Evaluating the synergistic neutralizing effect of anti-botulinum oligoclonal antibody preparations.
Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD50-500 MsLD50. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential.
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Eran
E
Diamant
Department of Infectious Diseases, Israel Institute for Biological Research, Ness Ziona, Israel.
Bat-El
BE
Lachmi
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Adi
A
Keren
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Ada
A
Barnea
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Hadar
H
Marcus
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Shoshana
S
Cohen
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Alon Ben
AB
David
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Ran
R
Zichel
D000704:Analysis of Variance; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology*; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology*; D001905:Botulinum Toxins / Q000037:antagonists & inhibitors / Q000276:immunology*; D003014:Clostridium botulinum / Q000737:chemistry*; D003470:Culture Media; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D009500:Neutralization Tests; D043025:Oligoclonal Bands / Q000276:immunology*
D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D003470:Culture Media; D007074:Immunoglobulin G; D043025:Oligoclonal Bands; D001905:Botulinum Toxins