Graduation Term

2024

Degree Name

Master of Science (MS)

Department

Department of Chemistry

Committee Chair

Jeremy D Driskell

Abstract

Developing innovative methods that leverage the distinct characteristics of gold nanoparticles (AuNPs) has the potential to significantly advance detection technologies for addressing biological threats and improving disease intervention. However, there are challenges associated with anchoring selective recognition elements, such as antibodies, onto the surface of AuNPs. Despite extensive research, current conjugation approaches for antibody-AuNPs are not universally applicable to all antibodies, are pH-sensitive, result in random orientations that reduce activity, and/or exhibit limited stability. Resolving these issues is crucial to realizing the full potential of AuNP-based assays and promoting their widespread adoption and implementation in the field of diagnostics. The main objective of this research project is to create a reliable strategy for immobilizing antibodies onto AuNPs to form highly efficient, oriented, and stable conjugates that can be used in AuNP-based immunoassays. The Driskell Group has previously and continuously studied the effects of localized protein charge and thiol functional groups on protein adsorption to AuNPs, and we aim to utilize this knowledge to design and synthesize antibody-AuNP bioconjugates that exhibit improved performance in bioassays.The primary idea put forth is that the specific, controlled conjugation of a polypeptide that incorporates multiple thiol groups and a substantial amount of positive charge in a localized area, to the Fc (fragment crystallizable) portion of an antibody, through the action of enzymes will lead to strong and oriented adsorption onto AuNPs. This conjugate will demonstrate resistance to clustering, exhibit long-term stability, and high levels of antigen-binding activity. The purpose of these studies is to develop highly active and stable antibody-AuNP conjugates, which are essential to advance AuNP-enabled platform technologies. In this work, a microbial transglutaminase (mTG)-assisted peptide conjugation to antibodies (IgGs) was performed and optimized. Various sequences of lysine (K), which introduces the positive charges, and cysteine (C), which introduces the free thiols were used as the peptide probes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to confirm that this conjugation occurs specifically to the Fc region of the antibody. Performing a deglycosylation procedure with an enzyme, PNGase F, on the native antibody before the conjugation of dansyl-tagged peptides reveals a brighter fluorescence at the heavy chain compared to the peptide-conjugated glycosylated (native) antibody. These results prove that mTG affords a site-specific conjugation of peptides to the Fc fragment of IgG antibodies and deglycosylation of the native IgG before peptide conjugation leads to a more efficient and effective mTG catalysis. The optimized conjugation procedure was applied to investigate the universality across a series of monoclonal antibodies. Results obtained here show that mTG can conjugate peptides to all the antibodies investigated confirming the universality of this procedure across monoclonal antibodies. The peptide-modified antibodies required purification to remove the enzymes (PNGase F and mTG) and the excess peptides to enable adsorption of the isolated peptide-modified antibodies on AuNP. The peptide-modified antibodies were purified using bovine serum albumin (BSA)-blocked Magne Protein G beads at high recovery (> 45%). BSA blocking of the cellulose-made, negatively charged Protein G functionalized magnetic beads played a crucial role in limiting non-specific interactions between the positively charged primary amines on lysine side chains of the conjugated peptides. Previous attempts to purify these modified antibodies using various dialysis and molecular weight cut-off approaches like Amicon filters resulted in very low recovery (< 10%). This work culminates in designing and optimizing a synthetic approach for conjugating peptides to the base (Fc region) of IgGs and the subsequent optimization of a purification procedure for the peptide-modified IgGs as the first step to the overall project which involves studying the impact of positive charges and free thiols on the orientation and affinity of antibodies on AuNP. KEYWORDS: Microbial transglutaminase; deglycosylation; bioconjugates; peptide-modified IgG; adsorption; fluorescence

Access Type

Thesis-Open Access

DOI

https://doi.org/10.30707/ETD2024.20240827063557904179.999967

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Available for download on Wednesday, August 19, 2026

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