Citation

Stoyanova V, Aleksandrov R, Lukarska M, Duhalov D, Atanasov V, Petrova S. Toxicon 2012; 60(5): 802-809.

Affiliation

Sofia University, Faculty of Biology, Department of Biochemistry, Laboratory of Enzymology, 8 "Dragan Tsankov" Blvd., 1164 Sofia, Bulgaria.

Copyright

(Copyright © 2012, Elsevier Publishing)

DOI

10.1016/j.toxicon.2012.06.003

PMID

22750218

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, haemolytic, 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 A(2) (PLA(2)) 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 (1.2x10(9) pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones - D(12), E(3), F(6), D(10) and G(5) exhibited the highest binding affinity for the toxic component. Clones A(1), D(12) and C(12) recognized preferentially vipoxin's acidic component. Clones E(3), G(5) and H(4) inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E(3), G(5), H(4), C(12), D(10) and A(11)) inhibited direct hemolytic activity of vipoxin and its pure PLA(2) subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A(2) activity for the vipoxin toxicity and its effective neutralization.

Language: en