Vipoxin is a potent postsynaptic heterodimeric
neurotoxin isolated from the
venom of the Bulgarian snake Vipera ammodytes meridionalis, whose
snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic,
anticoagulant,
convulsant, hypotensive, hyperglycemic etc.). The neutralization of
snake toxins calls for extensive research through the application of different approaches:
antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as
synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human
scFv antibodies against the
vipoxin's two subunits--basic and toxic
phospholipase A₂ (PLA₂) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized
vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 10⁹ pfu/ml) were analyzed for their binding (ELISA) and
enzyme-inhibiting abilities.
Single chain Fv-phage clones--D₁₂, E₃, F₆, D₁₀ and G₅ exhihest binding affinity for the toxic component. Clones A₁, D₁₂ and C₁₂ recognized preferentially
vipoxin's acidic component. Clones E₃, G₅ and H₄ inhibited the enzymatic activity of both
vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E₃, G₅, H₄, C₁₂, D₁₀ and A₁₁) inhibited direct hemolytic activity of
vipoxin and its pure PLA₂ subunit. The obtained specific
scFv antibodies will be used for
epitope mapping studies required to shed light on the role of the
phospholipase A₂ activity for the
vipoxin toxicity and its effective neutralization.