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

@entity72132 @entity13974 @entity25025
UNASSIGNED: @entity25025 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 @entity25025 , and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against @entity25025 . pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain @entity13974 (VH) and 21 primers for the light chain @entity13974 ( @entity13974 ). The VH and @entity13974 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*10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0*10(12) phage were used for panning with 4.1*10(6), 7.1*10(6), 2.1*10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound @entity72132 in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize @entity25025 strains that cause Pierce's disease of grapevine.
