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

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Protein biotinylation is crucial for various applications, including antibody library selection using phage and yeast display systems.
• Biotinylated antigens are essential for streptavidin-based magnetic bead selection in phage display and flow cytometry in yeast display.
• The Lightning-Link® Biotin kit offers a rapid, dialysis-free method for protein biotinylation, beneficial for limited protein samples.

Purpose of the Study:

• To investigate the specificity of antibodies generated against biotinylated proteins.
• To assess the potential for selecting antibodies that recognize neo-epitopes created by specific biotinylation methods.
• To evaluate the implications of such specific antibody binding in display-based selection strategies.

Main Methods:

• Utilized phage and yeast display libraries for antibody selection.
• Employed the Lightning-Link® Biotin kit for protein biotinylation.
• Performed antibody binding assays and deep sequence analysis to identify antibody specificities.

Main Results:

• A specific antibody clone was identified, exhibiting high affinity (~10nM) for a neo-epitope exclusively generated by Lightning-Link® biotinylation.
• This antibody did not recognize non-biotinylated proteins or proteins biotinylated with alternative methods (Thermo Fisher kit, Avitag).
• Deep sequencing revealed this specific antibody to be highly abundant in multiple selection experiments.

Conclusions:

• Caution is necessary when using modifying reagents like biotinylation kits, as antibodies may be selected against the modification rather than the target antigen.
• Deep sequencing analysis is invaluable for identifying unintended antibody specificities in display-based selections.
• The identified antibody may serve as a valuable tool for detecting proteins biotinylated specifically with the Lightning-Link® method.