A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.
This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.
Riaño-Umbarila
Lidia
L
Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca, Mexico.
Juárez-González
Victor Rivelino
VR
Olamendi-Portugal
Timoteo
T
Ortíz-León
Mauricio
M
Possani
Lourival Domingos
LD
Becerril
Baltazar
B
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology*; D000915:Antibody Affinity; D019020:Directed Molecular Evolution; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000276:immunology; D009500:Neutralization Tests; D019151:Peptide Library; D012604:Scorpion Venoms / Q000276:immunology* / Q000302:isolation & purification; D012605:Scorpions; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
D000906:Antibodies; D007128:Immunoglobulin Fragments; D009498:Neurotoxins; D019151:Peptide Library; D012604:Scorpion Venoms; C072990:toxin 2, Centruroides noxius