title: Llama-derived single domain antibodies specific for Abrus agglutinin.
abstract: 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.
authors.affiliation: Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, 4555 Overlook Ave SW, Washington, DC 20375, USA. ellen.goldman@nrl.navy.mil
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
authors.forename: Dan
authors.initials: D
authors.lastname: Zabetakis
authors.forename: Scott
authors.initials: S
authors.lastname: Walper
authors.forename: Jinny L
authors.initials: JL
authors.lastname: Liu
authors.forename: Rachael
authors.initials: R
authors.lastname: Bernstein
authors.forename: Alena
authors.initials: A
authors.lastname: Calm
authors.forename: James P
authors.initials: JP
authors.lastname: Carney
authors.forename: Thomas W
authors.initials: TW
authors.lastname: O'Brien
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Walker
authors.forename: Eric A E
authors.initials: EA
authors.lastname: Garber
mesh_terms: D000036:Abrin / Q000008:administration & dosage / Q000276:immunology*; D000818:Animals; D000941:Antigens / Q000008:administration & dosage / Q000276:immunology*; D002161:Camelids, New World / Q000276:immunology*; D002942:Circular Dichroism; D007114:Immunization; D037121:Plant Lectins / Q000276:immunology*; D061905:Single-Domain Antibodies / Q000276:immunology*
chemical_list: D000941:Antigens; D037121:Plant Lectins; D061905:Single-Domain Antibodies; C016940:abrus agglutinin; D000036:Abrin