Generation of Single-Chain Fv Fragments and Multivalent Derivatives scFv-Fc and scFv-CH3 (Minibodies)

Intermediate-sized antibody fragments, such as scFv-C_H 3 (minibodies) and scFv-Fc, retain high avidity for their antigen due to their nature of bivalency. In addition, the incorporation of the Fc region enables the generation of antibody fragments with a variety of biological functions and pharmacokinetic properties. This protocol describes the isolation of variable genes from hybridoma cells, gene assembly, cloning, and stable expression of these fragments in mammalian cells. These fragments are expressed as single peptide chains which bypass the challenges of coexpressing immunoglobulin heavy and light chains. Included is also a protocol to screen for high expressing clones, and suggestions for purification and biochemical characterization of the purified proteins.