title: Semiautomated panning of naive camelidae libraries and selection of single-domain antibodies against peptide antigens.
abstract: With the identification of vast numbers of novel proteins through genomic and proteomic initiatives, the need for efficient processes to characterize and target them has increased. Antibodies are naturally designed molecules that can fulfill this need, and in vitro methodologies for isolating them from either immune or naïve sources have been extensively developed. However, access to pure protein antigens for screening purposes is a major hurdle due to the limitations associated with recombinant production of eukaryotic proteins. Consequently, rational peptide design based on proteomic methodologies such as protein modeling, secondary sequence prediction, and hydrophobicity/hydrophilicity prediction, in combination with other bioinformatics data, is being explored as a viable solution to isolate specific antibodies against difficult antigens. Single-domain antibodies are becoming the ideal antibody format due to their structural advantages and ease of production compared to conventional antibodies and antibody fragments derived from conventional antibodies. For screening purposes, phage display technology is a well-established technique. With this technique, a repertoire of antibody fragments can be displayed on the surface of filamentous phages (f1, fd, M13) followed by screening against various antigenic targets. Furthermore, the technique can be expanded to a high-throughput scale using a magnetic-based, in-solution panning protocol which allows for the screening of multiple target antigens simultaneously. In this chapter, we describe a semiautomated panning method to screen a naïve Camelidae library against rationally designed peptide antigens, followed by preliminary characterization of isolated binders.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, Canada.
authors.forename: Jyothi
authors.initials: J
authors.lastname: Kumaran
authors.forename: C Roger
authors.initials: CR
authors.lastname: Mackenzie
authors.forename: Mehdi
authors.initials: M
authors.lastname: Arbabi-Ghahroudi
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D000941:Antigens / Q000737:chemistry / Q000276:immunology*; D057205:Automation, Laboratory; D002161:Camelids, New World / Q000235:genetics / Q000276:immunology; D061505:Cell Surface Display Techniques / Q000379:methods*; D019151:Peptide Library; D010455:Peptides / Q000276:immunology*; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000941:Antigens; D019151:Peptide Library; D010455:Peptides; D061905:Single-Domain Antibodies