RELATIONSHIP BETWEEN THE LOCALIZATION OF EPITOPES IN THE TOXINS OF Tityus serrulatus SCORPIONS AND THE NEUTRALIZING POTENTIAL OF THERAPEUTIC ANTIVENOMS

MARIA, W.S.; GRANIER, C.; VELARDE, D.T.; CHÁVEZ-OLÓRTEGUI, C.

Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG, Brazil and Institut de Biotechnologie et  Pharmacologie, Faculté de Pharmacie, Montpellier, France.

Administration of antivenom, prepared from hyperimmunized horse plasma is a recognized therapeutic means of circumventing poisoning by Tityus serrulatus (Ts) scorpion venom. The neutralizing potential of antivenoms has been assessed by studying neutralization of lethality, a procedure that causes the death of a large number of mice. It has therefore become increasingly important to develop alternative methods for assaying antivenoms.  Here we  address the issue of the identification of epitopes, by assessing  the capacity of overlapping peptides derived from the three T. serrulatus  toxins, Ts IV  (a-type),  Ts II and Ts VII (toxin-gboth b-type) to react with horse anti-Ts antivenoms of different potency.

Anti-Ts venom plasmas were obtained following conventional immunization schedules of the FUNED. Mice were used in the in vivo neutralization assays following the official resolution of Health Department from Brazil. Results were analyzed by the Probit test and neutralization is expressed as 50% effective dose (ED₅₀). Overlapping pentadecapeptides(24 for Ts VII, 25 for Ts II and 26 Ts IV toxins) frame shifted by 2 residues were prepared according to the protocol previously described by Molina et al.,1996 and bound to cellulose membranes. These membranes were probed by incubation with sera of immunized horses .

The results showed that  the same peptides are strongly recognized by the sera with high neutralizing potential and  indicate that the Spot-technique using synthetic peptides covalently bound to cellulose membrane should be used as a in vitro  technique to evaluate the potency of Brazilian Tityus antivenoms.

Supported by: CNPq, INSERM. 

CORRESPONDENCE TO:

Wany Selena Maria, Alameda dos Cariocas 280,  Vespasiano, MG, CEP: 33 200 000 , Brasil, Email: selena@funed.mg.gov.br