title: Hapten mediated display and pairing of recombinant antibodies accelerates assay assembly for biothreat countermeasures.
abstract: A bottle-neck in recombinant antibody sandwich immunoassay development is pairing, demanding protein purification and modification to distinguish captor from tracer. We developed a simple pairing scheme using microliter amounts of E. coli osmotic shockates bearing site-specific biotinylated antibodies and demonstrated proof of principle with a single domain antibody (sdAb) that is both captor and tracer for polyvalent Marburgvirus nucleoprotein. The system could also host pairs of different sdAb specific for the 7 botulinum neurotoxin (BoNT) serotypes, enabling recognition of the cognate serotype. Inducible supE co-expression enabled sdAb populations to be propagated as either phage for more panning from repertoires or expressed as soluble sdAb for screening within a single host strain. When combined with streptavidin-g3p fusions, a novel transdisplay system was formulated to retrofit a semi-synthetic sdAb library which was mined for an anti-Ebolavirus sdAb which was immediately immunoassay ready, thereby speeding up the recombinant antibody discovery and utilization processes.
authors.affiliation: Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, Texas, USA.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Sherwood
authors.forename: Andrew
authors.initials: A
authors.lastname: Hayhurst
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D001905:Botulinum Toxins / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006241:Haptens / Q000276:immunology*; D029024:Marburgvirus / Q000378:metabolism; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000276:immunology; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D019809:Streptavidin / Q000235:genetics / Q000378:metabolism
chemical_list: D006241:Haptens; D009698:Nucleoproteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D019809:Streptavidin; D001905:Botulinum Toxins