Multiplexing Nanobody Kinetics Measurements at the Single-Molecule Level


Single-molecule methods provide useful insights into the intermolecular variations and functional differences of individual molecules and have revealed much about the complexity of biological processes. Today it is possible to monitor binding kinetics at the single molecule level in a highly parallel fashion using zero-mode waveguide (ZMW) arrays. This allows monitoring the binding/unbinding of fluorescently-labelled molecules on millions of immobilised targets simultaneously, obtaining a full kinetic description of their interactions, offering a complementary picture to classical techniques but also uncovering important details that are missed in bulk studies. In our work, we use ZMW arrays to study the binding kinetics of antibody-antigen interactions at the single-molecule level. Moreover, we demonstrate that it is possible to couple these binding kinetics measurements with single-molecule protein sequencing to multiplex kinetic analysis to panels of nanobodies. This new approach to the study of nanobodies will help us understand the sequence/function relationship in nanobodies and open new directions in nanobody affinity maturation.