Oriented Surface Immobilization of Antibodies Using Enzyme-Mediated Site-Specific Biotinylation for Enhanced Antigen-Binding Capacity

The effectiveness of surface-immobilized antibodies is often diminished by improper antibody orientation and limited stability, impeding the analytical performance of biosensors. Here, we report a novel enzyme-mediated strategy to biotinylate the Fc region of an anti-horseradish peroxidase (anti-HRP) antibody with site-specificity that enables oriented immobilization on a streptavidin-functionalized surface. Microbial transglutaminase (mTG) catalyzes the covalent coupling between the amine functional group on a biotin analogue (NH₂-PEG₄-biotin) and the side chain of a privileged glutamine residue (Q295) located on the heavy chain Fc region of IgG antibodies. For comparison, an anti-HRP antibody was biotinylated using an amine-reactive biotin analogue (NHS-PEG₄-biotin) to covalently couple to lysine residues randomly located throughout the antibody. The antibody that reacted with a 40-fold excess of biotin reagent formed conjugates with a biotin-to-antibody ratio of 1.9 ± 0.3 and 5.0 ± 0.6 for the site-specific and random biotinylation strategies, respectively. Western blot analysis confirms that mTG-mediated biotinylation is restricted to the heavy chain, while lysine-targeted biotinylation is observed on both the heavy and light chains. The site-specific and randomly biotinylated antibodies were immobilized onto streptavidin-coated polystyrene 96-well plates to evaluate antigen (HRP) binding activity. The site-specific biotinylated antibody provided a 3-fold improvement in antigen binding capacity, sensitivity, and detection limit, that is attributed to the proper orientation of the antibody when immobilized through the Fc region. This chemo-enzymatic strategy is universally applicable to other antibodies for oriented antibody immobilization via site-specific linking chemistries without the need for protein engineering.