Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications

The single variable new antigen receptor domain antibody fragments (V(NAR)s) derived from shark immunoglobulin new antigen receptor antibodies (IgNARs) represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of V-NAR domains with a focus on a family of V(NAR)s specific for apical membrane antigen 1 (AMA-1) from Plasmodium falciparum. The V(NAR)s are compared to traditional monoclonal antibodies (mAbs) in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 degrees C, V(NAR)s have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that V-NAR domains are able to refold following heating to 80 degrees C. We also demonstrate that V-NAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark V-NAR domains for various diagnostic platforms and related applications.