Understanding aggregation-based assays: nature of protein corona and number of epitopes on antigen matters

The development of assays that exploit aggregation of gold nanoparticles (NPs) has been widely studied for detection of biomolecules in diagnostics. These assays are often based on antibody–antigen interactions to mediate aggregation of NPs. However, the protein parameters underlying the performance of these assays are not well understood. In this study, we systematically examine how the nature of the protein corona on the NPs, formed from either antibody or antigen, and how the number of binding sites or epitopes on the antigen affect aggregation. We selected two small antigen proteins: 13 kDa recombinant dengue viral envelope domain III protein with a polyhistidine tag (DIII-His), and 19 kDa vascular endothelial growth factor A (VEGFA), to form protein corona around NPs and study the aggregation induced by their monoclonal and polyclonal antibodies. We then reciprocated the systems to form protein corona with the antibodies and compared the aggregation induced by the antigens. We showed that the nature of the protein corona matters, as the corona formed from antigens had lower limits of detection and elicited greater degrees of NP aggregation compared to the corona formed from antibodies. Furthermore, the number of epitopes on the antigen matters, as polyclonal antibodies, which target multiple epitopes on the antigen, were able to induce aggregation for both antigen- and antibody-corona systems. In contrast, monoclonal antibodies that target a single epitope on the antigens induced aggregation for the antigen-corona system only. Our results showed that an understanding of the antibody–antigen system is crucial for establishing guidelines for rational selection of proteins in the design of aggregation-based assays with NPs.