Title: Evaluation of anti-hemagglutinin Hn-33 single domain antibodies: kinetics, binding epitopes, and thermal stability

Authors: George P. Anderson, Dan Zabetakis, Rachael D. Bernstein, Shuowei Cai, Bal Ram Singh, Ellen R. Goldman

Addresses: Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington DC 20375, USA. ' Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington DC 20375, USA. ' Nova Research Inc., 1900 Elkin St # 230, Alexandria, VA 22308, USA. ' Botulinum Research Center, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA. ' Botulinum Research Center, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA. ' Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington DC 20375, USA

Abstract: Botulinum Neurotoxin A (BoNT/A) is produced by Clostridium botulinum as a complex with Neurotoxin-Associated Proteins (NAPs) which protect the toxin from proteases and promote adsorption in the gut. Hemagglutinin 33 (Hn-33) makes up the largest fraction of NAPs in the BoNT/A complex. We characterised single domain antibodies (sdAb) which bind Hn-33; the dissociation constant (Kd) varied from 3.8 × 10⁻⁹ M to 1.3 × 10⁻¹⁰ M. Sandwich assays showed that the six sdAb bind two distinct epitopes on Hn-33. Circular dichroism was used to monitor sdAb|s secondary structure, thermal denaturing upon heating and rapid refolding upon cooling.

Keywords: botulinum neurotoxins; hemagglutinin; single domain antibody; thermal stability; luminex; surface plasmon resonance; circular dichroism; kinetics; binding epitopes.

DOI: 10.1504/TBJ.2011.041816

The Botulinum Journal, 2011 Vol.2 No.1, pp.59 - 71

Published online: 06 Aug 2011 *

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