title: Immunodiagnostic reagents using llama single domain antibody-alkaline phosphatase fusion proteins.
abstract: Naive libraries of single domain antibodies (sdAbs) enable rapid isolation of binders to nearly any target. These binders, however, lack the benefits bestowed by in vivo affinity maturation and typically have low affinity toward their targets. We expressed five low-affinity toxin binding sdAbs, previously selected from a naive library derived from variable regions of llama heavy chain-only antibodies, as fusions with a hyperactive mutant Escherichia coli alkaline phosphatase (AP) and examined the impact on apparent affinity and utility. AP spontaneously dimerizes in solution, effectively dimerizing the fused sdAbs, imparting avidity in place of the lower affinity monomeric interactions. The sdAb-AP fusion also combines the target recognition domain with a signal transduction domain, commonly used in enzyme-linked immunosorbent assays (ELISAs). The functional affinity of the sdAb-AP fusions, often increased by a factor of 10 over unfused sdAbs, and their utility as tracer reagents in ELISAs was dramatically improved, giving limits of detection of 300 ng/ml or less, whereas parental sdAbs gave no discernible signal at the toxin concentrations tested. The fusion of sdAbs to AP presents a valuable route to facilitate the implementation of sdAb-based immunoreagents rapidly selected from existing naive libraries toward new or emerging threats.
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
authors.forename: Marla D
authors.initials: MD
authors.lastname: Swain
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
authors.forename: Joseline
authors.initials: J
authors.lastname: Serrano-González
authors.forename: Jinny L
authors.initials: JL
authors.lastname: Liu
authors.forename: Dan
authors.initials: D
authors.lastname: Zabetakis
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000276:immunology*; D000818:Animals; D002161:Camelids, New World / Q000276:immunology*; D002772:Cholera Toxin / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology*; D012276:Ricin / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology*
chemical_list: D004768:Enterotoxins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C031818:enterotoxin B, staphylococcal; D012276:Ricin; D002772:Cholera Toxin; D000469:Alkaline Phosphatase