Microbial Transglutaminase Mediated Conjugation of Antibodies for Improved Orientation onto Gold Nanoparticles Across Various Amino Crosslinkers

Author

• Faith E. Breausche, Illinois State UniversityFollow

Graduation Term

Summer 2026

Degree Name

Master of Science (MS)

Department

Department of Chemistry

Committee Chair

Jeremy D. Driskell

Committee Member

Patrick C. Hillesheim

Committee Member

Jon A. Friesen

Abstract

Immunoassays and numerous biosensors are based on specific interactions between antibodies and their targets referred to as antigens. The sensitivity of those diagnostic assays can be improved by optimizing antibody orientation on the surface (e.g., gold nanoparticles, membranes, polystyrene) to increase the accessibility of antigen-binding sites. Historically, antibodies have been immobilized through randomly positioned lysine residues using chemical methods such as N-hydroxysuccinimide (NHS) activated ester. This approach, however, may lead to improper orientation and diminished antigen capture as the antigen-binding sites are blocked by the surface. To improve the antibody’s orientation upon immobilization, our group aimed for site-specific attachment of a payload to function as a chemical tether onto the fragment crystallizable (Fc) region using microbial transglutaminase (mTG). mTG has been reported to conjugate the primary amine of a payload (e.g., NH₂-PEG₄-Biotin) to a conserved glutamine residue (Q295) of an antibody. We have previously reported successful site-specific conjugation of biotin with mTG across various host species and subtypes of antibodies. Currently, we are investigating the adsorption of mTG modified antibodies onto a gold nanoparticle (AuNP) surface through various immobilization strategies, including biotin-streptavidin, copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), lipoic acid-Au, and polyhistidine tag-Ni²⁺ nitrilotriacetic acid. Preliminary results show improvement, across some of the aforementioned modification strategies, in antibody orientation upon binding to its complimentary functionalized AuNP surface when compared to randomly modified antibodies (NHS ester). This work demonstrates the flexibility of mTG to site-specifically install many established linking chemistries and provides new opportunities to improve the sensitivity of biosensors and immunoassays.

Access Type

Thesis-Open Access

Recommended Citation

Breausche, Faith E., "Microbial Transglutaminase Mediated Conjugation of Antibodies for Improved Orientation onto Gold Nanoparticles Across Various Amino Crosslinkers" (2026). Theses and Dissertations. 2369.
https://ir.library.illinoisstate.edu/etd/2369