Development of a generalisable tryptophan-optimised quenchbody biosensor based on a synthetic nanobody library
Quenchbodies are antibodies labelled with a fluorophore that increases in fluorescence intensity upon antigen binding, which makes them promising candidates for the development of diagnostic or other immunoassays requiring antigen quantification. Quenchbodies based on camelid nanobodies are particularly attractive for rapid development of immunoassays against molecular antigens of interest, due to their small size, ease of expression, high stability, rapid evolvability, and amenability to protein engineering. However, current nanobody-based quenchbodies display only modest fluorescence increases when binding to protein antigens (≤ 1.1-1.4-fold), with few examples reaching > 2-fold. Here we show that tryptophan residues in the nanobody CDRs are critical to fluorescence antigen detection, which subsequently informed the development of a synthetic convex-binding nanobody library that was used for the in vitro production of novel quenchbodies against human inflammatory cytokine interleukin