Measuring Nanobody Kinetics at the Single-Molecule Level

Summary: 

Antibodies are a paradigm for high-affinity, protein-based binding reagents and are extremely important in biotechnological, diagnostic, and therapeutic applications. Of special interest are nanobodies, recombinant variable domains from heavy-chain-only antibodies. Nanobodies have several advantages: their small molecular weight, superior solubility and stability and clearance rate. One particular use of nanobodies is their use in imaging, which requires tailoring affinity and specificity for their targets. Despite their benefits, nanobodies have only recently been used in single-molecule assays, e.g. PAINT imaging, which require careful tuning of their kinetic properties. In this work, we demonstrate the detection of nanobody-antigen binding at the single-molecule level. We first demonstrate the measurement of interaction kinetics between an immobilised GFP target and a LaG-16 antibody in solution, with similar results to bulk-derived kinetics constant. We then demonstrate the same kind of experiments in a more complex situation with multiple nanobodies.