Antibody Binding to Peptide Conjugated Gold Nanoparticles Preferring Dense Antigen Coating with Small Deflection Angle

Antibody–antigen interactions are fundamental to immune defenses against pathogen infection, where antibody binding affinity and mode control functional outcomes. While literature show optimal IgG binding on flat surfaces occurring at relatively large interantigen distances (d ≈ 10–16 nm), how nanoscale curvature affects IgG binding remains unclear. Here, we present a model antigen–nanoparticle system by displaying varying densities of His6 peptides on gold nanoparticles (GNPs). Using GNPs’ nanoscale size and strong fluorescence-quenching capability, we probe their binding with anti-His antibody via dynamic light scattering (DLS) and GNP-based fluorescence quenching assays. We find that DLS detects only stable, chelative complexes, whereas the GNP quenching assay captures both stable and dynamic interactions and quantifies their affinities. We show that antibody binding is highly sensitive to antigen density: reducing His6 density from 10% to 2% (d increasing from ∼3.3 to ∼6.3 nm) weakens affinity by over 120-fold. For antigen–GNP conjugates with d values within the antibody’s accessible spacing range, the antigen deflection angle θ dominates binding behavior: θ ≤ 16° favors strong chelative binding, whereas θ ≥ 37° permits mainly weak monovalent binding.