ANTAGONISM OF THE INTRACELLULAR ACTION OF BOTULINUM NEUROTOXIN TYPE-AWITH MONOCLONAL-ANTIBODIES THAT MAP TO LIGHT-CHAIN EPITOPES

mAbs were produced in mice against highly purified, renatured light ch ain (LC) of botulinum neurotoxin A (BoNT A) that was immobilised on ni trocellulose to avoid the undesirable use of toxoids. Subcutaneous imp lants of relatively high amounts (up to 10 mu g each) of LC allowed it s slow release into the systemic circulation and, thus, yielded much h igher antibody titres against the underivatized antigen than had hithe rto been obtained by conventional immunization. Seven stable hybridoma cell lines were established which secrete mAb of IgG(1) and IgG(2b) s ubclasses reactive specifically with BoNT A and LC, in native and dena tured states, without showing any crossreactivity with types B, E, F o r tetanus toxin. The pronounced reactivities of three mAbs towards ref olded LC or intact toxin, observed in immunobinding and precipitation assays, relative to that seen in Western blots imply a preference for conformational epitopes. Though mAbs 4, 5 and 7 failed to neutralize t he lethality of BoNT in vivo, administration intraneurally of mAb7 pre vented the inhibition of transmitter release normally induced by subse quent extracellular administration of BoNT A. Notably, the latter mAb reacted with a synthetic peptide corresponding to amino acids 28-53 in the N-terminus of the LC, a highly conserved region in Clostridial ne urotoxins reported to be essential for maintaining the tertiary struct ure of the chain. Most importantly when mAbs 4 or 7 were microinjected inside ganglionic neurons of Aplysia, each reversed, though transient ly, the blockade of acetylcholine release by the toxin; this novel fin ding is discussed in relation to the nature of the zinc-dependent prot ease activity of the toxin.