Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs

• DOI: 10.1007/978-1-61779-974-7_8
• Publication Date: 2012-10-19T18:00:53Z
• Authors: Cécile Vincke; Carlos Gutiérrez; Ulrich Wernery; Nick Devoogdt; Gholamreza Hassan...; Serge Muyldermans
• Keywords: single domain antibody; Camelus; DNA, Complementary; Genetic Vectors; Molecular Sequence Data; Restriction Mapping; Antibody engineering; Polymerase Chain Reaction; Epitopes; Escherichia coli; recombinant antibody; Animals; Bacteriophages; Amino Acid Sequence; Lymphocytes; Molecular Biology; Base Sequence; Single-Domain Antibodies; 3. Good health; Electroporation; Nanobody; RNA; Immunization; phage display; Immunoglobulin Heavy Chains; Camelids, New World

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.