title: Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera.
abstract: 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.
authors: Sofia University "St. Kliment Ohridski", Faculty of Biology, Department of Biochemistry, Laboratory of Enzymology, 8 "Dragan Tsankov" Blvd., 1164 Sofia, Bulgaria.
authors: Vishnya
authors: V
authors: Stoyanova
authors: Radoslav
authors: R
authors: Aleksandrov
authors: Maria
authors: M
authors: Lukarska
authors: Deyan
authors: D
authors: Duhalov
authors: Vasil
authors: V
authors: Atanasov
authors: Svetla
authors: S
authors: Petrova
mesh_terms: D000818:Animals; D002031:Bulgaria; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D009498:Neurotoxins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D054467:Phospholipases A2 / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000378:metabolism* / Q000494:pharmacology; D014757:Viper Venoms / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D017819:Viperidae / Q000378:metabolism*
chemical_list: D009498:Neurotoxins; D057127:Single-Chain Antibodies; D014757:Viper Venoms; C033134:vipoxin; D054467:Phospholipases A2