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

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 x 10(-11) to 1.6 x 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.

Fig. 1

Schematic representations of the processes used for (a) direct binding assays and (b) sandwich assays by Luminex

Fig. 2

Specific binding of selected soluble sdAb to cognate antigen. Clones DA5, DF1, DF5 and NA6 (panels A–D) are representatives of the many isolated sdAb that were highly specific for BoNT A Tx. Homogeneous direct-binding assays were performed by incubating sdAb with microspheres coated with BoNT A toxin, BoNT A toxoid and BoNT A complex toxoid, as well as other BoNT toxin serotypes

Fig. 3

Ability of soluble sdAb protein to bind to BoNT A Tx-coupled microspheres after exposure to heat. Both mouse and rabbit conventional antibody and sdAb were heated to 85 °C for variable amounts of time and then cooled prior to assays. Sample assay concentrations were 1 μg/mL and 10 μg/mL for sdAb and conventional antibody, respectively

Fig. 4

Titration of BoNT A Tx-coupled microspheres with sdAb clones DF5 and DA5 after four successive heating and cooling cycles. For these assays, 50 μL of sdAb clones (50 μg/mL in PBS) were heated from 25 °C to 85 °C at a rate of 6 °C/min and then held at 85 °C for 5 min and cooled back down to room temperature. Serial dilutions of cycled samples were then added to Luminex microspheres coated with BoNT A Tx

Fig. 5

Live BoNT A toxin sandwich assays using sdAb clones DF5, DA5, DA2 and A18 as captors paired with four different antibody tracers. The captor/tracer pairs were challenged with 1×10⁵pg of BoNT A toxin or BoNT A toxin complex. Specificity for BoNT A toxin or A toxin complex was evaluated by challenging the captor/tracer pairs with 1×10⁵pg each of the other six BoNT serotypes

Fig. 6

Live BoNT A toxin sandwich assays using sdAb clones DF5 and DA5 tracers paired with various antibody captors. The background values used were derived from signals obtained at the lowest BoNT A Tx concentration (0.46 ng/ml)

Fig. 7

CD melting curves of sdAb DA5 during heating cycles. The blue curve is the first heating cycle, where a spectrum was collected at each step (not shown), with only the value at 203 nM shown. The green, red and black curves are the second, fourth, and seventh heating cycles, respectively, where only the value at 203 nm was monitored during the heating cycle