Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB), ricin, and botulinum toxin A (BoNT/A) complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications.We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.Sharks, similar to camelids, possess unconventional heavy (H) chain antibodies, consisting of heavy chain homodimers in which each chain contains one single variable and five constant domains [1,2]. The conserved amino acid residues between the shark heavy chain antibody and those involved in forming the core of immunoglobulin and T-cell receptor variable regions, gave impetus for naming shark heavy chain antibody Immunoglobulin New Antigen Receptor (IgNAR or NAR) [2,3]. Structural analysis by electron microscopy, crystal structure, and 3D modeling revealed that there are three NAR isotypes. These isotypes are defined according to their pattern of inter-loop disulfide linkages within the variable region and the timing of their appearance during the animal's development [3-6].Using genetic engineering a single NAR variable (V) antigen-binding domain can be expressed as a separate soluble protein often referred to as a shark single domain antibody (sdAb). Shark sdAbs contain four conserved frame regions (FRs) and two complementarity-determining regions (CDRs), making them the smallest (~12 kD) Ig-based recognition units with full capacity for antigen bindin