PRODUCTION OF SCFV ANTIBODY FRAGMENTS FOLLOWING IMMUNIZATION WITH A PHAGE-DISPLAYED FUSION PROTEIN AND ANALYSIS OF REACTIVITY TO SURFACE-EXPOSED EPITOPES OF THE PROTEIN-F OF PSEUDOMONAS-AERUGINOSA BY CYTOFLUOROMETRY

To increase the possibilities of obtaining antibodies to surface-expos ed epitopes of Pseudomonas aeruginosa protein F, we immunized mice wit h cloned and expressed oprF gene as a gIII-fusion protein displayed on the M13 phage surface, The fusion protein elicited mouse antibodies r eacting with the purified protein F at a limit dilution of 1:10,000, R ecombinant clones expressing antibody fragments were constructed from the genes of selected B cells of hyperimmunized mouse after a first ro und of panning against the protein F, Expression of single chain Fv (S cFv) antibody fragments to the protein of P, aeruginosa was detected b y ELISA in 20 of 384 clones obtained after the first panning selection , The 20 positive clones recognizing different protein F epitopes as d emonstrated by ELISA were assayed by flow cytometry to identify antibo dy fragments reacting only with surface-exposed epitopes of the protei n F on whole bacteria; one of the 20 clones tested showed a level of r eactivity compatible with surface-exposed epitope that can lead to ult erior developments in targeting studies.