Enzyme-Mediated Site-Specific Conjugation of Antibodies to Nanoparticles

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PROJECT SUMMARY/ABSTRACT Despite extensive efforts, current strategies of antibody-gold nanoparticle (AuNP) conjugation chemistries are not universally applicable to all antibodies, are pH dependent, result in random orientation for diminished activity, and/or have limited stability. These issues must be addressed to realize the full potential of AuNP-enabled as- says and promote wide-spread acceptance and implementation in the diagnostics arena. The long-term goal of this research is to develop a strategy to immobilize antibody onto gold nanoparticles to form highly active, oriented, and stable conjugates for use in AuNP-enabled immunoassays. Our central hypothesis is that en- zyme-mediated, site-specific conjugation of a polypeptide to the Fc fragment of an antibody to incorporate multi- ple thiol groups and a high density of localized positive charge will promote oriented and robust adsorption onto gold nanoparticles. Our rationale for these studies is that successful completion would drive the novel design of highly active and stable antibody-gold nanoparticle conjugates critically needed to advance AuNP-enabled plat- form technologies. Driven by compelling preliminary data, we will test our hypothesis and progress toward our long-term research goal by completing the following Aims: 1) Enzyme-mediated Modification and Characteriza- tion of Antibodies; 2) In situ Analysis of Modified Antibody Adsorption onto AuNPs; and 3) Quantitative Analysis of Antigen-binding Activity for Antibody-AuNP Conjugates. In our previous and ongoing work, we have detailed the role of localized protein charge and thiol functional groups on the orientation and affinity, respectively, for the adsorption of proteins to gold nanoparticles. Under the first aim, a short, cysteine- and lysine-rich peptide will be conjugated to the site-specific Q295 that is conserved on the Fc fragment of the antibody using microbial transglu- taminase (mTG) and the conjugation will be confirmed by mass spectrometry and protein charge. Under the second aim, a competitive protein binding assay and nanoparticle tracking analysis, already validated in our lab, will be used to quantitatively evaluate the binding affinity of the peptide-modified antibody to gold nanoparticles. Under the last aim, a previously proven enzyme assay will be used to quantify the antigen-binding activity for bioconjugates formed with unmodified and peptide-modified antibodies. This strategy is innovative because of the use of a polypeptide as a crosslinking agent between the antibody and the AuNP surface and the use of an enzyme for site-specific conjugation of the peptide to the Fc region the antibody. The project is significant be- cause it enables the precise control of antibody adsorption onto gold nanoparticles without protein engineering and will lead to generalized design principles that provide a streamlined process to optimize immobilization chemistry to form robust and highly functional nanoparticle probes.