Resolving multivalent antibody binding states with defined spatial antigen-patterning

Multivalent interactions are fundamental to many biological systems, including how antibodies bind to their respective antigens. Still, their accumulated binding strength, i.e. avidity, resulting from binding and rebinding events of multiple interacting points, remains difficult to measure. Classical assay platforms like ELISA and SPR lack control over antigen positioning, limiting the resolution of the binding characteristics that shape biological outcomes at nanoscale. Here, we present PANMAP, a planar and plate-based assay inspired by ELISA that uses DNA origami to present antigens at defined nanoscale patterns, enabling direct measurements of spatially resolved binding events, termed avidity profiles. Using IgG antibodies as a model system, PANMAP distinguishes between monovalent and bivalent binding states by combining equilibrium absorbance measurements with a simple biophysical model. The avidity profiles reveal how the balance shifts between monovalent and bivalent interactions as a function of antibody concentration and antigen spacing, with intermediate separation distances favoring bivalent binding events and excluded at both near and far distances. This spatial profiling allows decoupling of affinity dependency from avidity profiles to reveal how spatial constraints influence binding equilibria. Our approach fills a longstanding gap in multivalent interaction measurement and offers a new tool for antibody engineering, development of multivalent reagents, therapeutic screening, and mechanistic immunology.