tailieunhanh - Báo cáo khoa học: 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 (·10 8 recombinants). The lib-rary 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 noxiusscorpion venom. | ềFEBS Journal 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 Lidia Riano-Umbarila Victor Rivelino Juarez-Gonzalez Timoteo Olamendi-Portugal Mauricio Ortiz-Leon Lourival Domingos Possani and Baltazar Becerril Department of Molecular Medicine and Bioprocesses Institute of Biotechnology NationalAutonomous University of Mexico Cuernavaca Mexico Keywords affinity maturation directed evolution human scFv library phage display scorpion toxin Correspondence B. Becerril Av. Universidad No. 2001 Colonia Chamilpa Cuernavaca 62210 Mexico Tel 52 7773 291669 E-mail baltazar@ Note The sequences reported have been deposited in the GenBank database under accession nos. AY781338 AY781339 AY781340 AY781341 and AY781342 corresponding to scFvs 3F 6F 610 A 6009F and C1. Received 9 March 2005 revised 21 March 2005 accepted 28 March 2005 doi 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 X 108 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 LD50 of Cn2 toxin when a 1 10 molar ratio of toxin-to-anti-body fragment was used. It was also able to neutralize 2 LD50 of the whole venom. These results pave the way for the future generation of recombinant human antivenoms. In recent years