Application of a synthetic phage antibody library (ETH-2) for the isolation of single chain fragment variable (scFv) human antibodies to the pathogenic isoform of the hamster prion protein (HaPrPsc).
To overcome the limitation represented by the poor immunogenicity of prion protein (PrP) for conventional monoclonal antibodies preparation, we adopted an antibody phage display strategy to isolate specific human single chain fragment variable (scFv) directed towards the pathogenic isoform of the hamster prion protein (HaPrPsc). Phage-displaying HaPrPsc reactive scFvs were obtained after three rounds of selection of the ETH- 2 synthetic antibody library on HaPrPsc-coated immunotubes and subsequent amplification in TG1 E. coli cells. These phage-antibodies bind in ELISA to HaPrPsc and do not cross-react with the recombinant hamster prion protein (rHaPrP). Sequence analyses of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry on lymphoid cells indicate that the selected scFv recognize distinct epitopes in the PrPsc molecule. The results of this study demonstrate that display of scFvs on filamentous phage offers the possibility of producing phage antibodies showing immunoglobulin-like functions using only in vitro procedures, thus overcoming limitations of conventional hybridoma technology.
Ascione
Alessandro
A
Section of Pharmacogenetics, Drug Resistance and Experimental Therapeutics, Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Roma, Italy.
Flego
Michela
M
Zamboni
Silvia
S
De Cinti
Emanuela
E
Dupuis
Maria Luisa
ML
Cianfriglia
Maurizio
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D001435:Bacteriophages / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D018448:Models, Immunological; D008969:Molecular Sequence Data; D019151:Peptide Library; D011328:Prions / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011328:Prions; D020033:Protein Isoforms; D011994:Recombinant Proteins