Antagonism of the intracellular action of botulinum neurotoxin type A with monoclonal antibodies that map to light-chain epitopes.
mAbs were produced in mice against highly purified, renatured light chain (LC) of botulinum neurotoxin A (BoNT A) that was immobilised on nitrocellulose to avoid the undesirable use of toxoids. Subcutaneous implants of relatively high amounts (up to 10 micrograms each) of LC allowed its slow release into the systemic circulation and, thus, yielded much higher antibody titres against the underivatized antigen than had hitherto been obtained by conventional immunization. Seven stable hybridoma cell lines were established which secrete mAb of IgG1 and IgG2b subclasses reactive specifically with BoNT A and LC, in native and denatured states, without showing any cross-reactivity with types B, E, F or tetanus toxin. The pronounced reactivities of three mAbs towards refolded 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 the lethality of BoNT in vivo, administration intraneurally of mAb7 prevented the inhibition of transmitter release normally induced by subsequent 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 neurotoxins reported to be essential for maintaining the tertiary structure of the chain. Most importantly, when mAbs 4 or 7 were microinjected inside ganglionic neurons of Aplysia, each reversed, though transiently, the blockade of acetylcholine release by the toxin; this novel finding is discussed in relation to the nature of the zinc-dependent protease activity of the toxin.
Cenci Di Bello
I
I
Department of Biochemistry, Imperial College of Science, Technology & Medicine, London, England.
Poulain
B
B
Shone
C C
CC
Tauc
L
L
Dolly
J O
JO
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology*; D001048:Aplysia; D001905:Botulinum Toxins / Q000037:antagonists & inhibitors* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D009101:Multiple Myeloma; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D009498:Neurotoxins / Q000037:antagonists & inhibitors* / Q000276:immunology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009498:Neurotoxins; D010455:Peptides; D001905:Botulinum Toxins