Affinity study of scFv human antibodies against crotoxin using mutants evoluted in silico, constructed and expressed in bacteria

• Grant number: 11/03801-9
• Support Opportunities: Scholarships in Brazil - Master
• Start date: August 01, 2011
• End date: July 31, 2013
• Field of knowledge: Biological Sciences - Immunology - Immunochemistry
• Principal Investigator: Irene Fernandes
• Grantee: Tamires Souza de Oliveira
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Our goal in this project is to study the affinity of mutant human antibody scFv anti-crotoxin, seeking to understand the mechanisms of action of crotoxin and a possible alternative treatment for poisoning by the bite of snakes of the genus Crotalus. Crotalus poisoning, although accounting for only 8% of cases, it has the highest mortality. The venom has several toxins, crotoxin (CTX) primarily responsible for its neurotoxicity. The injuries caused by snake venoms have been included in the list of Neglected Tropical Diseases of WHO. Three clones human anti-crotoxin scFv were selected and sequenced from a phage library by Cardoso et al, 2000. Clone 6, which showed the highest neutralizing capacity on the lethality induced by CTX and whole venom was expressed and partially characterized during the Scientific Initiation project's proponent (with a grant FAPESP). A mutant has also been obtained by site directed mutagenesis and is being expressed. In this project, we intend to study the affinity of scFv mutants. For this, two other mutants generated by in silico studies will be constructed and expressed in E. Coli. The following are purified and tested for their affinity (by surface plasmon resonance - Biacore). (AU)