Retrieval of phage displayed scFv fragments using direct bacterial elution.
Phage displayed repertoires of antibody fragments, either single chain Fv (scFv) or Fab, have become a real alternative to traditional hybridoma technology in the generation of monoclonal antibodies. The steps usually taken in the selection from such repertoires were analysed and the necessity of chemical elution of bound phage-Abs and precipitation of amplified phage particles questioned. By using a semi-synthetic scFv library as a source, phage antibodies recognising a panel of seven antigens were isolated utilising direct bacterial elution of bound phage. Selections against two antigens were subsequently performed with bacterial or chemical elution in parallel and the resulting pools of phage antibodies compared. It is demonstrated that direct bacterial elution can be used when selecting from phage displayed antibody repertoires but that the enrichment of high affinity binders might be jeopardised. In addition, a simplified and more rapid scheme for amplification and use of phage displayed repertoires is described.
Wind
T
T
Department of Biostructural Chemistry, Institute of Molecular and Structural Biology, Aarhus University, Denmark. wind@biobase.dk
Stausbøl-Grøn
B
B
Kjaer
S
S
Kahns
L
L
Jensen
K H
KH
Clark
B F
BF
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry* / Q000821:virology; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region