Binding kinetics of antiricin single domain antibodies and improved detection using a B chain specific binder.
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.
Anderson
George P
GP
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375, USA.
Bernstein
Rachael D
RD
Swain
Marla D
MD
Zabetakis
Dan
D
Goldman
Ellen R
ER
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002161:Camelids, New World / Q000378:metabolism; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007700:Kinetics; D019151:Peptide Library; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012276:Ricin / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D019151:Peptide Library; D012276:Ricin