, 2005). However, the combined venoms were more efficient to be recognized by serum with high neutralizing potency. We assumed that the complexity of the antigen used in the ELISA is not favorable for establishing a correlation between the antigenic reactivity and the neutralizing properties, probably due to the existence of a limited spectrum of neutralizing antibodies. Therefore, we evaluated simpler antigens, in the form of peptides, which could mimic epitopes including the neutralizing ones. The epitope-mapping Spot technique was used (Maria et al., 2005, Alvarenga et al., 2010b and Machado de Ávila et al., 2004), thereby allowing a systematic search for continuous epitopes. These regions,

besides being antigenic, may also correspond to neutralizing epitopes because they are related either to the catalytic site or to the mechanism of action of the toxins (Murakami find more et al., 2005, Alvarenga et al., 2010a and Alvarenga et al., 2010b; Felicori et al., 2009; de Moura et al., 2011). Taking into consideration the recognition of the beta-catenin inhibitor three different dermonecrotic proteins by horse antivenoms with high neutralizing potency,

nine reactive peptides were selected (i.e., three from each protein). Some reactive peptide regions of LiD1 had been previously identified (Felicori et al., 2006 and Felicori et al., 2009), confirming the immunogenicity of some regions. However, for the first time such mapping was produced with toxins from three different Loxosceles species. Among the mapped antigenic

regions, an analysis of the recognition frequency by the different sera was done. When the serum was tested at low dilution, the recognition frequency of some epitopes was the same in sera with high or low neutralizing potency. When the serum dilution was increased, the low neutralizing potency sera were not able to recognize Rebamipide the peptides, whereas the high neutralizing potency sera were able to recognize the peptides, suggesting that the test conditions may influence the discrimination between the different sera. Some sequences appeared to be best candidates for such differentiation (e.g., peptides 2 and 3). Peptide 3 (164DFSGPYLPSLPTLDA178) from SMase-D I was not recognized by any low neutralizing potency serum. This region has been reported to be a highly conserved region in SMase-D from L. laeta ( Murakami et al., 2005). Peptide 3 corresponds to a variable loop, which is five residues shorter than sequences from other species. As reported by de Giuseppe et al. (2011), this loop exposes the active site. Therefore, peptide 3 seems to be an important region for the identification of high neutralizing potency sera. Peptide 2 (22EFVNLGANSIETDVS36), which is present in SMase-D from L. intermedia and L. gaucho venoms, corresponds to a conserved region suggesting a structural and functional homology between the toxins.