title: Selecting and purifying autonomous human variable heavy (VH) domains.
abstract: Antibodies are invaluable macromolecules effectively utilized as detection reagents and therapeutics. Traditionally, researchers have relied upon the entire immunoglobulin molecule, however advances in protein engineering have ushered the use of antibody fragments as equally important biological tools such that at present, the downstream application generally dictates the antibody format employed. We provide herein robust and proven protocols for the isolation of autonomous human antibody variable heavy domains (VH). The strategy utilizes combinatorial phage-displayed libraries targeting human VH domain positions previously shown to promote autonomous behavior, and selection against a specified antigen. Subsequently, autonomous VH domains are characterized and chosen using standard biophysical methods.
authors.affiliation: Terrence Donnelly Center for Cellular and Biomolecular Research and Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada.
authors.forename: Raffi
authors.initials: R
authors.lastname: Tonikian
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D000915:Antibody Affinity / Q000276:immunology; D061505:Cell Surface Display Techniques; D015723:Gene Library; D006801:Humans; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D013205:Staphylococcal Protein A