Biotin binder (BB) and BB-LucYY5: A sensitive and quantitative platform for biotin-labelled protein analysis

Biotin is a key small molecule in life sciences because its strong interaction with streptavidin enables highly sensitive and robust detection of proteins and nucleic acids. However, the tetrameric structure required for stable binding of biotin to streptavidin limits its flexibility as a detection molecule. In this study, we developed a luciferase-based assay to analyse biotinylated proteins using a novel biotin binder (BB) derived from the single-chain variable fragment (scFv) of a high-affinity rabbit monoclonal antibody. Fourteen monoclonal antibody clones were isolated using the ISAAC method, and three clones with high specificity for biotinylated lysine peptides were selected. Surface plasmon resonance analysis identified clone 49 as having the highest binding affinity, and its scFv and IgG were designated BB and BB-IgG, respectively. BB-IgG enabled the highly sensitive detection of biotin-labelled proteins by immunoblotting, immunoprecipitation, immunostaining, and electron microscopy of animal cells, plants, and malarial parasites. Furthermore, BB fused to a stable luciferase (BB-LucYY5) allowed the quantitative analysis of biotinylated proteins in vitro and in immunoprecipitated samples. These findings demonstrated that the BB system provides a versatile platform for detecting biotin-labelled proteins, and that BB-LucYY5 offers a quantitative assay for their analysis.