Efficient construction of a highly useful phage-displayed human antibody repertoire

• Published in: Biochemical and biophysical research communications Vol. 336; no. 4; pp. 1207 - 1213
• Main Authors: Rojas, Gertrudis, Lamdan, Humberto, Padron, Sheila, Munoz, Yasmiana, Ayala, Marta, Gavilondo, Jorge V.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.11.2005
• Subjects: Amino Acid Sequence; Antibody library; Antigens - metabolism; Humans; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - metabolism; Immunoglobulin Variable Region - genetics; Lymphocytes - metabolism; Molecular Sequence Data; Peptide Library; Phage display; Protein Binding; Repertoire size; Surface Plasmon Resonance; Antibody library; Phage display; Repertoire size
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2005.09.005

We have constructed a highly useful phage-displayed human antibody repertoire with limited cloning efforts. Our strategy was to maximize diversity during the first steps of library construction through the use of various lymphoid sources from several donors, inclusion of different immunoglobulin isotypes, and performance of multiple separate amplification reactions with all possible combinations within a complex primer set. The resulting variable region collections were cloned to form a moderate size library, composed by 4.25 × 10 8 single chain antibody fragments. This repertoire was successfully used to retrieve binders to seven model antigens: six proteins and one 12 aa peptide. Binding affinities reached nanomolar and even subnanomolar range. Sequence diversity and V-gene usage variability among binders were proven. Our approach was not focused on absolute library size, but on a high quality sampling of variable regions from the human antibody repertoire.