Graduation Term

Summer 2025

Degree Name

Master of Science (MS)

Department

Department of Chemistry

Committee Chair

Jeremy D. Driskell

Committee Member

Jon A. Friesen

Committee Member

Lisa F. Szczepura

Abstract

Immobilization strategies used for fixing antibodies to solid phase surfaces significantly contribute to the antigen capture capacity and influences the performance and sensitivity of immunoassays. Traditional methodologies result in the random orientation and/or the weak attachment of immobilized antibodies. Different formats of immunoassays scope in applicability to many fields of research and everyday use. Improving the efficiency of immobilizing antibodies in immunoassays is a beneficial endeavor to increase assay sensitivity. Here, we investigated the use of enzyme-mediated conjugation of antibodies to facilitate a highly oriented immobilization. Microbial transglutaminase (mTG) catalyzes the covalent bond formation between a plethora of primary amine containing substrates to the side chain of glutamine residues found within a specific chemical environment. In regards to IgG antibody conjugation, mTG targets one specific glutamine residue, Q295, located on the Fc region away from the antigen binding sites. We explored site-specific biotinylation via mTG using an NH2-PEG4-Bt linker on a rat IgG1 anti-HRP antibody for directed and oriented immobilization to streptavidin functionalized surfaces. For immunoassay performance analysis, site-specific biotinylations were compared to random biotinylations achieved with an NHS-PEG4-Bt linker that conjugated to any primary amine accessible on the anti-HRP antibody surface. With optimized reaction conditions, mTG biotinylations were quantified as a ratio of approximately 1.9 biotins per antibody, with a theoretical maximum of 2, after removing a sterically hindering glycan at N297 with PNGase F enzymatic deglycosylation. Random biotinylations were quantified as a ratio of approximately 5 biotins per antibody where the randomness of conjugation was shown to not hinder the antigen binding event through localized surface plasmon resonance. The site-specific nature of mTG biotinylation to only the heavy chain of the IgG antibody was confirmed with Western blot analysis. Oriented immobilization provided a 3-fold increase in antigen binding capacity compared to random immobilization. This increase in orientation and loading capacity by site-specific biotinylation contributed to the immunoassay’s low detection limit of 9.3 pM HRP antigen and high sensitivity relative to other immunoassay performances achieved with random biotinylations, traditional ELISAs, and Fc binding orientation facilitators like protein G. Further investigations with the use of mTG mediated conjugation are being explored to demonstrate the enzyme’s universality to IgGs of different host species as well as subtypes. Additionally, biotinylation linkers are being optimized in terms of length with different PEG units to establish a maximum increase in antigen binding capacity and therefore improved immunoassay performance.

Access Type

Thesis-Open Access

DOI

https://doi.org/10.30707/ETD.1763755358.78493

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