title: Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.
abstract: There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9) M to 7.0×10(-10) M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5) pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.
authors.affiliation: Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington, DC, United States of America.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Walper
authors.affiliation: Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington, DC, United States of America.
authors.forename: Jinny L
authors.initials: JL
authors.lastname: Liu
authors.affiliation: Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington, DC, United States of America.
authors.forename: Daniel
authors.initials: D
authors.lastname: Zabetakis
authors.affiliation: Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington, DC, United States of America.
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
authors.affiliation: Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Washington, DC, United States of America.
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
mesh_terms: D000818:Animals; D002161:Camelids, New World / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007114:Immunization; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D039841:Leukocyte L1 Antigen Complex / Q000276:immunology*; D019151:Peptide Library; D061905:Single-Domain Antibodies / Q000008:administration & dosage / Q000276:immunology*; D014615:Vaccinia / Q000276:immunology* / Q000473:pathology / Q000517:prevention & control; D014616:Vaccinia virus / Q000276:immunology* / Q000472:pathogenicity
chemical_list: D007143:Immunoglobulin Heavy Chains; D039841:Leukocyte L1 Antigen Complex; D019151:Peptide Library; D061905:Single-Domain Antibodies