Abstract

A method to obtain polyvalent anti-Bitis and polyvalent-anti-Naja antibodies was developed by immunizing horses with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambica crude venoms. Antibody production was followed by the ELISA method during the immunization procedure. Once the desired anti-venom antibody titers were attained, horses were bled and the immunoglobulins were separated from the sera by (NH₄)₂SO₄ precipitation, cleaved with pepsin and filtered through a 30 kDa ultrafiltration membrane. F(ab´)₂ fragments were further purified by Q-Fast Flow chromatography, concentrated by molecular ultrafiltration and sterilized by filtration through 0.22 μm membranes. The resulting F(ab´)₂preparations were rich in intact L and in pieces of H IgG(T) chains, as demonstrated by electrophoresis and Western blot and exhibited high antibody titers, as assayed by the ELISA method. In addition, the preparations possess a significant capacity to neutralize the lethality of venoms, as estimated by ED₅₀ determination in mouse assay and are free of toxic substances, pyrogen and bacterial or fungal contaminations.

Key words: African snake venoms, envenoming, antivenoms, immunoglobulin IgG(T), F(ab`)₂ fragments, immunotherapy.

Abbreviation

ELISA, Enzyme-linked immunosorbent assay; SDS-PAGE,sodium dodecylsulfate - polyacrylamide gel electrophoresis; ED₅₀, half maximum effective dose; LD₅₀, half maximum lethal dose.