Abstract / Summary
ABSTRACT Antibody precomplexation is a well‐known method to enhance the valency and affinity of hemagglutinin (HA), thus allowing their use in various tests, such as hemagglutination assays. How antibody precomplexation regulates the valency of HAs from monovalent to multivalent and thus enhances their affinity, however, remains elusive. Here, we analyze the binding of antibody‐precomplexed HA constructs to glycan‐containing supported lipid bilayers (SLBs) with controlled receptor density to assess the effects of valency on affinity. Although precomplexation of HA increases hemagglutination with red blood cells (RBCs), mass photometry data shows a heterogeneous mixture of components with variable HA valencies. The Δ D n /‐Δ F n ratios ( i . e ., the acoustic ratios) from quartz crystal microbalance with dissipation monitoring (QCM‐D) confirm the heterogeneity of complexes also at the surface. Using QCM‐D combined in situ with spectroscopic ellipsometry (SE), we observed an avidity enhancement of precomplexed HA from the micromolar to the nanomolar range. Despite the distribution in valencies, precomplexed HA shows superselective binding responses to varying surface receptor densities. Orientation of the binding sites and the rigidity of the precomplexed HA structures affect the binding in different ways depending on the presentation of the receptors, i.e., 2D on a flat surface and 3D in solution.