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

Marla D. Swain, George P. Anderson, Dan Zabetakis, Rachael D. Bernstein, Jinny L. Liu, Laura J. Sherwood, Andrew Hayhurst, Ellen R. Goldman

Research output: Contribution to journalArticle

24 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)339-348
Number of pages10
JournalAnalytical and Bioanalytical Chemistry
Volume398
Issue number1
DOIs
StatePublished - Sep 2010
Externally publishedYes

Fingerprint

Single-Domain Antibodies
New World Camelids
Neurotoxins
Binders
Toxoids
Assays
Bacteriophages
Heating
Serogroup
Protein Refolding
Surface plasmon resonance
Microspheres
Surface Plasmon Resonance
Screening
Proteins
Thermodynamic stability
Libraries
Display devices

Keywords

  • Botulinum neurotoxin
  • Camelid
  • Immunoassay
  • Single-domain antibody

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

Cite this

Swain, M. D., Anderson, G. P., Zabetakis, D., Bernstein, R. D., Liu, J. L., Sherwood, L. J., ... Goldman, E. R. (2010). Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin. Analytical and Bioanalytical Chemistry, 398(1), 339-348. https://doi.org/10.1007/s00216-010-3905-3

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin. / Swain, Marla D.; Anderson, George P.; Zabetakis, Dan; Bernstein, Rachael D.; Liu, Jinny L.; Sherwood, Laura J.; Hayhurst, Andrew; Goldman, Ellen R.

In: Analytical and Bioanalytical Chemistry, Vol. 398, No. 1, 09.2010, p. 339-348.

Research output: Contribution to journalArticle

Swain, MD, Anderson, GP, Zabetakis, D, Bernstein, RD, Liu, JL, Sherwood, LJ, Hayhurst, A & Goldman, ER 2010, 'Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin', Analytical and Bioanalytical Chemistry, vol. 398, no. 1, pp. 339-348. https://doi.org/10.1007/s00216-010-3905-3
Swain, Marla D. ; Anderson, George P. ; Zabetakis, Dan ; Bernstein, Rachael D. ; Liu, Jinny L. ; Sherwood, Laura J. ; Hayhurst, Andrew ; Goldman, Ellen R. / Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin. In: Analytical and Bioanalytical Chemistry. 2010 ; Vol. 398, No. 1. pp. 339-348.
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