Document Type

Article

Publication Date

2025

Publication Title

Langmuir

Keywords

biopolymers, immobilization, immunology, nutrition, peptides and proteins

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-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.

Funding Source

This work was funded by the National Institutes of Health – NIGMS (Awards 1R15GM146167-01 and 1R15GM146167-01S1). Partial support was also provided by Illinois State University, Department of Chemistry. This article was published Open Access thanks to an agreement between Milner Library and ACS.

DOI

10.1021/acs.langmuir.5c00656

Comments

First published in Langmuir (2025): https://doi.org/10.1021/acs.langmuir.5c00656

Supporting information available at https://pubs.acs.org/doi/10.1021/acs.langmuir.5c00656?goto=supporting-info

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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