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

ABSTRACT: 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

SUBMITTER: Beitello E 

PROVIDER: S-EPMC12044683 | biostudies-literature | 2025 Apr

REPOSITORIES: biostudies-literature