Quantifying Bound and Active Antibodies Conjugated to Gold Nanoparticles: A Comprehensive and Robust Approach To Evaluate Immobilization Chemistry

[Image: see text] Gold nanoparticles (AuNPs) functionalized with antibodies have the potential to improve biosensing technology because of the unique optical properties of AuNPs and the specificity of antibody–antigen interactions. Critical to the development and optimization of these AuNP-enabled sensing technologies is the immobilization of the antibody onto the AuNP. The development of novel immobilization strategies that optimize antibody loading and orientation in an effort to enhance antibody activity, and therefore assay performance, has been the focus of many recent studies. However, few analytical methods exist to accurately quantify the activity of conjugated antibodies and reliably compare different immobilization strategies. Herein, we describe an enzyme-mediated assay to quantify the fraction of the immobilized antibodies that is accessible for antigen binding. Anti-horseradish peroxidase (anti-HRP) antibody is mixed with AuNPs to allow for conjugation, and the unbound, excess antibody is quantified with a modified Bradford assay to determine antibody loading onto AuNPs. The conjugates are then mixed with excess HRP to saturate all accessible binding sites, and bound HRP is quantified based on enzymatic reaction rate. This analytical scheme was used to compare two common immobilization strategies, nonspecific adsorption and protein A-mediated immobilization. We found that the antibody surface coverage is greater for direct adsorption than protein A-mediated binding; however, 23 ± 6% of the directly adsorbed antibodies were active, whereas 91 ± 19% of the antibodies bound through protein A were active. In addition to establishing this method as quantitatively precise and accurate, our results emphasize the need to quantify both antibody loading and antibody activity upon conjugation to gain greater insight into differences in immobilization chemistries and identify optimum protein conjugation strategies to maximize immunoassay performance.