title: Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands.
abstract: Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (1) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (2) expression, purification, and characterization of the multivalent peptide-VT ligands; (3) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (4) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield custom peptide-VT ligands that appear to maintain the antigenic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, ON, Canada, K1A 0R6.|College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Norah A
authors.initials: NA
authors.lastname: Alturki
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, ON, Canada, K1A 0R6.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Henry
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, ON, Canada, K1A 0R6.|School of Environmental Sciences, University of Guelph, Guelph, ON, Canada.
authors.forename: C Roger
authors.initials: CR
authors.lastname: MacKenzie
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, ON, Canada, K1A 0R6. mehdi.arbabi@nrc-cnrc.gc.ca.|School of Environmental Sciences, University of Guelph, Guelph, ON, Canada. mehdi.arbabi@nrc-cnrc.gc.ca.|Department of Biology, Carleton University, Ottawa, ON, Canada. mehdi.arbabi@nrc-cnrc.gc.ca.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Arbabi-Ghahroudi
mesh_terms: D000818:Animals; D002161:Camelids, New World; D061505:Cell Surface Display Techniques; D015870:Gene Expression; D059014:High-Throughput Nucleotide Sequencing; D008024:Ligands; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D061905:Single-Domain Antibodies / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies
keywords: Camelid; NGS; Peptide; Phage display; Single-domain antibody; VHH