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Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin

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Abstract

Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders. All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated selected sdAb as capture and tracer elements demonstrated that all of the sdAb were able to recognize soluble (“live”) BoNT A Tx and BoNT Ac Tx with virtually no cross-reactivity with other BoNT serotypes. The isolated sdAb did not exhibit the high degree of thermal stability often associated with these reagents; after the first heating cycle most of the binding activity was lost, but the portion of the protein that did refold and recover antigen-binding activity showed only minimal loss on subsequent heating and cooling cycles. The binding kinetics of selected binders, assessed by both an equilibrium fluid array assay as well as surface plasmon resonance (SPR) using toxoided material, gave dissociation constants (K D ) in the range 2.2 × 10−11 to 1.6 × 10−10 M. These high-affinity binders may prove beneficial to the development of recombinant reagents for the rapid detection of BoNT A, particularly in field screening and monitoring applications.

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Acknowledgments

Marla D. Swain is a National Research Council (NRC) Postdoctoral Fellow. We thank Tetracore Inc for providing the monoclonal anti-BoNT B antibody. This work was supported by JSTO-CBD/DTRA Project # 8.10033_07_NRL_B, JSTO-CBD/DTRA Contract # HDTRA 1-07-C-0018 and NIH construction grant C06 RR012087.

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Authors and Affiliations

  1. Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC, 20375, USA

    Marla D. Swain, George P. Anderson, Dan Zabetakis, Jinny L. Liu & Ellen R. Goldman

  2. Nova Research Inc., 1900 Elkin Street, Suite 230, Alexandria, VA, 22308, USA

    Rachael D. Bernstein

  3. Department of Virology and Immunology, Southwest Foundation for Biomedical Research, 7620 NW Loop 410, San Antonio, TX, 78227, USA

    Laura J. Sherwood & Andrew Hayhurst

Authors
  1. Marla D. Swain
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  2. George P. Anderson
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  3. Dan Zabetakis
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  4. Rachael D. Bernstein
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  5. Jinny L. Liu
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  6. Laura J. Sherwood
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  7. Andrew Hayhurst
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  8. Ellen R. Goldman
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Correspondence to Ellen R. Goldman.

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Swain, M.D., Anderson, G.P., Zabetakis, D. et al. Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin. Anal Bioanal Chem 398, 339–348 (2010). https://doi.org/10.1007/s00216-010-3905-3

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  • DOI: https://doi.org/10.1007/s00216-010-3905-3

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