title: A novel approach to human anti-HLA mABs production: use of phage display libraries.
abstract: The production of human monoclonal antibodies was previously limited to very laborious and time-consuming processes involving EBV-transformation and/or hybridoma generation. Due to the development of molecular cloning techniques, it is now possible to produce human monoclonal antibody fragments quickly by panning phage display libraries against predefined antigenic specificities. Therefore, we tested this technology for producing human single chain Fv fragments (scFvs) against HLA-DR1 purified molecules immobilized on solid phase. Enrichment of DR1-specific phages was measured through five selection rounds of a synthetic library and revealed a 100-fold amplification. Soluble antibody fragments were then expressed and 7 out of 48 clones were found to secrete scFvs which specifically bind to DR1 molecules in ELISA. Further analysis revealed binding of the scFvs also to DR3 but not to DR5 or DR7 molecules correlating with the presence of particular polymorphic aminoacid residues in the DR beta chain. Western blot analysis indicated that the 7 scFvs react with the DR1 alpha/beta-dimer but not with free alpha- or beta- chains. This study shows that the innovative approach of phage display libraries can efficiently provide scFv fragments as useful reagents for the identification and dissection of HLA polymorphic epitopes.
authors.affiliation: Servizio di Immunogenetica, Istituto Nazionale per la Ricerca sul Cancro c/o Centro di Biotecnologie Avanzate, Genova, Italy.
authors.forename: M P
authors.initials: MP
authors.lastname: Pistillo
authors.forename: J
authors.initials: J
authors.lastname: Hammer
authors.forename: E
authors.initials: E
authors.lastname: Bono
authors.forename: F
authors.initials: F
authors.lastname: Sinigaglia
authors.forename: F
authors.initials: F
authors.lastname: Bottero
authors.forename: A
authors.initials: A
authors.lastname: Gho
authors.forename: G B
authors.initials: GB
authors.lastname: Ferrara
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics*; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D015798:HLA-DR1 Antigen / Q000276:immunology*; D015803:HLA-DR3 Antigen / Q000276:immunology; D015805:HLA-DR5 Antigen / Q000276:immunology; D015809:HLA-DR7 Antigen / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D015798:HLA-DR1 Antigen; D015803:HLA-DR3 Antigen; D015805:HLA-DR5 Antigen; D015809:HLA-DR7 Antigen; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011994:Recombinant Proteins