Binding Kinetics of Antiricin Single Domain Antibodies and Improved Detection Using a B Chain Specific Binder

Abstract

Single domain antibodies are the recombinantly expressed binding fragments derived from heavy chain antibodies found in camels and llamas. These unique binding elements offer many desirable properties such as their small size ( approximately 15 kDa) and thermal stability, which makes them attractive alternatives to conventional monoclonal antibodies. We created a phage display library from llamas immunized with ricin toxoid and selected a number of single domain antibodies. Phage selected on ricin were found to bind to either ricin A chain or the intact molecule; no ricin B chain binders were identified. By panning on B chain, we identified binders and have characterized their binding to the ricin B chain. While they have a poorer affinity than the previously described A chain binders, it was found that they performed dramatically better as capture reagents for the detection of ricin, providing a limit of detection in enzyme linked immunosorbent assay (ELISA) below 100 pg/mL and excellent specificity for ricin versus the highly related RCA 120 (1 to 10 000). We also reevaluated the previously isolated antiricin single domain antibody binding kinetics using surface plasmon resonance and found their K(d)s matched closely to those previously obtained under equilibrium binding conditions measured using the Luminex flow cytometer.

Stepwise Engineering of Heterodimeric Single Domain Camelid VHH Antibodies That Passively Protect Mice from Ricin Toxin

“…Finally, the V H Hs identified in this study may also prove useful in ricin toxin detection. Indeed, Anderson et al (36) developed a ricin-specific immunoassay using camelid V H Hs that can differentiate between ricin and the closely related protein RCA I. With this assay, they were able to achieve sensitive ricin detection (Ͻ100 pg/ml) using an anti-RTB V H H, B4, with an affinity for ricin of 2 nM.…”

Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications

“…Ideally, antibodies that bind to different epitopes on either the A or B subunit are required for efficient detection of ricin. Anderson et al [22] reported on high affinity sdAbs against both RTA and RTB and their combined use in detection platforms that included sandwich ELISA and Luminex immunoassay. From the panel of six antibodies evaluated in this study, three epitopes, two from the B subunit and one for the A subunit, were identified.…”

“…Pelat et al [20] has reported on the generation and characterization of phage library-derived human single-chain Fv (scFv) fragments with the capacity to neutralize biological activity of ricin. Similarly Anderson et al [21,22] and Walper et al [23] have generated and characterized phage-library derived single-domain antibodies (sdAbs) against RTA and RTB.…”

Selection and evaluation of single domain antibodies toward MS2 phage and coat protein

“…CD measurements were performed using a Jasco J-815 CD spectropolarimeter equipped with a PTC-423S peltier temperature control system, as described previously (Anderson et al, 2010).…”

“…Each variable domain has three CDRs, which have the greatest sequence variability and also form the antigen-binding pockets that determine the affinity and specificity of each antibody for its antigen (Muyldermans, 2001). In combination with phage display technology and biopanning, immune sdAb libraries containing high affinity binder repertoire provide a facile route to select highly specific and strong affinity antibodies for sensitive detection of bio-threat agents (Anderson et al, 2010;Conway et al, 2010;Graef et al, 2011;Sherwood et al, 2007;Walper et al, 2012).…”