Generation of VNAR Libraries from Immunized Sharks and Selection of Target-Specific Clones

Cartilaginous fishes (sharks, skates, rays, and chimeras) are the most phylogenetically distant lineage relative to mammals in which somatically rearranging immunoglobulins (Igs or antibodies) have also been found. Alongside their conventional (heavy-light chain) isotypes, IgM and IgW, sharks produce the novel isotype, IgNAR, a heavy-chain homodimer. Naturally lacking light chains, antigen binding is mediated by two highly soluble and independently functioning variable domains, or VNARs, each having a molecular weight of approximately 12 kDa. The small size, high affinity for antigen, and extreme structural stability of single-domain VNARs make them an emerging prospect for use in therapeutic, diagnostic, and research applications. In this chapter, we detail the immunization protocol we use to raise an antigen-specific IgNAR response in the nurse shark (Ginglymostoma cirratum), the subsequent cloning of the variable domains from this isotype, and the selection of antigen-specific VNARs by phage display.