Developing protein interaction-based technologies such as biosensors requires a clear understanding of receptor-target kinetics. Nanobodies, which are camelid-derived single-domain antibodies, are ideal biosensor receptors due to their high specificity, stability, and ease of production. During biosensor development, multiple nanobody variants are often tested against the same target to identify the best binders. Surface plasmon resonance (SPR) is a robust, label-free method for measuring these interactions, but its many experimental variables can complicate the establishment of a streamlined protocol specially for non-high-throughput instruments. Here, we present an SPR workflow that enables the comparative analysis of nanobody variants binding to lysozyme as a model target on a Biacore T100 instrument. These protocols cover the steps from protein expression and purification to final affinity ranking. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of lysozyme-specific nanobodies Support Protocol 1: Protein identification Basic Protocol 2: SPR workflow for the characterization of nanobody variants Support Protocol 2: SPR planning and assay development.
Díaz‐Galicia et al. (Fri,) studied this question.