in Florida, and present in both Texas and California. Identification of the pathogen is done with DNA based methods which are effective but they are expensive and time consuming. Simpler, antibody based detection methods for the pathogen are not available because there are no antibodies available to use for this purpose. With recombinant DNA technology, we have made a ‘library’ of antibodies that recognize the pathogen. Our antibody library has more than 10 million unique antibodies and the genes that encode them. We have screened this library of antibodies for antibodies that bind to specifically chosen proteins that are present on the surface of ‘Ca. Liberibacter asiaticus’, and a number of antibodies that recognize different proteins from CaLas were selected. We have demonstrated a technology to produce antibodies against any protein target encoded by ‘Ca. Liberibacter asiaticus’. Our results will be of interest to the regulatory and research communities concerned with this pathogen. Future applications will include advanced diagnostic methods for citrus greening and the development of immune labeling reagents for in planta applications.

Technical Abstract: ‘Ca. Liberibacter asiaticus’ is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of ‘Ca. Liberibacter asiaticus’. The primary library has more than 107 unique antibodies and the genes that encode them. We have screened this library of antibodies for antibodies that bind to specifically chosen proteins that are present on the surface of ‘Ca. Liberibacter asiaticus’. These proteins were used as ‘bait’ for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule expressing protein KpsF; a protein component of the type IV pilus (CapF); and two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and ‘Ca. Liberibacter asiaticus’ infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for production of scFvs. Thus far, screening of these scFvs is more efficient when using phage bound, rather than soluble scFvs. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by ‘Ca. Liberibacter asiaticus’. Future applications will include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.