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 alpha-bungarotoxin (alpha-BT), but not Naja naja kaouthia alpha-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 alpha-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 alpha-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.

A novel in vitro potency assay of antisera against Thai Naja kaouthia based on nicotinic acetylcholine receptor binding

“…An in vitro assay based on PSNT binding to nAchR was studied by Stiles 25 , 26 and Alape-Giron et al . 12 , 27 .…”

Characterization of α-conotoxin interactions with the nicotinic acetylcholine receptor and monoclonal antibodies

“…Our repeated attempts to develop antibodies against sublethal doses of native unconjugated α-conotoxin GI were not productive. However, sublethal doses of various snake venom α-neurotoxins have been very effective for the generation of mAbs [28,35].…”

Characterization of multiple nicotinic acetylcholine receptor‐binding proteins and phospholipases A2 from the venom of the coral snake Micrurus nigrocinctus nigrocinctus

“…The N-terminal segment of Mnn-4 (Table 1) is 35% identical with those of N. n. oxiana neurotoxin II and N. n. atra ~-cobrotoxin, while the N-terminal part of Mnn-3C is 42% identical with that of the long-chain ~-neurotoxin 3.9.4 from N. melanoleuca [14]. Mnn-3C was also recognized in an ELISA by polyclonal antibodies to the long-chain ~-neurotoxin N. n. oxiana neurotoxin I, but not by five MAbs to this toxin [11].…”

“…Mouse antisera to Naja naja atra ~-cobrotoxin and N. n. oxiana neurotoxins I and II were generated as described [10,11]. Antisera against notexin and ,8-bungarotoxin were prepared in rabbits by s.c. injection of native toxins (1 /zg) emulsified in Freund's complete adjuvant (Difco, Detroit, MI).…”

“…Horse M. nigrocinctus antivenom was produced as described [12]. Ascites fluids containing MAbs were produced in pristane-primed BALB/c mice [5,11].…”