Induction of neutralizing antibodies against Tityus serrulatus scorpion toxins by immunization with a mixture of defined synthetic epitopes

We have used the Spot method of multiple peptide synthesis to prepare sets of immobilized overlapping peptides of uniform size (15 mer), covering the complete amino acid sequences of TsNTxP a non-toxic and immunogenic protein and TsIV, an alpha -type toxin that is the major lethal component of the v enom of scorpion Tityus serrulatus. Anti-TsNTxP antibodies binding to pepti des, revealed three antigenic regions, one in the N-terminal, the second in the central part and the other in the C-terminal part of TsNTxP. One pepti de epitope in the C-terminal part of TsIV was identified with anti-TsIV neu tralizing rabbit antibodies. Anti-peptide antibodies were raised against th ese four peptides all together covalently coupled to keyhole limpet hemocya nin (KLH) and found to neutralize in vitro the toxic effects of the T serru latus venom. Quantities of venom equivalent to 13,5 LD50 were effectively n eutralized by I ml of the anti-peptide serum. The antigenic specificities o f the anti-peptides were compared by an indirect enzyme-linked immunosorben t assay (ELISA) using synthetic peptides and crude venoms from T. serrulatu s, T. bahiensis, T. cambridgei, T. stigmurus, Androctonus autralis Hector a nd Centruroides sculpturatus to coat the microtitration plates. The anti-pe ptide antibodies had a comparable high reactivity with the crude venom of T . serrulatus, moderate binding to T. bahiensis, T. cambridgei, T. stigmurus and Centruroides sculpturatus venoms but were unable to recognize the veno m of Androctonus autralis Hector. These results show that by using peptides derived from the sequence of scorpion toxins, the generation of anti-pepti de antibodies able to neutralize the cognate venom appears to be an alterna tive strategy for the easy preparation of antivenoms. (C) 2001 Published by Elsevier Science Ltd.