title: Effective use of nitrocellulose-blotted antigens for phage display monoclonal antibody selection.
abstract: The combinatorial phage display library approach to antibody repertoire cloning offers a powerful tool for the isolation of specific antibodies to defined target antigens. Panning strategy is often a very critical point for selecting antibody displayed on the surface of bacteriophages. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well defined antigen source. However, when the antigen is difficult to purify by means of laborious and time-consuming chromatography procedures, panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental complications. In the present work, we successfully generated a mouse monoclonal antibody fragment from a phage display library directed against protein E7 of HPV18 avoiding antigen purification as for immunizing mice as for antibody library selection. Our work demonstrates the feasibility of phage antibody selections on antigens transferred to a nitrocellulose membrane as solid support, using one-dimensional polyacrylamide gel electrophoresis system as the only practice to separate a given antigen present in bacterial crude cell lysate.
authors.affiliation: Institute of Microbiology, Catholic University, Rome, Italy. Francesco.paronisterbini@edu.rm.unicatt.it
authors.forename: Francesca
authors.initials: F
authors.lastname: Bugli
authors.forename: Francesco
authors.initials: F
authors.lastname: Paroni Sterbini
authors.forename: Rosalia
authors.initials: R
authors.lastname: Graffeo
authors.forename: Flavia
authors.initials: F
authors.lastname: Caridi
authors.forename: Raffaella
authors.initials: R
authors.lastname: Iantomasi
authors.forename: Riccardo
authors.initials: R
authors.lastname: Torelli
authors.forename: Luca
authors.initials: L
authors.lastname: Masucci
authors.forename: Paola
authors.initials: P
authors.lastname: Cattani
authors.forename: Giovanni
authors.initials: G
authors.lastname: Fadda
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000941:Antigens / Q000737:chemistry*; D015153:Blotting, Western; D003101:Collodion / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D052161:Human papillomavirus 18; D006801:Humans; D051379:Mice; D019151:Peptide Library
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D019151:Peptide Library; D003101:Collodion