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.

Duplex DNA was attached to semiconductor nanoparticles providing selective detection of thrombin. Using the method reported here, semiconductor nanoparticles can have selective sensory functions for a host of additional analytes in the future. The system uses one DNA strand that selectively binds an analyte (thrombin), while the complementary DNA strand contains a redox-active metal complex. The accessibility of the metal complex to the nanoparticle surface is increased upon thrombin binding due to unravelling of the duplex DNA secondary structure. Increased interactions between the metal complex and the nanoparticle surface will decrease nanoparticle emission intensity, through charge transfer. Initially, water-soluble nanoparticles with carboxylate-terminated monolayers showed thrombin-specific responses in emission intensity (-30% for 1:1 nanoparticle to DNA, +50% for 1:5). Despite the selective responses, the thrombin binding isotherms indicated multiple binding equilibria and more than likely nanoparticle aggregation. The need for a nonaggregative system comes from the potential employment of these sensors in live cell or living system fluorescence assays. By changing the nanoparticle capping ligand to provide an ethylene glycol-terminated monolayer, the binding isotherms fit a two-state binding model with a thrombin dissociation constant of 3 nM in a physiologically relevant buffer. This article demonstrates the need to consider capping ligand effects in designing biosensors based on semiconductor nanoparticles and demonstrates an initial DNA-attached semiconductor nanoparticle system that uses DNA-analyte binding interactions (aptamers).