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Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display

Author:
Qing Yuan, Ramon Jordan, Ronald H. Brlansky, Olga Istomina, John Hartung
Source:
Journal of microbiological methods 2015 v.117 pp. 148-154
ISSN:
0167-7012
Subject:
Citrus, DNA libraries, DNA primers, Pierce's disease, Vitis, Xylella fastidiosa, antibodies, bacteriophages, clones, crops, detection, enzyme-linked immunosorbent assay, genes, grapes, phage vectors, polymerase chain reaction, strains, technology
Abstract:
Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (VH) and 21 primers for the light chain variable region (VL). The VH and VL were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×107 independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×1012 phage were used for panning with 4.1×106, 7.1×106, 2.1×107 phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine.
Agid:
61475
Handle:
10113/61475