Characterization of monoclonal antibodies against Naja naja oxiana neurotoxin I

Abstract

Seven monoclonal antibodies (mAbs) were developed against neurotoxin I (NT-1), a protein from central Asian cobra (Naja naja oxiana) venom which binds specifically to nicotinic acetylcholine receptor (AchR). All of the mAbs cross-reacted with another long-chain post-synaptic neurotoxin, Bungarus multicinctus α-bungarotoxin (α-BT), but not Naja naja kaouthia α-cobratoxin, in an enzyme-linked immunosorbent assay (e.l.i.s.a.). Short-chain post-synaptic neurotoxins like Naja naja atra cobrotoxin, Laticauda semifasciata erabutoxin b, or N. n. oxiana neurotoxin II did not cross-react with the NT-1 mAbs, but an antigen(s) found in Dendroaspis polylepis, Acanthophis antarcticus and Pseudechis australis venoms was immunoreactive. The e.l.i.s.a. readings for dithiothreitol-reduced NT-1 and NT-1 mAbs ranged from 13 to 27% of those for native toxin but reduced α-BT was not immunoreactive. Synthetic NT-1 peptides were used in epitope-mapping studies and two, non-contiguous regions (Cys15-Tyr23 and Lys25-Gly33 or Pro17-Lys25 and Asp29-Lys37) were recognized by the NT-1 mAbs. The NT-1 mAbs individually inhibited 31-71% of α-BT binding to AchR in vitro and afforded a slight protective effect in vivo with a toxin:antibody mole ratio of 1:1.5. This report is the first to describe mAbs which recognize and protect against a heterologous, long-chain, post-synaptic neurotoxin from snake venom.