Mecanismos moleculares da neutralização da atividade letal das toxinas escorpiônicas por anticorpos monoclonais e policlonais

Abstract

The major goal of this thesis is the immunochemical study of the scorpion toxins. Four manuscripts were presented concerning this subject. In the first report, a detoxified immunogen was prepared by conjugation of a toxic fraction (TstFG50), of the T. serrulatus venom, with bovine serum alburnin (BSA). The immunogen was inject in mice for inducing antibodies. In vivo protection assays showed that immunized mices resist to the challenge with a dose of twice LD50 of the TstFG50. We used the Spot method to characterize epitopes of protective antibodies. It is likely that these epitopes correspond to the neutralizing epitopes. Usually, they correspond to regions of the toxins that are known to be involved in the toxin active sites.In the second manuscript, monoclonal antibodies (mAbs) against Tityus serrulatus venom were produced and characterized. The capacity of mAbs to neutralize the TstFG50 was determined by in vitro neutralization assays. Only mAbTsl neutralized 50% of the toxic effects produced by scorpion venom and showed 35% inhibition of the binding of 125I-TsVII. To map the epitope recognized by the protective mAbTsl, the Spot method was used. The neutralizing properties of mAbTs1 might be explained by spatial vicinity of epitope residues with pharmacophore residues.In the following paper, the Spot method was used to characterize the binding of the peptides of the Tsll, TslV and TsVIl with horse anti-Ts antisera for therapeutic use. Ali antisera tested showed reactivity with several peptides from ali three toxins. The reactive peptides were synthesized, coupled to KLH and used as antigens to coat the microtitration by ELISA. The mixture of the N-terminal peptides of Tsll, and TsVIl and of the C-terminal of TslV were found to give a linear relationship with the neutralizing triter of horse's serum of low neutralizing potency. However, high neutralizing antivenoms did not show the expected response in peptide ELISA. This observation is discussed in the context of the occurrence of continuous and discontinuous epitopes at the toxins.Finally, in the last paper we conducted an immunochemical study of the anatoxin Amm VIII. This protein was previously isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus. Despite 87% identity with AaH II, potent a -toxin, Amm VIII is not toxic to mice. However, antisera against this protein protect mice from AaH II lethal action. Here, we report that the Amm VIII protein elicits only antibodies that recognize discontinuous-type epitopes. Anti-Amm VIII antibodies were also found to cross-react towards several of the discontinuous-continuous peptides designed from the AaH II structure, pointing to a possible involvement of the corresponding discontinuous epitopes in the in vitro AaH II neutralizing capacity displayed by anti-Amm VIII antibodies. Altogether, our results show that it is possible to design antibody-reactive peptides from discontinuous parts of scorpion toxins. The position of the reactive segments in the structural context of scorpion toxins highlights the antigenic properties of the Amm VIII anatoxin and reasonably explains the capacity of anti-Amm VIII antibodies to neutralize the potent a scorpion toxin AaH II.