PRODUCTION AND IMMUNOLOGICAL CHARACTERIZATION OF A NEUTRALIZING MONOCLONAL ANTIBODY SPECIFIC TO DERMONECROTIC PROTEIN FROM Loxosceles intermedia SPIDER VENOM

Oliveira,J.C.R., Bohórquez,K., Nascimento,A.L., Martins,M.S., Alvarenga,L.M., Granier, C., Chávez-Olortegui, C.

Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil. Departamento de Parasitologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. Institud de Biotechnologie et Pharmacologie, Université de Montpellier, France.

A group of related proteins of 35 kDa with sphingomyelinase activity has been described as the major toxic components and is involved in the dermonecrotic, lethal activity and induction of C-mediated haemolysis. Studies confirmed the importance of monoclonal and polyclonal antibodies for the neutralization of the dermonecrosis activity of Loxosceles venoms. We have prepared monoclonal antibodies by immunization of mice with crude venom of L. intermedia. One monoclonal antibody was very efficient to neutralize the dermonecrotic effects of L. intermedia. The antigenic specificity of this monoclonal antibody was compared by an indirect enzyme-linked immunosorbent assay and immunoblotting using crude venoms from L. intermedia, L. laeta (Brazil), L. laeta (Perú) and L. gaucho to coat the microtitration plates or nitrocellulose membranes. The antibody had reactivity with L. intermedia but was unable to recognize the other Loxosceles venoms. To determine the specific antigenic region(s) of dermonecrotic protein, we have used the Spot method of multiple peptide synthesis to prepare sets of immobilized overlapping peptides of uniform size (12 mer), covering the complete aminoacid sequence of a dermonecrotic protein. Anti-dermonecrotic monoclonal antibody binding to continuous peptides, revealed epitopes in the N-terminal part of the dermonecrotic protein. This study confirm the hypothesis that effective serum therapy against loxoscelism should contain antibodies against all medically important venom species.

Supported by: FAPEMIG, CNPqand INSERM

CORRESPONDENCE TO:

Júlio Cézar Rodrigues de Oliveira,Rua João Gualberto filho 931 , Belo Horizonte ,MG, CEP: 31035570, Brasil,Email: oliveirajcr@uol.com.br