Single‐molecule validation and optimised protocols for the use of secondary nanobodies in multiplexed immunoassays

Recently developed secondary nanobodies or single-domain antibodies present a powerful tool for immunodetection. Unlike traditional antibodies, their monovalence enables pre-association with primary antibodies prior to sample staining, presenting a straightforward affinity-based antibody labelling solution. This not only simplifies and streamlines immunoassays, it also supports multiplexed techniques where conflicts in the species of the desired primary antibodies preclude standard indirect immunostaining. Despite these advantages, the use of secondary nanobodies remains sparse, due perhaps to a lack of evaluation on their suitability for assays requiring quantification and an assessment of optimal protocols for their use. Here, we propose a set of experiments spanning total internal reflection fluorescence and confocal microscopies that can be used to validate secondary nanobody binding, specificity, and their propensity for mis-targeted binding in multiplex assays. Using these tools, we analysed the binding properties of commercially available secondary nanobodies and outline optimised protocols for their robust use.