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-exposed epitopes of Pseudomonas aeruginosa protein F, we immunized mice with cloned and expressed oprF gene as a gIII-fusion protein displayed on the M13 phage surface. The fusion protein elicited mouse antibodies reacting with the purified protein F at a limit dilution of 1:10,000. Recombinant clones expressing antibody fragments were constructed from the genes of selected B cells of hyperimmunized mouse after a first round of panning against the protein F. Expression of single chain Fv (ScFv) antibody fragments to the protein of P. aeruginosa was detected by ELISA in 20 of 384 clones obtained after the first panning selection. The 20 positive clones recognizing different protein F epitopes as demonstrated by ELISA were assayed by flow cytometry to identify antibody fragments reacting only with surface-exposed epitopes of the protein F on whole bacteria; one of the 20 clones tested showed a level of reactivity compatible with surface-exposed epitope that can lead to ulterior developments in targeting studies.
Department of Microbiology and Immunology, Université de Montréal, Québec, Canada.
P
P
Kermani
L
L
Péloquin
J
J
Lagacé
D000818:Animals; D000954:Antigens, Surface / Q000276:immunology; D017104:Bacteriophage M13 / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids; D018272:Porins / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology*
D000954:Antigens, Surface; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D018272:Porins; D011993:Recombinant Fusion Proteins