Mapping of an epitope recognized by a neutralizing monoclonal antibody specific to toxin Cn2 from the scorpion Centruroides noxius, using discontinuous synthetic peptides

The Na+-channel-affecting toxin Cn2 represents the major and one of the mos t toxic components of the venom of the Mexican scorpion Centruroides noxius Hoffmann. A monoclonal antibody BCF2 raised against Cn2 has been shown pre viously to be able to neutralize the toxic effect of Cn2 and of the whole v enom of C. noxius. In the present study the epitope was mapped to a surface region comprising the N- and C-terminal segments of Cn2, using continuous and discontinuous synthetic peptides, designed on the basis of the sequence and a three-dimensional model of Cn2. The study of peptides of varying len gth resulted in the identification of segments 5-14 and 56-65 containing re sidues essential for recognition by BCF2. The peptide (abbreviated SP7) wit h the highest affinity to BCF2 (IC50 = 5.1 mu M) was a synthetic heterodime r comprising the amino acid sequence from position 3-15 (amidated) of Cn2, bridged by disulfide to peptide from position 54-66, acetylated and amidate d. Similar affinity was found with peptide SP1 [heterodimer comprising resi dues 1-14 (amidated) of Cn2, bridged with synthetic peptide 52-66 (acetylat ed)]. SP1 and SP7 were used to induce anti-peptide antibodies in mouse and rabbit. Both peptides were highly immunogenic. The sera obtained were able to recognize Cn2 and to neutralize Cn2 in vitro. The most efficient protect ion (8.3 mu g Cn2 neutralized per mt of serum) was induced by rabbit anti-S P1 serum.