Isolation of Antigen-Specific Nanobodies

Recombinant Nanobodies, derived from camelid heavy-chain antibodies, are a unique form of monoclonal antibodies composed of a single antigen-binding domain. Nanobodies are the smallest intact antigen-binding entities that can be derived from an in vivo affinity-matured antibody. They are small (15 kDa, 2.2 nm diameter, 4 nm height), soluble, stable, robust, and easy to produce in various host cells. Nanobodies bind their cognate antigens with nanomolar affinities and often interact with epitopes that are less accessible to/or less immunogenic for classical antibodies. Nanobodies are easily tailored for various applications. We have developed a robust technology to identify antigen-specific Nanobodies. Here, we describe, in detail, the protocols for the generation of Nanobody phage display libraries and for the isolation and characterization of antigen-specific Nanobodies from such libraries.