Llama Single Domain Antibodies Specific for the 7 Botulinum Neurotoxin Serotypes as Heptaplex Immunoreagents

Abstract

BackgroundThere are currently 7 known serotypes of botulinum neurotoxin (BoNT) classified upon non-cross reactivity of neutralizing immunoglobulins. Non-neutralizing immunoglobulins, however, can exhibit cross-reactivities between 2 or more serotypes, particularly mosaic forms, which can hamper the development of highly specific immunoassays, especially if based on polyclonal antisera. Here we employ facile recombinant antibody technology to subtractively select ligands to each of the 7 BoNT serotypes, resulting in populations with very high specificity for their intended serotype.Methods and FindingsA single llama was immunized with a cocktail of 7 BoNT toxoids to generate a phage display library of single domain antibodies (sdAb, VHH or nanobodies) which were selected on live toxins. Resulting sdAb were capable of detecting both toxin and toxin complex with the best combinations able to detect 100s-10s of pg per 50 µL sample in a liquid bead array. The most sensitive sdAb were combined in a heptaplex assay to identify each of the BoNT serotypes in buffer and milk and to a lesser extent in carrot juice, orange juice and cola. Several anti-A(1) sdAb recognized A2 complex, showing that subtype cross-reactivity within a serotype was evident. Many of our sdAb could act as both captor and tracer for several toxin and toxin complexes suggesting sdAb can be used as architectural probes to indicate BoNT oligomerisation. Six of 14 anti-A clones exhibited inhibition of SNAP-25 cleavage in the neuro-2A assay indicating some sdAb had toxin neutralizing capabilities. Many sdAb were also shown to be refoldable after exposure to high temperatures in contrast to polyclonal antisera, as monitored by circular dichroism.ConclusionsOur panel of molecularly flexible antibodies should not only serve as a good starting point for ruggedizing assays and inhibitors, but enable the intricate architectures of BoNT toxins and complexes to be probed more extensively.

Assembly and Function of the Botulinum Neurotoxin Progenitor Complex

“…Nanobodies are the smallest antigen-binding fragments (~12-15 kDa) of naturally occurring heavy-chain-only antibodies (VHH) present in camelids (Hamers-Casterman et al 1993;Muyldermans et al 2001) and they have been exploited as inhibitors for BoNTs (Conway et al 2010;Dong et al 2010;Mukherjee et al 2012;Tremblay et al 2010). VHH provided additional sites to facilitate a more compact crystal packing Lam et al 2009), which significantly improved the diffraction quality of crystals to 2.7 Å resolution.…”

Selection of Recombinant Antibodies from Antibody Gene Libraries

“…In principle, the antigens are immobilized to a solid surface, such as nitrocellulose [ 63 -65 ], column matrices [ 15 , 16 ] or mostly on plastic surfaces with high protein binding capacity such as polystyrene tubes or microtiter wells [ 37 , 66 -68 ]. Alternatively, panning can also be performed on antigens immobilized on magnetic beads [ 69 ] or in solution with biotinylated antigens followed by a "pulldown" using streptavidin-coated magnetic beads [ 36 , 70 , 71 ]. After incubation of the antibody phage library with the antigen, stringent washing steps are performed to remove the vast excess of nonbinding antibody phage.…”

Disorders of the Neurologic System and Special Senses

“…Interestingly, a new rapid typing test is based on llama single-domain antibodies. 76 Treatment is mainly supportive and follows the guidelines for tick paralysis. Nutritional support is more important because the clinical course is longer.…”