Efficient construction of a highly useful phage-displayed human antibody repertoire.
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 4.25x10(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.
Rojas
Gertrudis
G
Recombinant Antibodies Laboratory, Pharmaceuticals Division, Center for Genetic Engineering and Biotechnology, Playa, La Habana, Cuba. gertrudis.rojas@cigb.edu.cu
Lamdan
Humberto
H
Padron
Sheila
S
Munoz
Yasmiana
Y
Ayala
Marta
M
Gavilondo
Jorge V
JV
D000595:Amino Acid Sequence; D000941:Antigens / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008214:Lymphocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D020349:Surface Plasmon Resonance
D000941:Antigens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library