Isolation and characterization of Clostridium difficile toxin-specific single-domain antibodies

Camelidae single-domain antibodies (VHHs) are a unique class of small binding proteins that are promising inhibitors of targets relevant to infection and immunity. With VHH selection from hyperimmunized phage display libraries now routine and the fact that VHHs possess long, extended complementarity-determining region (CDR3) loop structures that can access traditionally immunosilent epitopes, VHH-based inhibition of targets such as bacterial toxins are being explored. Toxin A and toxin B are high molecular weight exotoxins (308 kDa and 269 kDa, respectively) secreted by Clostridium difficile that are the causative agents of C. difficile-associated diseases in humans and in animals. Here, we provide protocols for the rapid generation of C. difficile toxin A- and toxin B-specific VHHs by llama immunization and recombinant antibody/phage display technology approaches and for further characterization of the VHHs with respect to toxin-binding affinity and specificity and the conformational nature of their epitopes.