Selection of human antibody fragments on the basis of stabilization of the variable domain in the presence of target antigens.
Here we report a novel method for selecting human antibody fragments from nonimmunized variable domain libraries. The antibody fragments are selected on the basis of stabilization of the variable domain fragment (F(v)) in the presence of target antigens ("open sandwich selection"). One variable domain is displayed on phages and another is prepared as soluble molecules. These two reagents are mixed with the biotinylated target molecule and ternary complexes are captured by using streptavidin-conjugated magnet beads. After extensive washing, enriched clones are eluted by using target antigen. Some of the clones selected after 3 rounds are prepared as soluble domains, which then undergo another selection process. We obtained several human antibody fragments specific for human soluble erythropoietin receptor by using this method. Our method minimizes several of the disadvantages associated with human antibody selection through a phage-display system, such as construction of a large-scale library, deletion of genes during selection, and nonspecific binding.
Watanabe
Hideki
H
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Aoba-ku, Sendai 980-8579, Japan.
Tsumoto
Kouhei
K
Asano
Ryutaro
R
Nishimiya
Yoshiyuki
Y
Kumagai
Izumi
I
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000941:Antigens / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D007700:Kinetics; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017467:Receptors, Erythropoietin / Q000276:immunology
D000941:Antigens; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D017467:Receptors, Erythropoietin