Single domain antibodies (sdAb) that bind ricin with high affinity and specificity were selected from a phage display library derived from the mRNA of heavy chain antibodies obtained from lymphocytes of immunized llamas. The sdAb were found to recognize three distinct epitopes on ricin. Representative sdAb were demonstrated to function as both capture and tracer elements in fluid array immunoassays, a limit of detection of 1.6 ng/mL was obtained. One sdAb pair in particular was found to be highly specific for ricin. While polyclonal antibodies cross react strongly with RCA120, the sdAb pair had minimal cross reactivity. In addition, the binders were found to be thermal stable, regaining their ricin binding activity following heating to 85 degrees C for an hour. Cycles of thermally induced unfolding of the sdAb and their subsequent refolding upon cooling was monitored by circular dichroism. As several of the sdAb were observed to bind to ricin's A chain, cell free translation assays were performed to monitor the ability of the sdAbs to inhibit ricin's biological activity. One of the sdAb (C8) was particularly effective and blocked ricin's biological activity with an effectiveness equal to that of a mouse antiricin antibody. These results indicate that antiricin sdAb have great potential for both diagnostic and therapeutic applications.

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.

Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations.

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 Correspondence to: Ellen R. Goldman, ellen.goldman@nrl.navy.mil. Electronic supplementary material The online version of this article

Phage-displayed single domain antibodies (sdAb) were compared to monomeric solubly expressed sdAb and llama polyclonal antibodies for the detection of ricin. SdAb are comprised of the variable domain derived from camelid heavy chain only antibodies (HcAb). Although HcAb lack variable light chains, they as well as their derivative sdAb are able to bind antigens with high affinity. The small size of sdAb (∼16 kDa), while advantageous in many respects, limits the number of labels that can be incorporated. The ability to incorporate multiple labels is a beneficial attribute for reporter elements. Opportunely, sdAb are often selected using phage display methodology. Using sdAb displayed on bacteriophage M13 as the reporter element gives the potential for incorporating a very high number of labels. We have demonstrated the use of both sdAb and phage- displayed sdAb for the detection of ricin using both enzyme linked immunosorbent assays (ELISAs) and Luminex fluid array assays. The phage-displayed sdAb led to five to ten fold better detection of ricin in both the ELISA and Luminex assays, resulting in limits of detection of 1 ng/mL and 64 pg/mL respectively. The phage-displayed sdAb were also dramatically more effective for the visualization of binding to target in nitrocellulose dot blot assays, a method frequently used for epitope mapping.

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