RESUMEN

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.

Asunto(s)

Anticuerpos/genética , Anticuerpos/inmunología , Evolución Molecular Dirigida , Fragmentos de Inmunoglobulinas/inmunología , Biblioteca de Péptidos , Venenos de Escorpión/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Humanos , Fragmentos de Inmunoglobulinas/genética , Datos de Secuencia Molecular , Neurotoxinas/inmunología , Pruebas de Neutralización , Venenos de Escorpión/aislamiento & purificación , Escorpiones , Alineación de Secuencia , Resonancia por Plasmón de Superficie