Specific binder for Lightning-Link® biotinylated proteins from an antibody phage library.

Many applications required protein biotinylation. We routinely use biotinylated proteins to select single chain antibodies from phage and/or yeast display libraries. During phage selection the biotinylated antigens are bound to streptavidin coupled magnetic beads, while during yeast display, the biotinylated antigens are used during flow cytometry for both analysis and sorting. The Lightning-Link® Biotin kit, a rapid straightforward biotinylation kit that avoids the need for dialysis, is particularly useful when the amount of available protein is limiting. During routine screening of antibody libraries we identified a specific clone that bound a universal neo-epitope generated only when antigens are biotinylated with the commercial Lightning-Link® kit, with an affinity of ~10nM. Non-biotinylated proteins, and those biotinylated using alternative methods - the Thermo Fisher commercial kit or in vivo biotinylation using the Avitag (Ashraf et al., 2004) - were not recognized by this antibody. Using deep sequence analysis, the specific antibody was identified as being the most abundant in a number of different selections. This indicates the need for caution when using such modifying reagents, because of the possibility of selecting antibodies against the modification, rather than the target protein, and also highlights the value of deep sequencing analysis during display based selections. Furthermore, this antibody may have great utility in the analysis of proteins biotinylated using this method.