Single-Domain Antibodies for Intracellular Toxin Neutralization

Ricin is a plant-derived toxin with a history as a biothreat agent. The toxin’s enzymatic subunit, ricin toxin A chain (RTA), is a ribosome-inactivating protein that, when delivered into the cytoplasm of mammalian cells, arrests protein synthesis with extraordinary efficiency. Once within the cytoplasm, RTA is shielded from circulating toxin-neutralizing antibodies. Here, we describe methods we developed to neutralize RTA within the cytoplasm of Vero cells using DNA-based delivery of alpaca-derived single-domain antibodies (VHHs) targeting RTA’s active site. We describe the design of the VHH expression vectors, assessment of transient expression of VHHs in Vero cells by enzyme-linked immunosorbent assay and western blotting, and cytotoxicity studies. While the protocols here are specific to ricin, they are easily modified for other toxins or even intracellular pathogens such as viruses.